bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of cycloheximide and actinomycin D on the amino acid transport system of tetrahymena
Authors: Blum J. J. 
Published: February 2005

AbstractTetrahymena pyriformis were grown in axenic culture to late logarithmic and stationary phases, resuspended in an inorganic medium, and the rates of transport of α‐aminoisobutyric acid (AIB) and of the decarboxylation of L‐[1‐14C]leucine and L‐[1‐14C]tyrosine were measured. There was a rapid loss of each of these measures of amino acid transport in both late log phase and stationary phase cells. Addition of actinomycin D to the washed cells caused a small increase in the rate of loss of capacity to decarboxylate tyrosine and leucine. Addition of cycloheximide to the washed cells caused a reduction in the rates of loss of capacity to transport AIB and to decarboxylate leucine and tyrosine except that in late log phase cells cycloheximide markedly increased the rate of loss of capacity to decarboxylate leucine. When cells that had been pretreated with chlorpromazine to reduce their amino acid transport capacity were washed and resuspended in proteose peptone the capacity to decarboxylate tyrosine and leucine increased to control values within 1.5 hours. Addition of actinomycin D reduced the rate of recovery of transport capacity, but addition of cycloheximide caused transport capacity to decrease further. These results raised the possibility that there were two amino acid transport systems in this cell. The finding that AIB and N‐methylaminoisobutyrate are both taken up by Tetrahymena, the latter at one‐eighth the rate of the former, but that neither one alters the rate of uptake of the other provides preliminary support for this possibility. The present results further suggest that the transport system(s) has a short lifetime and that the balance between rate of synthesis and rate of loss of the transport system is controlled in part by the presence of exogenous amino acids.
Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands
Authors: Haraguchi Kenneth H. , Knox Robert J. , Weimar Virginia L. , Anderson Ronald A. 
Published: February 2005

AbstractRecycled mesodermal growth factor (R‐MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R‐MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is shown here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R‐MGF has yielded several components, none of which has a greater biological potency than the parent R‐MGF. In contrast, two components, MGF‐I and ‐II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R‐MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10–15 m̈g/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 m̈g of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R‐MGF in vivo may also reflect synergistic action of more than one R‐MGF component. Procedures for isolating gram quantities of R‐MGF and for the purification of different R‐MGF components by ion exchange chromatography are detailed.
Glycosaminoglycan changes involved in polymorphonuclear leukocyte activation in vitro
Authors: Vannucchi Simonetta , Fibbi Gabriella , Cappelletti Renzo , Rosso Mario Del , Chiarugi Vincenzo 
Published: February 2005

AbstractThe changes of cell coat and endocellular glycosaminoglycans associated with adhesion of polymorphonuclear leukocytes to tissue culture dishes were studied by electrophoresis on cellulose acetate. Polymorphonuclear leukocytes are coated by chondroitin 4 sulphate, while undersulphated chondroitin 4 sulphate and heparan sulphate are present in the cytoplasm. Upon adhesion polymorphonuclear leukocytes shed into the culture medium chondroitin 4 sulphate and concomitantly hyaluronic acid and heparan sulphate are exposed at the cell surface and shed into the culture medium. The role of these compounds in polymorphonuclear leukocyte physiology is discussed.
Inhibition of the serum‐dependent, amiloride‐sensitive sodium transport pathway in human fibroblasts by extracellular divalent cations
Authors: Villereal Mitchel L. 
Published: February 2005

