bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of protein phosphorylations in variant A431 cells with different growth responses to epidermal growth factor
Authors: Buss Janice E. , Chouvet Claire , Gill Gordon N. 
Published: February 2005

AbstractGrowth of a selected variant of A431 cells (clone 29) is stimulated by epidermal growth factor (EGF) in contrast to the growth inhibition caused by EGF in an unselected clone, A4318. Twelve phosphoproteins from each clone were compared to determine whether unique EGF‐dependent substrate phosphorylations might explain the cells' differing growth responses to EGF. Treatment of both clone 29 and A4318 cells with EGF increased phosphorylation of the EGF receptor/kinase and six cellular proteins identified on 2‐dimensional poly‐acrylamide gels. Four of these proteins (the EGF receptor/kinase and proteins of 36, 70, and 81 kd) contained phosphotyrosine in both clone 29 and A4318 cells, indicating that the same modification of several proteins occurred in cells which have totally different growth responses to EGF. Two proteins were identified whose phosphorylation was EGF dependent and which were unique to clone 29 cells; however, EGF increased phosphorylation of only serine residues in these proteins. This indicates that these proteins are not primary targets of the EGF‐dependent tyrosine‐specific protein kinase, but rather are substrates for serine‐specific kinase(s) activated as a consequence of EGF:receptor interaction. cAMP, which inhibited growth of both clones, was utilized to compare the effects of EGF when the growth response of both cell lines was similar. In the presence of cAMP, EGF increased A4318 cellular phosphotyrosine content and the phosphorylation of the same phosphotyrosine‐containing proteins of both clone 29 and A4318 cells. The in vivo activity of a second tyrosine‐specific protein kinase, p60v‐src in B77 Rous sarcoma virus (RSV)‐transformed newborn rat kidney (NRK) cells, was also unaffected by cAMP. Thus cAMP did not block the in vivo activity of two tyrosine‐specific kinases or the tyrosine phosphorylation of three specific protein substrates. A threshold model of tyrosine kinase activity is proposed as an alternative explanation for the differing growth responses to EGF.
Gene amplification as a mechanism of reversion at the HPRT locus in V79 chinese hamster cells
Authors: Zownir Olga , Fuscoe James C. , Fenwick Raymond , Morrow John 
Published: February 2005

AbstractSpontaneous phenotypic revertants of hypoxanthine phosphoribosyl‐transferase (HPRT) temperature‐sensitive V79 Chinese hamster cells were selected by plating a temperature‐sensitive mutant in HAT medium at 39°C. The incidence of such revertants was approximately 2 × 10−4 per cell. The majority of the revertants examined had increases of between three‐ and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39°C. When the revertants were cultivated in the absence of HAT, they recovered their HAT‐sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied:the Km for both PRPP and hypoxanthine was significantly different in the temperature‐sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind Ill restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5‐fold in one revertant and 1.4‐fold in the other. Reintroduction into non‐selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized.We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.
Furosemide‐sensitive potassium efflux in cultured mouse fibroblasts
Authors: Jayme David W. , Slayman Carolyn W. , Adelberg Edward A. 
Published: February 2005

AbstractTransfer of LM(TK−) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50–70% inhibited by furosemide or bumetanide. The diuretic‐sensitive component of K+ efflux requires both Na+ and Cl−, and is presumably mediated by a K+, Na+, Cl− cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic‐sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic‐sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low‐K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl−.
Receptors for pea and lentil lectins on human lymphoid cells: Demonstration of distinct lectin‐defined cell subclasses
Authors: Boldt David H. 
Published: February 2005

