bluuebunny/crossref_metadata_2025 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

The 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

The 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

Human 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

Chick 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

Three 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

Exposure 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

Hydrocortisone 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

The 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

This 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

Colchicine 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

Theophylline, 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

Calf 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

Human 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

The 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

Retinol 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.
Emergence of hamster fibroblast tumors in nude mice—evidence for in vivo selection leading to loss of growth factor requirement
Authors: Pérez‐Rodriguez R. , Chambard J. C. , Van Obberghen‐Schilling E. , Franchi A. , Pouysségur J.
Published: February 2005

The Chinese hamster lung fibroblast cell line (CC139) has high anchorage dependence for growth and has retained the high serum dependence of secondary cultures of adult fibroblasts. This cell line is tumorigenic in nude mice; however, the resulting tumor cells have different properties than those of the cell line injected. The tumor‐derived cells had strongly reduced or even lost both the high anchorage and the high serum dependence of CC139 cells. This finding suggests that an in vivo selection is necessary for CC139 cells to acquire the malignant phenotype. After mutagenesis, which increases the frequency of CC139 colony formation in agarose up to 8‐fold, we selected and analyzed 15 anchorage‐independent colonies. No correlation between the colony‐forming ability in agarose and serum‐growth factor requirement for DNA synthesis was observed. Each of these clones were injected into nude mice and the growth factor dependence of the ensuing tumor cells was compared to that of corresponding injected cells. All of the anchorage‐independent colonies with the exception of one (A71), had acquired in vivo a stable phenotype allowing for partial or total escape of growth factor requirement. A71, the only clone which maintained the same growth factor requirement after two passages in vivo (A71 T1 and A71 T2) had already gained, in vitro, the minimal growth factor “relaxation” compatible with in vivo growth. A71 and A71 T1 tumor cells arrested in G0/G1 can reinitiate DNA synthesis in the presence of mouse plasma, low concentrations of serum, or thrombin. The fact that none of the tumors analyzed (more than 20) were found to have retained the high serum dependence of CC139 cells strongly suggests that the partial loss of serum growth factor requirement acquired in vivo is an essential malignant character for bypassing the hormonal growth restraints imposed by the host upon CC139 cells.
Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP
Authors: Evain Danièle , Binet Elisabeth , Anderson Wayne B.
Published: February 2005

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E1, (−) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production.

Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP‐, and fluoride‐stimulated adenylate cyclase activities show a progressive increase with the retinoic acid‐induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation.

Treatment of the retinoic acid‐generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural‐like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E1, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.
Effects of serum deprivation on the initiation of DNA synthesis in the second generation in rat 3Y1 cells
Authors: Okuda Atusyuki , Kimura Genki
Published: February 2005

Rat 3Y1 cells arrested at early S by hydroxyurea traversed the remainder of S and G2 and completed mitosis after removal of the drug, irrespective of the absence of serum from the culture medium. When cells were deprived of serum for a period between early S and mitosis after removal of hydroxyurea, the cells delayed entry into S in the presence of serium in the second generation for the time length approximately equal to that of serum deprivation. When mitotic cells, which had been continously exposed to serum after removal of hydroxyurea, were deprived of serum for the next 24 hours and then were reexposed to serum, the cells delayed entry into S for more than 24 hours (more than the time length of serum deprivation). On the other hand, the cells already deprived of serum between early S and G2 in the first generation were less delayed in entry into S after postmitotic 24‐hour serum deprivation than were the cells exposed to serum between early S and G2 in the first generation. These results suggest that serum‐dependent events continue to occur in the first generation for on‐time entry into S in the next generation, and that these premitotic events (the potential for entry into S) decay if serum is absent for a long period of time after mitosis.
Selective isolation of stable and unstable dedifferentiated variants from a rat hepatoma cell line
Authors: Moore Emma E. , Weiss Mary C.
Published: February 2005

