bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sodium‐potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: Kinetic parameters, substrate specificity, and effects of phytohemagglutinin
Authors: Segel George B. , Kovach George , Lichtman Marshall A. 
Published: February 2005

AbstractWe have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood.The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+‐ATPase activity were characterized. The Na+, K+‐ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by iptimal concentrations of Na+ and K+ (Na+, K+‐ATPase) was 1.5 nmol of Pi hydrolyzed, μ g protein−1, 30 min−1 (range 0.9‐2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The Km for K+ was approximately 1.0 mM and the Km for Na+ was approximately 15 mM.Active Na+ and K+ transport and ouabain‐sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+‐ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+‐ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+‐ATPase when studied at the Km for ATP, Na+, K+ or Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.
Transformed cells have lost control of ribosome number through their growth cycle
Authors: Stanners C. P. , Adams M. E. , Harkins J. L. , Pollard J. W. 
Published: February 2005

AbstractPrevious studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus‐transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in post‐mitotic pre‐DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability.The rate of protein synthesis in the stationary phase of the transformed cells fell to 25‐30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribosome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L‐73 and LR‐73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells.A model for growth control of normal cells and its derangement in transformed cells is presented.
Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication‐stimulating activity (MSA)
Authors: Knauer Daniel J. , Smith Gary L. 
Published: February 2005

AbstractA temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus‐infected chicken embryo fibroblasts arrested by serum starvation at the non‐permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2‐deoxyglucose uptake, 3H‐thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication‐stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum‐free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.
MSA stimulation of AIB transport is independent of K+ accumulation in myoblasts
Authors: Merrill Gary F. , Dulak Norman C. , Florini James R. 
Published: February 2005

AbstractEnhanced uptake of amino acids is frequently associated with hyperplasia in cultured cells; we have recently shown (Merrill et al., '77) that this is true of the stimulation of rat myoblast proliferation in culture by Temin's Multiplication Stimulating Activity (MSA). In many cases the asymmetric distribution of Na+ and K+ across the cell membrane profoundly affects uptake of certain amino acids, so we investigated the possibility that enhanced Na+‐dependent AIB transport was the result of an MSA‐induced increase in K+ accumulation. MSA stimulated the rate of uptake of the potassium analog 86Rb+ 15‐25% within ten minutes; this rate remained elevated for at least seven hours. Effects were limited to the ouabain‐sensitive component of Rb+ uptake. (Our simultaneous measurements of 86Rb+ and 42K+ uptake demonstrated that Rb+ provides a useful qualitative but not an exact quantitative index of K+ uptake.) The stimulation of K+ uptake by MSA did not cause a large increase in total cellular K+ of the myoblasts; after five hours, MSA‐treated cells contained only about 20% more K+ than did corresponding controls (1.21 ± 0.02 vs. 1.01 ± 0.02 μmoles/mg protein, respectively). To investigate whether this small increase in K+ content could be amplified by the cell to account for the 50‐150% stimulation of AIB uptake, we preincubated cells with ouabain for various times and then measured total cell K+ and AIB uptake in the same culture dishes. Under conditions in which the MSA‐stimulated increase of total cell K+ was prevented by ouabain, a substantial stimulation of AIB uptake was still observed. We conclude that MSA stimulation of AIB transport is independent of increased accumulation of K+ in rat myoblasts.
Inhibiton by glucocorticoids and choleragen of the conditional growth of poorly adherent mononuclear phagocytes of newborn hamster liver and lung (Hormonal control of macrophage growth)
Authors: Nozawa Ryushi T. , Yokota Takeshi 
Published: February 2005

AbstractConditions for in vitro growth of mononuclear phagocytes from newborn hamster liver and lung were studied. In the primary cultures of liver and lung, round cells outgrew and frequently floated off into the culture medium. They were separated from fibroblast‐like cells adherent to plastic by collecting the medium. The round cells were identified as mononuclear phagocytes on the criteria of phagocytic capacity of heat‐killed bacteria and IgG‐coated erythrocytes, fine cell structure and cytochemistry. The phagocytes that had not been activated previously proliferated for about ten generations in F12 medium supplemented with 10% fetal calf serum depending on a growth factor produced by hamster brain, liver or lung cells. Without the factor, the cells quickly cytolysed. Mononuclear phagocytes from blood had the same characteristics of growth and cytochemistry, but had fewer IgG receptors at the cell surface than similar cells from the liver and lung.The effects of a variety of chemical compounds on the growth of the liver and lung cells were studied. Insulin stimulated their growth by 20‐30%, but was not replaceable for the growth factor. Glucocorticoids, dexamethasone and hydrocortisone, inhibited the growth of the phagocyates at the physiological concentrations: 3 × 10−9 M and 2 × 10−8 M for 50% inhibition, respectively. Indomethacin, non‐steroid anti‐inflammatory reagent, at 10−8 M to 10−6 M gave no effect. Choleragen that increases the intracellular cyclic AMP level, inhibited the growth at a concentration as low as 5 pg/ml. These data suggest that the growth of mononuclear phagocytes is controlled not only by a growth factor produced by other cells but also by glucocorticoids.
Immortalization of normal liver functions in cell culture: Rat hepatocyte‐hepatoma cell hybrids expressing ornithine carbamoyltransferase activity
Authors: Widman Lawrence E. , Golden Jonathan J. , Chasin Lawrence A. 
Published: February 2005

