bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microtubule‐disrupting drugs increase the frequency of conversion of a rat mammary epithelial stem cell line to elongated, myoepithelial‐like cells in culture
Authors: Paterson Frieda C. , Rudland Philip S. 
Published: February 2005

AbstractRat mammary (Rama) 25 cuboidal epithelial stem cells convert at a low frequency to elongated, Thy‐1‐positive, myoepithelial‐like cells in culture; one such cell line is termed Rama 29. Addition of increasing concentrations of the microtubule‐disrupting drug colchicine to sparse cultures of Rama 25 dramatically increases the percentage of colonies containing elongated cells and the percentage of Thy‐1‐positive cells when the drug is removed. Similar results on the formation of elongated cell colonies are obtained with other microtubule disruptors, such as vinblastine, vincristine, demecolcine, and nocodazole. The inactive analogues of colchicine β‐ and δ‐lumicolchicine and the microfilamental‐disruptors cytochalasin B and D are without effect on the formation of elongated cell colonies and Thy‐1‐positive cells. For a given concentration of colchicine the percentage of elongated cell colonies and Thy‐1‐positive cells increases the longer the cells are exposed to the drug (range 8–96 hr) and the longer the drug‐treated cultures are subsequently grown in drug‐free medium. Colchicine fails to display this morphological change on Rama 29 elongated cells and on Rama 600 epithelial cells from a rat mammary metastasizing tumor. Immunofluorescent localization of antisera to tubulin confirms that colchicine disrupts the microtubules in all three cell lines at similar concentrations (0.1 to 1 μM) to those required to increase the percentage of elongated cell colonies in Rama 25. The DNA synthesis inhibitor cytosine arabinoside fails to inhibit this conversion process, and time‐lapse cinematographic studies confirm that the conversion of a cuboidal to an elongated cell can take place without cell division. However, cell division may sometimes be required for subsequent stabilization events. Treatment of Rama 25 cells with colchicine under the same conditions also increases the abundance of elongated cell (Rama 29)‐associated polypeptides, and elongated cell clones isolated after such treatment show an overall pattern of protein synthesis very similar to that of Rama 29.
Diacylglycerols and mezerein activate neutrophils by a phorbol myristate acetate‐like mechanism
Authors: O'Flaherty Joseph T. , Redman Jimmy F. , McCall Charles E. , Wykle Robert L. , Schmitt Jeffery D. 
Published: February 2005

Abstractrac‐1‐O‐Myristoyl‐2‐O‐acetylglycerol, rac‐1‐O‐palmitoyl‐2‐O‐acetylglycerol, and rac‐1‐O‐oleoyl‐2‐O‐acetylglycerol acted like phorbol myristate acetate and mezerein in stimulating human neutrophil aggregation. Responses to these agents were equally influenced by cytochalasin B, extracellular calcium and magnesium, arachidonate antimetabolites, and procedures that rendered the cells desensitized to other agonists. The compounds also inhibited the binding of [3H]‐phorobol myristate acetate to its receptor on neutrophils. Thus, these agents are biologically homologous. They act by binding to a common receptor. This receptor may function physiologically as a transducer for endogenous glycerides that form in cells challenged by other stimuli.
Interactions between cyclic AMP‐ and phorbol ester‐dependent phosphorylation systems in S49 mouse lymphoma cells
Authors: Kiss Zoltá;n , Steinberg Robert A. 
Published: February 2005

AbstractHigh‐resolution two‐dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP‐dependent protein kinase or the guanyl nucleotide‐binding “Ns” factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild‐type and mutant cells: Phosphorylations shown previously ot be responsive to activation of intracellular cyclic AMP‐dependent protein kinase were stimulated by TPA in wild‐type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild‐type cells, and two novel phosphoprotein species appeared in response to TPA only in wild‐type cells. The latter two classes of TPA‐mediated responses specific to wild‐type cells could be evoked in adenylate cyclase‐deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: (1) TPA stimulates adenylate cyclase in wild‐type cells causing increased phosphorylation of endogenous substrates by cyclic AMP‐dependent protein kinase, (2) activated cyclic AMP‐dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA‐dependent phosphoproteins, and (3) cyclic AMP‐dependent events facilitate TPA‐dependent phosphorylation of some substrate proteins.
Chloroquine allows the secretion of internalized 125I‐epidermal growth factor from fibroblasts
Authors: Wakshull E. , Cooper Janet L. , Wharton Walker 
Published: February 2005

AbstractIncubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse‐chase paradigm in which cell surface receptors were labelled with 125I‐EGF at 4°C, it was found that when 100 μM chloroquine was present in the 37°C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor‐mediated endocytosis and subcellular distribution of 125I‐EGF‐receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I‐EGF released from the cells could rebind to surface receptors and be re‐internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I‐EGF whose rebinding was inbibited. Thus, the 125I‐EGF whose rebinding was inhibited. Thus, the 125I‐EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I‐EGF and mechanisms of its preferential rebinding are discussed.
Effect of sodium butyrate on actin distribution in rat 3Y1 fibroblasts in monolayer culture
Authors: Yamada Koji , Sasaki Masafumi , Kimura Genki 
Published: February 2005

AbstractWe studied the effect of sodium butyrate, a potent G1/G2‐arresting agent, on actin distribution in rat 3Y1 fibroblasts in monolayer culture by fluorescence microscopy of cells stained with 7‐nitrobenz‐2‐oxa‐1, 3‐diazole phallacidine (NBD‐Ph). When randomly proliferating cells were arrested mainly in G1 phase with butyrate, a reversible overaccumulation of cellular net protein occurred. In the G1‐arrested cells, actin markedly accumulated at the margin of cells, and a network structure of actin stress fibers appeared. When density‐arrested cells were replated sparsely and rearrested in the G1, early S, and G2 phases with butyrate or hydroxyurea, the actin network was observed extensively in the cells arrested in the G1 and G2 phases with butyrate. These results agree with our previous results indicating the existence of some physiological similarity between cells in the G1 and G2 phases and suggest that actin distribution somehow depends on the phases of the cell cycle. The actin profiles observed by the NBD‐Ph staining were confirmed by transmission electronmicroscopy (TEM) of negatively stained whole cells. TEM further revealed that electron‐dense amorphous materials were present at crossing points in the network but rarely present on interconnecting microfilament bundles.
Differential induction of glucose‐regulated and heat shock proteins: Effects of pH and sulfhydryl‐reducing agents on chicken embryo cells
Authors: Whelan Sandra A. , Hightower Lawrence E. 
Published: February 2005

AbstractGlucose‐regulated and heat shock proteins are two subsets of eukaryotic stress proteins that can be induced differentially, simultaneously, and reciprocally. Two new inducers, low extracellular pH and 2‐mercaptoethanol, that stimulate chicken embryo cells to synthesize glucose‐regulated proteins rapidly were found. Two classes of cellular targets for mercaptoethanol were defined operationally, one dependent on and the other independent of protein synthesis. A new inducer of heat shock proteins, high extracellular pH, was found as well. Inductions by low and high extracellular pH were inhibited by actinomycin D but were insensitive to cycloheximide. Inductions of glucose‐regulated and heat shock proteins are discussed in terms of changes in intracellular pH and sulfhydryl oxidation states.
Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts
Authors: Subramaniam M. , Shanmugam G. 
Published: February 2005

AbstractGene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum‐arrested and serum‐ stimulated cells. Addition of platelet‐derived growth factor to serum‐arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000‐dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5‐6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.
Hydrogen peroxide toxicity may be enhanced by heat shock gene induction in Drosophila
Authors: Love Jack D. , Vivino Alfred A. , Minton Kenneth W. 
Published: February 2005