This paper describes the selective isolation of dedifferentiated variants from a well‐differentiated rat hepatoma cell line (Fao). The well‐differentiated cells express the gluconeogenic enzymes, FDPase and PEPCK, and so can grow in a glucose‐free medium. By using glucose‐free medium in conjunction with the BudR‐visible light suicide technique it was possible to isolate two different classes of dedifferentiated variants from a mutagenized population of Fao cells. The variants of the first class resemble those previously described in that they display (1) an altered cellular morphology, (2) a pleiotrophic loss of all (or most) of the hepatic functions routinely analyzed in this laboratory, and (3) an extremely low reversion frequency (⩽ 10−8). The variants of the second class are characterized by an unstable phenotype and uncoordinated expression of the hepatic functions. Unstable variants can give rise to stable dedifferentiated variants, suggesting that the unstable variants may actually represent an intermediate in a two‐step dedifferentiation process.
Modulation of cell competence for induction of differentiation in myeloid leukemic cells
Authors: Symonds Geoff , Sachs Leo
Published: February 2005

It is shown that the competence of differentiation‐defective clones of mouse myeloid leukemic cells (MGI−D− clones) to be induced for some differentiation‐associated properties can be modulated. For certain inducers this was effected by changing the type of serum and for another inducer by removing serum from the culture medium. All the clones which could not be induced to differentiate by the differentiation‐inducing from of the normal macrophage and granulocyte inducing proteins MGI (MGI‐2), lipopolysaccharide (LPS), or dexamethasone (Dex) in serum‐free medium or in medium with horse, calf, rabbit, or sheep serum (nonpermissive serum) could be induced, by all three inducers, for the differentiation‐associated properties of lysozyme and Fc and/or C3 rosettes in syngeneic or allogeneic mouse serum or rat serum (permissive serum). The competence for induction of differentiation was altered merely by washing the cells and changing the type of serum, and addition of nonpermissive serum blocked the induction in permissive serum. This increased competence was associated with an increase in the ability for cap formation by concanavalin A. Removal of serum allowed some MGI−D− clones to be induced for lysozyme, Fc, and C3 rosettes by insulin. However, neither the changes in type of serum nor the removal of serum modulated in these clones the inducing effect of the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate. In contrast to the MGI−D− clones, changing the serum type or removing the serum did not modulate inducibility by MGI‐2, LPS, Dex, or insulin in other clones of myeloid leukemic cells that could be induced by MGI‐2 to differentiate either partly (MGI+D− clones) or completely (MGI+D+ clones) in horse or calf serum. The results indicate that by the appropriate modulation all MGI−D− clones tested could be induced for some differentiation‐associated properties; that there are factor(s) present in permissive sera which allow MGI−D− clones to be induced by MGI‐2, LPS, and Dex; that there are other factor(s) in nonpermissive sera which block this induction; and that in serum‐free medium cells of the appropriate clone can become susceptible to induction of these properties by insulin.
Isolation of hamster intestinal epithelial cells using hypoosmotic media and PVP
Authors: Carter Julia H. , Carter H. , Nussbaum J. , Eichholz Alexander
Published: February 2005

Vibration of hamster small intestinal segments in hypotonic media containing PVP is a rapid method for obtaining quantitative yields of viable intestinal epithelial cells. This preparation of epithelial cells offers a unique system for the study of epithelial cell function in vitro. The method for cell separation combines hypoosmotic swelling of cells, which separates them at the desmosomes, with mechanical agitation which releases the cells from the lamina propria. No chemical agents known to affect cell proteins and cell surfaces are employed in this procedure. Only a short time is elapsed between in vivo and in vitro conditions, i.e., a preparation time of approximately 75 minutes.