AbstractSodium influx in serum‐deprived human fibroblasts in a nominally Ca‐free, Mg‐free medium is significantly higher (17.8 ± 1.9 μmole/g prot/min) than that measured in a medium containing 1.8 mM Ca and 1 mM Mg (10.9 ± 0.7 μmole/g prot/min), and is stimulated dramatically (44.1 ± 6.1 μmole/g prot/min) by the addition of 10% fetal bovine serum (FBS), suggesting that an enhanced influx of Ca ions is not a necessary condition for serum activation of the amiloride‐sensitive Na influx pathway. The addition of 2 mM ethylenediaminetetraacetic acid (EDTA) to serum‐deprived cells in a low Ca, low Mg medium also results in a dramatic stimulation of Na influx (40.4 ± 3.7 μmole/g prot/min), while the addition of EDTA to cells assayed in a low Ca, low Mg medium in the presence of FBS has no significant effect on Na influx (45.3 ± 4.1 μmole/g prot/min). Thus, the stimulatory effects of FBS and EDTA are not additive. Kinetic analysis in the presence of varying amiloride concentrations indicate that the EDTA‐stimulated Na influx occurs via the amiloride‐sensitive Na pathway. The activation of Na influx in cells rinsed free of Ca and Mg Can be readily reversed by the addition of Ca or Mg to the assay medium. The Ca concentration required to give 50% inhibition of Na influx is 52 ± 7.6 μM (n = 3) for cells assayed in serum‐free medium and 272 ± 29 μM (n = 3) for cells assayed in the presence of 10% FBS. At physiological Ca concentrations (1.8 mM) the Na influx is maximally inhibited by Ca both in the presence and absence of serum. Since Na influx in 1.8 mM Ca medium is 2.5‐fold higher in the presence of serum than in its absence, these data suggest that the serum‐induced change in the K, for Ca modulation of the amiloride‐sensitive Na transport pathway is not sufficient to explain the serum stimulation of Na influx in human fibroblats.
High intracellular pH accompanies mitotic activity in murine lymphocytes
Authors: Gerson Donald F. , Kiefer Hansruedi 
Published: February 2005

AbstractIntracellular pH is crucial to the control of many cellular processes such as glycolysis, protein synthesis, and cell division. To study the relation between intracellular pH and mitotic activity in actively dividing Con A‐ or LPS‐stimulated splenic lymphocytes, a method was developed to determine intracellular pH using a fluorescence‐activated cell sorter. The new method uses the pH‐sensitive fluorochrome, 4‐methylumbelliferone. Results obtained with it not only correspond qualitatively with the results obtained using 14C‐dimethyloxazolidinedione (DMO) but also clearly show the active and inactive subpopulations. The intracellular pH of mitogen‐stimulated murine lymphocytes increases from pH 7.15 to pH 7.45 when the population has greatest mitotic activity. The intracellular pH of three virus‐transformed lymphocyte cell lines is higher by ∼ 0.5 pH unit when the cells are in exponential growth compared to stationary phase.
Ribosomal proteins are synthesized preferentially in cells commencing growth
Authors: Tushinski Robert J. , Warner Jonathan R. 
Published: February 2005

AbstractMouse 3T3 cells, in stationary phase because of serum deprivation, have only half the ribosome content of growing cells. Furthermore, the proportion of protein synthesis devoted to ribosomal proteins is only half that in growing cells. On addition of serum the synthesis of each ribosomal protein increases threefold, demonstrating the coordination of the synthesis of the ribosomal proteins. Half that increase is due to a general increase in total protein synthesis; half is due to a differential increase in ribosomal protein synthesis. The latter is abolished by a concentration of actinomycin D which blocks only ribosomal RNA transcription. The results are discussed with reference to a general hypothesis of growth regulation proposed by Stanners et al. (1979).
The use of flurescent dyes to measure membrane potentials: A response
Authors: Smith Thomas C. 
Published: February 2005

AbstractThe use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related to the application of the dyes have been reviewed. These problems include: (1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, (2) the effect of altered energy metabolism on the fluorescent response of the dyes and on Em, and (3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.
Transcription of chick genes by mammalian RNA polymerase II in chick erythrocyte‐mammalian cell heterokaryons
Authors: Zuckerman Steven H. , Linder Stig , Ringertz Nils R. 
Published: February 2005

AbstractThe introduction of chick erythrocyte nuclei into mammalian cell cytoplasms results in their reactivation as evidenced by the de novo transcription of chick genes and the synthesis of both globin and constitutive proteins. In the present study, chick erythrocytes have been fused to L6 rat myoblasts and to alphaamanitin‐resistant variants of L6 to determine whether the chick or the mammalian RNA polymerase II was responsible for transcription of chick genes. Heterokaryons formed by fusing chick erythrocytes with alpha‐amanitin‐resistant L6 myoblasts synthesize both chick globin and chick constitutive proteins in the continued presence of 5 μg/ml alpha amanitin ten days postfusion. Both the synthesis of globin and other chick polypeptides occurs at levels comparable to those observed for untreated heterokaryons. Synthesis occurs under conditions in which insignificant chick RNA polymerase II activity can be detected irv wild‐type heterokaryons by autoradiography. These results demonstrate that RNA polymerase II is one of the mammalian proteins that is selectively taken up by the chick nucleus during reactivation in the presence of alpha amanitin. Furthermore, the mammalian RNA polymerase II alone can account for the transcription of both differentiation specific and constitutive genes in the chick nucleus.
Effects of urea treatment on the divalent cation‐independent cell aggregation of 3T3MIT fibroblasts
Authors: Wright Thomas C. , Robinson John M. , Karnovsky Morris J. 
Published: February 2005