AbstractLectins are useful probes for studying cell surface glycoconjugates. Pea (PL) and lentil (LL) lectin each requires for binding a fucosyl‐ and two α‐mannosyl residues in core regions of glycopeptides, but differences in outer chain carbohydrates may alter their relative binding affinities. We used binding studies with [125I]‐PL and LL and flow cytometry with fluorescein‐conjugated (FITC)‐PL and ‐LL to study their interactions with peripheral lymphocytes. Binding of both lectins to lymphocytes was saturable, reversible, and inhibited by α‐methyl mannose. Scatchard analyses were consistent with two classes of receptors for each lectin. Flow cytometric analyses demonstrated that cell to cell receptor densities varied. Sixty‐five percent of lymphocytes bound PL (mean 2 × 106 receptors/cell) and 45% bound LL (mean 3 × 106 receptors/cell). Competition studies demonstrated mutual inhibition, but flow cytometry revealed persistent FITC‐PL or ‐LL binding despite 20‐fold molar excess of the other lectin. Distributions of receptors for PL and LL on lymphocytes were as follows: (1) 45% of lymphocytes bound both PL and LL; (2) 20% of lymphocytes bound PL alone; (3) 35% of lymphocytes bound neither PL nor LL. Despite similar binding requirements for PL and LL and overlap between their receptors on lymphocytes, there appear to be subsets of receptors specific for each lectin. These results may reflect abilities of PL and LL to discriminate subtle carbohydrate differences on lymphocyte surfaces.
Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides
Authors: Bethlenfalvay N. C. , Lima J. E. , Waldrup T. 
Published: February 2005

AbstractOpossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D‐glucose and optimum inorganic phosphate requirement for D‐glucose utilization at pH 7.4 and 8.1. D‐Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH‐dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 μmol, at pH 8.1, 9.5 μmol/ml cells/hr were utilized. Red cells suspended in leukocyte‐free plasma utilized D‐glucose at a rate of 3.0 μmol/ml/hr at pH 7.5. Seven percent of D‐glucose flowed through the pentose phosphate pathway; this rate increased 11‐fold by methylene blue stimulation. The amount of D‐glucose recycled through the pentose phosphate pathway increased 300‐fold in the presence of the redox dye.
Adhesion site composition of murine fibroblasts cultured on gelatin‐coated substrata
Authors: Haas Robert , Banerji S. Sheila , Culp Lloyd A. 
Published: February 2005

AbstractFibroblasts in vivo adhere to a collagenous extracellular matrix. We present here a combined morphological and biochemical analysis of the adhesion sites of fibroblast‐like cells cultured in vitro on gelatin‐coated plastic, for comparison with earlier model studies using serum (plasma‐fibronectin [pFn])‐coated plastic. Scanning electron microscopy shows that cell adhesion to the gelatin is quite similar to that on plastic, but with some morphological differences reminiscent of those caused by higher concentrations of fibronectin adsorbed to the substratum. Measurement using 125I‐radiolabeled pFn shows the level of substratum‐bound pFn adsorbed from serum in the growth medium is, however, comparable on gelatin or plastic; thus, differences due to pFn must be attributed to the quality of the adsorbed protein, not its absolute quantity. Gel electrophoretic analysis of cellular adhesion sites formed on the two substrata shows their compositions to be qualitatively similar, suggesting again that the same fundamental adhesion processes are involved. However, three protein bands do change; notably, cellular fibronectin is increased on gelatin. These three proteins are also the most resistant to saline extraction, suggesting their intrinsic importance in the adhesion sites. The nature of the growth substratum thus appears to modulate a fundamentally unvarying morphology and adhesion site composition of the cells that adhere to it.
Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells
Authors: Ebsworth Nicolette M. , Taylor‐Papadimitriou Joyce , Rozengurt Enrique 
Published: February 2005

AbstractThe purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L‐cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP‐elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP‐elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP‐stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid‐insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose‐response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP‐stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP‐elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.
Prostaglandin synthesis by cells comprising the calf pulmonary artery
Authors: Menconi Michael , Hahn Gerald , Polgar Peter 
Published: February 2005

AbstractProstaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PG12) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGl2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGl2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10−12 M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10−12M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a “quiescent” nongrowth state.
Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells
Authors: Smith Robert J. , Larson Sandra , Stred Susan E. , Durschlag Roberta P. 
Published: February 2005