Although the technique yields a pure population of epithelial cells, the cells are of different morphologies, are removed from different areas of the crypts and villi, and therefore presumably have different functions. Examination of the intestinal tissue remaining after several vibration intervals by light and scanning electron microscopy indicates that the sequences of release of cells is removal of: (1) cells from the villus based, (2) cells from the lower one‐half to two‐thirds of the villi, (3) cells from the villus tips (and some crypts), and (4) cells from the crypts. When pools of a + b cells are compared to pools of c + d cells, it is found that villus cells can be characterized by: (1) processes, such as monosaccharide absorption, associated with the brush border, and (2) synthesis of components (e. g., glycoproteins) of the brush border. Surprisingly, disaccharide hydrolytic activity is found in cells which transport monosaccharides poorly. The subpopulations of cells synthesize proteins equally.
Activation of transglutaminase during cell cycle in CHO cells
Authors: Scott Karen F. Frasier , Russell Diane Haddock
Published: February 2005

Transglutaminase (TGase) activity was measured during cell cycle progression in Chinese hamster ovary (CHO) cells synchronized by release of quiescent cultures and in CHO cells synchronized by mitotic shake off. In cells released from quiescent cultures, a greater than 2‐fold increase in TGase activity occurred within 3 h of stimulation, followed by a rapid decline and a second and third peak of activity at 6 and 9 h after stimulation. The increase in TGase activity was not inhibited at any time measured by the administration of cycloheximide. Further, there was a significant increase in TGase activity detectable at 1, 5, and 6 h in CHO cells exposed to actinomycin D at time zero. TGase activity in CHO cells was not increased by the addition of either dibutyryl cAMP or 8‐bromo‐cAMP whereas ornithine decarboxylase (ODC) activity was increased at all times measured by the administration of cAMP analogs. These data suggest that TGase and ODC are regulated by separate hormonal pathways during cell cycle progression. In addition, ODC is induced in CHO cells by a transcriptional mechanism whereas TGase appears to be activated. In order to determine if the biphasic increase in TGase exhibited in G1 of cell cycle was the result of two populations of cells progressing through G1, we assessed TGase activity in CHO cells synchronized by mitotic shake off, which produces a greater than 95% cell synchrony. Again, there were two major peaks of TGase activity at 3 and 5 h, respectively. However, there was a marked difference in the expression of TGase within the first 2 h after release from mitosis. During this period, there was a marked and significant decrease in TGase activity within 1 h to a value about 35% of the time zero value. Another G1‐specific marker, ODC, also exhibited a marked decline in activity within the first hour, followed by a single peak of expression between 4 and 5 h in these cells. Both enzymes have high activity during G2 phase of cell cycle as previously reported for ODC. Both enzymes are rapidly decreased after release from mitosis. The peak expression of both TGase and ODC suggest they catalyze important enzymatic events required for cell cycle progression.
Modulation of synthesis of specific proteins in endothelial cells by copper, cadmium, and disulfiram: An early response to an angiogenic inducer of cell migration
Authors: Hannan G. N. , McAuslan B. R.
Published: February 2005

Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10−3 M CuSO4 or 10−5 M CdCl2 four cell‐associated polypeptides (Mr = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell‐associated protein (Mr = 32,000 and 40,000 daltons) was induced.

Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO4 (10−6 M) enhanced the synthesis in Type 2 cells of a protein (Mr = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell−associated polypeptides, does not involve a sulphydryl−containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.
Effect of insulin on folic‐acid transport in cultured fibroblasts
Authors: Eilam Y. , Ariel M. , Grossowicz N.
Published: February 2005

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of 3H‐folic acid was linear with time during 15 seconds and reached a plateau level at 2–3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of the experiments (up to 10 minutes). Insulin caused a two‐fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 m̈M insulin. A lag period of 7–10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentration (above 5 × 10−6 M) a linear component was observed. Insulin increased the slope of the linear component and the Vmax of the saturable component while the Km remained unaltered.
Effect of glycosaminoglycans on fibronectin‐mediated cell attachment
Authors: Klebe Robert J. , Mock Penny J.
Published: February 2005