AbstractNormal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver‐specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H‐ornithine into protein arginine, and can be selectively grown in arginine‐free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue‐specific phenotypes.
Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution
Authors: Vlodavsky I. , Fielding P. E. , Johnson L. K. , Gospodarowicz D. 
Published: February 2005

AbstractBinding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistribution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non‐overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such a density‐dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cell independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization.The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.
Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts
Authors: Pledger W. J. , Thompson W. J. , Epstein P. M. , Strada S. J. 
Published: February 2005

AbstractA rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10−6 M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D.When BHK cells become quiescent by maintenance in 0.5% serum conditions for 48 hours, a rapid (15‐60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10−8 to 10−6 M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis.Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (≃ 20 μM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3‐4 S form and 5‐6 S form. In quiescent cells, the 5‐6 form greatly predominates relative to the 3‐4 form. Addition of serum to quiescent cells results in a rapid appearance of increased 3‐4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3‐4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.
Growth and G1 arrest of sarcoma virus transformed cells in serum free media
Authors: Moskowitz Merwin , Cheng David K. S. 
Published: February 2005

AbstractRous‐sarcoma transformed BHK cells can be continuously cultured in a medium containing Eagle's Minimal Essential Medium, iron and biotin. The rate of cell multiplication increased when serine, or serine plus other non‐essential amino‐acids were added to the medium. With biotin deleted from the medium there is a reduction in DNA synthesis and most cells are blocked in G1.
Early inhibition of phospholipid synthesis in dimethyl sulfoxide (DMSO) treated friend erythroleukemia (FL) cells
Authors: Harrel L. , Lacour F. , Friend C. , Durbin P. , Semmel M. 
Published: February 2005

AbstractPhosphate metabolism in Friend erythroleukemia cells undergoing DMSO‐induced differentiation was studied. Thirty minutes after the cells were exposed to DMSO in medium at pH 7, an inhibition of 39% in the incorporation of phosphate into phospholipids was observed. This decrease was not due to a change in the precursor pools since phosphate uptake and the phosphorylation of the organic soluble compounds were only inhibited 13%.Inhibition of phospholipid synthesis preceded inhibition of RNA and protein synthesis and reached a maximum after 24 hours of DMSO treatment. At this time, the phospholipid content of the cells was also decreased as compared to that of the control untreated cells. Phospholipid synthesis remained at a level significantly lower than in the controls over the 4‐day observation period, at which time 85% of the treated cells were benzidine positive.Separation of the different phospholipids by chromatography on thin layer silicate gel plates showed that, after one hour of DMSO treatment, more than 80% of the radioactivity was in phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. Phosphatidylethanolamine was the most inhibited. Incorporation of inositol into phospholipid was also significantly decreased. However, there was little variation in the phospholipid composition of the treated and non‐treated cells other than a decrease in the percent of sphingomyelin after 48 hours of DMSO treatment.These changes in phospholipid metabolism may initiate the first step in the complex differentiation process. The phospholipids are important components of membranes and the inducers are known to influence their fluidity.
On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol
Authors: Moris Réjean , Giguére Louis 
Published: February 2005

AbstractWe have found that tryptose phosphate broth (TPB) prevents the inhibitory effect of chloramphenicol (CAM) on the cell proliferation of chick embryo fibroblasts. Study of growth parameters indicated that no lag or adaptation period appeared necessary for TPB‐exposed chick cell populations to grow in the presence of CAM suggesting that a particular cell type was not selected. TPB did not prevent the inhibitory effect of CAM on the mitochondrial protein‐synthesizing system. This was supported by cytochrome oxidase activity measurements, studies on the incorporation of 35S‐methionine into mitochondrial proteins, electron microscopic observation of alterations in mitochondrial structure. Oxygen consumption was reduced by 95% and cyanide, 2‐4‐dinitrophenol, and salicylhydroxamic acid do not significantly affect the residual respiration. Analyses of reduced‐minus‐oxidized‐cytochrome spectra of CAM‐treated chick cells demonstrate the disappearance of the absorption bands of cytochromes aa3, b559, c1, and c. The presence of a type b cytochrome with maxima at 552 and 557 nm was observed. The results obtained indicate that long‐term cultures of CAM‐treated chick embryo cells cultivated in the presence of TPB grow with mitochondria devoid of a functional respiratory chain.
The cyclic AMP‐dependent initiation of DNA synthesis by T51B rat liver epithelioid cells
Authors: Boynton A. L. , Whitfield J. F. 
Published: February 2005