AbstractRecent evidence suggests that low dose exposure of cells to hydrogen peroxide and/or induction of heat shock protein (HSP) synthesis will render cells resistant to the lethal effects of a subsequent high dose hydrogen peroxide stress. We explored this possibility in the Drosophila melanogasterSchneider tissue culture line 2. It was found that chronic low dose exposure (1 mM H2O2for 3 days) resulted in marked potentiation of the toxic effects of a subsequent high dose exposure (50 mM H2O2 for 1 h), as assessed by impairment of uridine incorporation and cell proliferation. Cells preexposed to low dose H2O2 exhibited enhanced heat shock gene transcription upon exposure to high dose H2O2, as compared to cells that did not receive low dose preexposure. Transcriptional induction of the heat shock genes by a mild non‐toxic heat shock resulted in marked enhancement of the anti‐proliferative effects of a subsequent H2O2 exposure. Thus, low dose hydrogen peroxide exposure or mild heating results in subsequent enhancement of high dose hydrogen peroxide toxicity; this effect correlates with enhanced heat shock gene expression. Possible mechanisms are discussed.
Effects of maturational agents on expression and secretion of two partially characterized high molecular weight milk‐related glycoproteins in MCF‐7 breast carcinoma cells
Authors: Abe Miyako , Kufe Donald 
Published: February 2005

AbstractWe have previously defined a human mammary epithelial antigen using a murine monoclonal antibody (MAb), designated DF3, prepared against a membrane‐enriched fraction of a human breast carcinoma. MAb DF3 detects a cell surface antigen with a molecular weight (mw) of approximately 300 Kd and a higher mw species also detectable in human milk. These findings and the demonstration that butyric acid (BA) increases DF3 antigen expression suggested that MAb DF3 reacts with a differentiation antigen detectable in human breast carcinoma cells. The results of the present study demonstrate that MAb DF3 reacts with two mucin‐like high mw glycoproteins (330 and 450 Kd) present in MCF‐7 breast carcinoma cells. The results also demonstrate that the intracellular content and secretion of DF3 antigen is increased by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and 1‐β‐D‐arabinofuranosylcytosine (ara‐C). Other known inducers of differentiation including retinoic acid (RA), hexamethylene bisacetamide (HMBA), 1,25‐dihydroxy vitamin D3 (1,25(OH)2D3) and certain polar solvents decrease DF3 antigen expression. Furthermore, the results demonstrate that DF3 antigen is secreted and that the extent coincides with changes in intracellular content. Finally, actinomycin D and cycloheximide inhibit the increases in DF3 antigen expression following TPA treatment thus suggesting that newly synthesized RNA and protein are required for induction of this antigen. Thus, the monitoring of DF3 antigen expression may provide a marker for studying maturation of human breast cancer cells.
Altered endocytosis in a mutant of LM fibroblasts defective in cell–cell fusion
Authors: Li Wande , Ryser Hugues J.‐P. , Shen Wei‐Chiang 
Published: February 2005

AbstractMutants of LM fibroblasts selected for their decreased ability to undergo polyethylene glycol‐induced cell‐to‐cell fusion (F40 subline) were examined for possible alterations of their ability to carry out endocytosis. Both fluidphase endocytosis of inulin and horseradish peroxidase and nonreceptor mediated adsorptive endocytosis of poly(L‐lysine) were reduced to 60% of control values. Comparable results were obtained when the uptake of poly(L‐lysine) was measured as internalization of surface‐bound label in label‐free medium or following continuous exposure. Accelerated breakdown of internalized label was ruled out as a cause for decreased label accumulation. Accelerated exocytosis is an unlikely cause, and it is suggested that the decreased uptake is due to a decrease in the constitutive membrane vesiculation process that leads to the formation of endocytotic vesicles. The capacity of F40 cells to degrade internalized horseradish peroxidase and poly(L‐lysine) was not impaired, nor was their susceptibility to the cytotoxic action of methotrexate‐poly(L‐lysine). This drug conjugate must be degraded inside cells and release small molecular methotrexate in order to be cytocidal. These data suggest that only the first step of nonspecific endocytosis is impaired, while the subsequent steps that require fusion of endosomes to lysosomes proceed normally. Since the formation of primary endosomes requires membrane fusion through the external aspect of the plasma membrane and in that respect resembles cell‐cell fusion, we propose the hypothesis that the observed decrease in endocytosis is related to the decreased ability of F40 cells to fuse with each other, and reflects a decreased efficiency of fusion processes at the external face of the plasma membrane.
Highly adherent mutants of SV40‐transformed mouse fibroblasts: Genetic and biochemical characterization
Authors: Benoni Désiré , Bradley W. E. C. 
Published: February 2005