In this study, the role of glycosaminoglycans in fibronectin‐mediated cell attachment to collagen has been investigated. While it has been established that fibronectin possesses binding sites for several glycosaminoglycans, it was found that only dextran sulfate and macromolecular heparin could decrease the initial rate of cell attachment to collagen. Low molecular weight heparin was inactive. This study suggests that the glycosaminoglycan binding site of fibronectin plays a role in the mechanism of cell attachment.
Immunological characterization of metallothioneins in mouse LMTK cells and in a variant resistant to cadmium
Authors: Maiti Indu , Mbikay Majambu , Marengo Claude , Thirion Jean‐Paul
Published: February 2005

A cadmium‐resistant variant isolated from mouse fibroblast LMTK cells can grow in the presence of 40 μM Cd2+. This variant retained its properties in the absence of selecting agent. Induction of metallothionein was measured in cell extracts by radioimmunoassay. The maximum amount of metallothioneins in the cells was reached after 36 hours. The cadmium resistant variant produced two times more metallothionein than the wild‐type when exposed to 10–20 μM Cd2+. By Ouchterlony double diffusion, the metallothioneins from cultured cells formed a line of parital identity with the mouse liver serotype and a line of complete identity with one of the two mouse kidney serotypes. These observations raise the possibility of a tissue‐specific expression of metallothionein genes.
Commitment and proliferation kinetics of human lymphocytes stimulated in vitro: Effects of colchicine on mitogen response
Authors: Hall David J. , O'leary James J. , Rosenberg Andreas
Published: February 2005

We have examined the effects of colchicine on concanavalin A (Con A)‐ and phytohemagglutinin (PHA)‐stimulated human peripheral blood lymphocytes and from the time course of proliferation have extracted the relative size of the responding cell population, the rate of entry of this population into S‐phase, and the length of the lag period. Additions of colchicine at any time did not appear to influence the size of the responding population nor did it greatly affect the duration of the lag period. Only the rate at which the cell population enters initial S‐phase is a function of the time of previous exposure to colchicine. Colchicine does not appear to inhibit the commitment of stimulated lymphocytes to enter the cell cycle. Rather, it merely serves to decrease the biochemical processes responsible for fixing a maximal rate of entry into S‐phase.
Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation
Authors: Logan William J. , Klip Amira , Gagalang Enriqueta
Published: February 2005

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na+‐dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α‐amino isobutyric acid (AIB), and by α‐methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid‐deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6‐fold stimulation after 6 hours. The basal and stimulated transport were equally Na+‐dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9‐fold in deprived cells. Amino acid‐deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline‐supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline‐deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly+ or L exclusively were ineffective. The stimulation of the transport‐rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid‐depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.
The use of fluorescent dyes to measure membrane potentials: A critique
Authors: Johnstone R. M. , Laris P. C. , Eddy A. A.
Published: February 2005

Under controlled conditions, fluorescent cyanine dyes can be used to mesure membrane potentials of cell suspensions. Similar changes in membrane potential can be followed both with fluorescent dyes and electrophysiological probes in response to changes in the ion composition of the medium. Recent reports that attempt to abrogate the use of the cyanine dyes in measurements of the membrane potential are misleading.
Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber
Authors: Kerrick W. G. L. , Bolles L. L.
Published: February 2005

The Ca2+ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca2+‐activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca2+ (10‐5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca2+ insensitive tension. In contrast, pretreatment with low Ca2+ (10‐8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca2+‐sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca2+‐activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP‐dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca2+‐activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.
Residual radiation injury exhibited in long‐term bone marrow cultures
Authors: Reincke Ursula , Hannon Eileen C. , Hellman Samuel
Published: February 2005

Residual radiation injury was demonstrated in long‐term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose‐response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.
Variation of the carbohydrates of glycoproteins of cells growing on different surfaces
Authors: Warren L. , Blithe D. L. , Cossu G.
Published: February 2005

The carbohydrate components of some glycoproteins of hamster cells differ as a function of their growth on various substrates; glass, plastic, or plastic coated with collagen. This observation is interpreted as an effect of the environment on cellular structure at the molecular level. The basis of the change and its possible significance are discussed.