AbstractThe brief rise in the cellular cyclic AMP content which occurs late in the prereplicative phases of rat hepatocytes in vivo and T51B rat liver epitheloid cells in vitro seems to be necessary for the initiation of DNA synthesis. Thus, the extracellular calcium‐deprivation in T51B rat liver cells in culture which induces a late G‐1 block is rapidly reversible (cells surge into S phase within one hour) either by creating a cyclic AMP surge by the addition of calcium or 3‐isobutyl‐1‐methyl xanthine (a cyclic 3′, 5′‐nucleotide phosphodiesterase inhibitor) or by the exogenous addition of low concentrations of cyclic AMP itself (i.e., 10−8‐10−5 M). On the other hand, prevention of the calcium‐induced cyclic AMP surge by imidazole (a cyclic 3′, 5′‐nucleotide phosphodiesterase activator) blocked the initiation of DNA synthesis by the calcium‐deprived T51B cells.
Concanavalin A is mitogenic for resident peritoneal macrophages
Authors: Wang Ai‐Lan , Basch Ross S. 
Published: February 2005

AbstractCon A, a known T‐cell mitogen, is also mitogenic for resident peritoneal macrophages. The stimulated cells morphologically resemble macrophages and are actively phagocytic. The concentration of con A (30 μg/ml) required to stimulate 3H‐TdR incorporation is ten times that required for T‐cell activation. Con A must be present throughout the entire culture period to produce the maximum effect, and con A‐depleted supernatant fluids from con A‐stimulated cells cannot replace the con A requirement. Stimulation of 3H‐TdR incorporation occurs after a 48‐hour lag period and is maximal on the fifth to seventh day of culture. At the peak of the response, 20–30% of the macrophages can be stimulated to incorporate 3H‐TdR, but little or no increase in the total number of cells present in the culture occurs. This and pulse‐chase experiments indicate that only a single cycle of replication occurs in the stimulated cells. Con A‐responsive peritoneal macrophages appear to be a distinct subpopulation and might play a different role in the interaction with T cells and B cells in the immune response than the con A‐non‐responsive cells.
Properties and separation of T lymphocyte growth stimulatory activity (TL‐GSA) and of granulocyte‐macrophage colony stimulatory activity (GM‐CSA) produced separately from two human T lymphocyte subpopulations
Authors: Wu A. M. 
Published: February 2005

AbstractWe have previously identified two stimulatory activities affecting blood cell maturation in PHA‐stimulated human lymphocyte conditioned medium (PHA‐LyCM). One was granulocyte‐macrophage colony stimulatory activity (GM‐CSA), and the other was T lymphocyte growth stimulatory activity (TL‐GSA) in suspension culture. In this paper we have shown that although both activities can be produced from purified non‐adherent human T lymphocytes, they are produced from two distinct subpopulations. The production of these activities was greatly enhanced by T cell mitogens. Both protein factors were relatively heat stable (56°, 30 minutes), were sensitive to trypsin treatment and were specific for primate blood cells. These two activities were fractionated by means of ammonium sulfate precipitation, Sephadex G‐150 gel filtration, DEAE cellulose and Con A‐Sepharose column chromatographies. MW of the major peak estimated from the elution volume of gel filtration in the presence of 0.5 M NaCl was 40,000 for GM‐CSA and 13,000 for TL‐GSA. Results from Con A‐Sepharose column showed that while about 70% of TL‐GSA was bound to Con A, less than 25% of GM‐CSA was bound. These observations show that the majority of TL‐GSA and GM‐CSA were separable by these two conventional column chromatographic methods.
Regulation of the G1 → S phase transition in chick embryo fibroblasts with α‐keto acids and L‐alanine
Authors: Groelke John W. , Baseman Joel B. , Amos Harold 
Published: February 2005

AbstractTemporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum‐stimulated DNA synthesis in G1‐arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H‐thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4° C while exposed to the drug. Several α‐keto acids (pyruvate, oxaloacetate, α‐keto‐butyrate) at 0.5‐1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L‐alanine (D‐alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Clucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin‐stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for α‐keto acid or L‐alanine. A serum component(s) with a molecular weight of about 100 permitted insulin‐stimulated DNA synthesis in inhibited cells.