AbstractGrowth of nontransformed 3T3MIT fibroblasts in media containing 200 mM urea leads to the rapid acquisition of the transformed adhesive phenotype as evidenced by an increased rate of divalent cation‐independent cell aggregation. The increased rate of divalent cation‐independent cell aggregation of urea treated 3T3MIT cells shares many properties with the high rate of aggregation of transformed cells including a sensitivity to treatment with trypsin or hyaluronidase and a reduction in the presence of exogenously added hyaluronic acid. Reversal of the urea‐induced increase in aggregation occurs within 24 hours in the absence of urea and can be blocked by 0.2 μg/ml cycloheximide. In the presence of cycloheximide, low rates of aggregation can be restored by the addition of urea‐conditioned supernatents. The results of these experiments suggest that the loss of an aggregation‐inhibitory activity during growth in media containing 200 mM urea is responsible for the increased rate of divalent cation‐independent cell aggregation. After removal of this aggregation‐inhibitory activity, the normally lowly adhesive 3T3MIT cells become phenotypically transformed with regards to the rate of divalent cation‐independent cell aggregation.
Amiloride, protein synthesis, and activation of quiescent cells
Authors: Lubin Martin , Cahn Frederick , Coutermarsh Bonita A. 
Published: February 2005

AbstractAmiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.
Intracellular distribution of transglutaminase activity during rat liver regeneration
Authors: Remington John A. , Russell Diane Haddock 
Published: February 2005

AbstractTransglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic‐particulate, and cytoplasmic‐soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic‐particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic‐particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat liver cells.
Properties and fate of plasma fibronectin bound to the tissue culture substratum
Authors: Haas Robert , Culp Lloyd A. 
Published: February 2005

AbstractPlasma fibronectin (pFn) is a serum protein which, when adsorbed to a glass or plastic substratum, mediates the adhesion of fibroblasts in culture. We have studied some of the details of its adsorption and subsequent fate. By using 125l‐labeled pFn, we show that a substratum incubated with pFn adsorbs approximately 0.4 μg/cm2 pFn (a monomolecular layer), and one incubated with medium containing serum adsorbs approximately 7 ng/cm2 pFn (a 12‐fold enrichment relative to a random selection of the soluble proteins). SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) suggests the bound serum proteins (eluted with SDS) are primarily BSA and β‐globulins. The bound pFn adheres so tightly, though, that most resists elution, as assayed (1) with pFn radioiodinated before binding, (2) with pFn radioiodinated after binding, or (3) by the cell spreading activity of the bound pFn retained after SDS treatment. Under culture conditions, there is a continuous “turnover” of substratum‐bound pFn: soluble pFn can bind to a serum‐coated substratum, while bound pFn is gradually removed by incubation with serum proteins. The presence of fibroblasts increases the rate of this removal several‐fold. By SDS‐PAGE the material removed (as well as that eluted from the substratum with SDS after cell detachment) is intact pFn or large (possibly proteolytically generated) fragments. Thus, pFn binds preferentially to the tissue culture substratum, but can be removed subsequently by the combined action of cells and other serum proteins.
Complementary recessive 25‐hydroxycholesterol‐resistant somatic cell mutants – assay of 25‐hydroxycholesterol binding activity
Authors: Sinensky M. , Logel J. , Torget R. 
Published: February 2005

AbstractComplementation analysis of recessive 25‐hydroxycholesterol‐resistant mutants of the CHO‐KI cell shows the existence of at least two complementation groups, one of which is missing a binding activity for 25‐hydroxycholesterol. Both complementation groups are shown to be refractory to inhibition of cellular HMG‐CoA reductase activity and in the inhibition of biosynthesis of this enzyme by 25‐hydroxycholesterol.
Glycopeptide hormone production by cultured human diploid fibroblasts
Authors: Milsted Amy , Day Debra‐Lynn , Cox Rody P. 
Published: February 2005