AbstractGlutamine is synthesized in skeletal muscle, released to the circulation, and transported to other tissues, where it may provide important substrate for gluconeogenesis, ammoniagenesis, and energy‐yielding pathways. With the ultimate goal of delineating the factors that control glutamine production and release by skeletal muscle, we have studied the regulation of two key enzymes, glutamine synthetase and glutaminase, in the L6 line of rat skeletal muscle cells grown in monolayer culture. The cultured myotubes were found to have glutamine synthetase and phosphate‐dependent glutaminase activities. Glutamine synthetase activity was increased following incubation (1) in glutamine‐free medium (threefold); (2) in medium containing high glutamic acid concentrations (fourfold); and (3) in medium supplemented with dexamethasone (threefold). In each case the increase in glutamine synthetase activity required several hours to reach a maximum and was prevented by cycloheximide, suggesting that the change occurred through increased enzyme biosynthesis. No substances tested were found to affect glutaminase activity. We conclude that glutamine synthetase in cultured skeletal muscle is responsive to substrate, product, and hormonal regulation.
Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40‐transformed human fibroblasts
Authors: Smith Janet W. , Tupper Joseph T. 
Published: February 2005

AbstractPrevious studies indicate that although normal and Simian virus (SV40)‐transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40‐transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+‐K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80–90% of total cell Ca++ in both cell types.
Adenosine‐resistant chinese hamster fibroblast variants with hyperactive adenosine‐deaminase: An analysis of the protection against exogenous adenosine afforded by increased activity of the deamination pathway
Authors: Fernandez‐Mejia C. , Debatisse M. , Buttin G. 
Published: February 2005

AbstractThe activity of purine salvage and interconversion enzymes was examined in two sublines of Chinese hamster cells–RA11 and RA41–isolated on the basis of their resistance to adenosine concentrations toxic to wild‐type CCL39 cells. Adenosine deaminase (ADA) activity was found to be two times higher in RA11 and three times higher in RA41 than in CCL39. Inhibition of ADA activity by coformycin reduced the level of adenosine resistance but did not restore wild‐type sensitivity, indicating that a second defect contributes to the adenosine‐resistant phenotype of these variants; evidence was indeed obtained for the presence in both lines of additional alterations protecting them against the lethal depletion of phosphoribosylpyrophosphate (Ishii and Green, 1973) imposed by adenosine to wild‐type cells. To gain better insight into the influence of ADA hyperactivity on adenosine resistance, a procedure was developed for the specific isolation of variants with increased levels of ADA activity. Cell lines with 3–5 times and then 100–500 times the wild‐type ADA activity were stepwise recovered. These investigations confirmed that amplification of ADA can efficiently contribute in protecting cells against high concentrations of exogenous adenosine. The variants isolated by this procedure again manifested, in addition to amplification of ADA activity, another alteration decreasing their sensitivity to adenosine. A possible mechanism accounting for the frequent isolation of variants that coexpress ADA hyper‐activity and a second defect contributing protection against adenosine toxicity are considered.
Factors modifying 3‐aminobenzamide cytotoxicity in normal and repair‐deficient human fibroblasts
Authors: Boorstein Robert J. , Pardee Arthur B. 
Published: February 2005

Abstract3‐Aminobenzamide (3‐AB), an inhibitor of poly(ADP‐ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3‐AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3‐AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3‐AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3‐AB for 36–48 hours following MMS treatment. During this same interval, 3‐AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3‐AB. Human mutant cells defective in DNA repair differed in their responses to 3‐AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3‐AB than control cells, while Huntington's disease cells had a near‐normal response. Among UV‐sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3‐AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3‐AB could be dependent on inability of the mutant cells to repair damage by other processes.
Flat revertants derived from kirsten murine sarcoma virus‐transformed cells produce transforming growth factors
Authors: Salomon David S. , Zwiebel James A. , Noda Makoto , Bassin Robert H. 
Published: February 2005

AbstractTwo flat cellular revertant cell lines, F‐2 and C‐11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki‐MuSV)‐transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non‐transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F‐2 and C‐11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum‐free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F‐2 and C‐11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.