AbstractMutants of SV40‐transformed mouse fibroblasts have been isolated that have greatly increased cell‐substratum adherence. The adherent phenotype (COL−) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild‐type and mutant cells were found with respect to the protein content of the substrate‐attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+‐chelating agent. However, the mutants yielded 2.5‐10‐fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6‐thioguanine, which suggests X‐linkage.
A comparison of the platelet‐derived growth factor‐dependent tyrosine kinase activity in sparse and confluent fibroblasts
Authors: Kazlauskas Andrius , Dicorleto Paul E. 
Published: February 2005

AbstractConfluent (density‐inhibited) human foreskin fibroblasts require a higher concentration of platelet‐derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity‐inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180‐kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3PO4, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine‐specific antibody. The extent of PDGF‐dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF‐induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF‐induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that (1) PDGF‐dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and (2) density‐dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity of the PDGF receptor.
Electrophysiological differences between bone cell clones: Membrane potential responses to parathyroid hormone and correlation with the cAMP response
Authors: Ferrier Jack , Ward Angela 
Published: February 2005

AbstractElectrophysiological measurements on three clonally derived bone cell populations showed a positive correlation between longer‐term hyperpolarizing membrane potential responses to parathyroid hormone (PTH) and an intracellular cAMP response to PTH. One clone (RCJ 1.20) had no sustained electrophysiological response and no cAMP response to PTH. Another clone (ROS 17/2.8) had both a sustained hyperpolarizing response and a cAMP response to PTH. The third clone (RCB 2.2) initially had both an electrophysiological response and a cAMP response to PTH, but both responses were lost after prolonged growth in culture. Application of dibutyryl cAMP to RCJ 1.20 and ROS 17/2.8 cells produced both transient and sustained hyperpolarizing responses. Application of isobutylmethylxanthine produced a sustained hyperpolarization. These results suggest that the hyperpolarizing response to PTH is related to a cAMP‐mediated increase in Ca2+ conductance, which leads to an increase in Ca2+‐activated K+ conductance. The pronounced membrane potential spikes and fluctuations that occur in some of the clonal lines were shown to be unrelated to the hyperpolarizing response to PTH. This was demonstrated by the lack of correlation between the occurrence of the spikes or fluctuations and the occurrence of the hyperpolarizing response to PTH in the various cell lines, by the lack of effect of PTH on the spikes and fluctuations, and by the lack of effect on the hyperpolarizing response to PTH of verapamil and quinine, both of which significantly reduce the spikes and fluctuations.
Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding
Authors: Eide Brock L. , Krebs Edwin G. , Ross Russell , Pike Linda J. , Bowen‐Pope Daniel F. 
Published: February 2005

AbstractPreincubation of Swiss 3T3 cells with the tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) at 37°C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125l‐platelet‐derived growth factor (125I‐PDGF), and does not affect the affinity of 125I‐PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I‐PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand‐binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.
The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation
Authors: Koury Mark J. , Bondurant Maurice C. , Mueller Thomas J. 
Published: February 2005

AbstractErythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units‐erythroid (CFU‐e), develop into erythrocytes. Biochemical studies of this hormone‐directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU‐e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia‐inducing Friend virus (FVA). Using these FVA‐infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48‐hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS‐polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electro‐metrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase‐specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP‐mediated terminal differentiation of FVA‐infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP‐mediated differentiation in these cells.