AbstractHuman diploid fibroblasts in culture were examined for production of glycopeptide hormones. Forty‐one percent of the strains produced human chorionic gonadotropin (hCG) under normal growth conditions. Constitutive hCG synthesis was apparently unrelated to donor age, length of time in culture, or number of passages. Follicle stimulating hormone (FSH) was not found in any strain investigated. Only one cell strain produced free α‐chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p < 0.01) in all constitutive strains, but had no effect on non‐hCG‐producing fibroblast strains. Sodium butyrate (Bu) was effective in increasing hCG synthesis in only one constitutive strain, derived from a newborn foreskin. HU treatment had no apparent effect on cell structure. All Bu‐treated strains, both those producing hCG and the nonproducers, showed morphological alterations; cells were flattened and they contained ordered arrays of refractile granules. It is suggested that hCG synthesis in cultured human diploid fibroblasts may result from a localized chromosomal event in which the loci responsible for this hormone are activated. Human diploid fibroblasts in culture are shown to be amenable to the study of gene expression and its modulation.
Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation: Dissociation from effects on transepithelial resistance and cyclic AMP levels
Authors: Thomas S. Randall , Schultz Stanley G. , Lever Julia E. 
Published: February 2005

AbstractChanges in transepithelial electrical resistance and cyclic nucleotide levels were monitored accompanying chemical induction of domes in a clonal subline of MDCK kidney epithelial cells. Confluent cell monolayers grown on nitrocellulose filters exhibited a relatively high mean transepithelial resistance (387 ohms · cm2). Hexamethylene bisacetamide, a potent inducer of dome formation (Lever, 1979b), stimulated significantly increased transmonolayer resistance as well as elevated levels of intracellular cyclic AMP. By contrast, dimethylformamide, an equally potent inducer of dome formation in MDCK cells, did not appreciably alter either resistance values or cyclic nucleotide levels. These results suggest that induction of dome formation in epithelial cell cultures by compounds generally known as inducers of differentiation may involve multiple and separate mechanisms.
Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats
Authors: Kilberg M. S. , Vida T. A. , Barber E. F. 
Published: February 2005

AbstractThe present report shows that System A‐mediated 2‐aminoisobutyric acid (AIB) uptake is elevated in hepatocytes Isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, β, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.
Permselectivity changes in malaria (Plasmodium falciparum) infected human red blood cell membranes
Authors: Kutner S. , Ginsburg H. , Cabantchik Z. I. 
Published: February 2005

AbstractThe development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite‐dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that (i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; (ii) permeation of the fluorescent anion transport substrate NBD‐taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized exythrocytes, as compared with noninfected cells; (iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; (iv) permeation of anionic probes was temperature dependent (Ea:11 ± 1 kcal/mole); and (v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.
The adhesive glycoprotein laminin is an agglutinin
Authors: Kennedy Dorothy W. , Rohrbach David H. , Martin George R. , Momoi Takashi , Yamada Kenneth M. 
Published: February 2005

AbstractThe glycoprotein laminin appears to function in the attachment of various epithelial cells to basement membranes. We examined whether its putative cell‐adhesive activity could be analyzed in a simple, one‐component model system—the agglutination of erythrocytes. Laminin is a potent agglutinin of aldehyde‐fixed sheep and human erythrocytes, with half‐maximal agglutination of 0.8 μg/ml in a standard hemagglutination assay. Inhibitors of this hemagglutinating activity include gangliosides and certain charged phospholipids. The spectrum of molecules is similar but not identical to inhibitors of the hemagglutinating activity of the adhesive glycoprotein fibronectin. Laminin is much less biologically active in three other assays for fibronectin biological activity involving cell spreading on tissue culture substrates, attachment of fibroblastic cells to type I collagen, and restoration of normal morphology to transformed fibroblasts. The adhesive glycoproteins laminin and fibronectin therefore differ markedly in biological activities in several specific adhesion assays; however, they resemble one another in binding to heparin, collagen, and cell surfaces and in their agglutinin activity.