bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cellular mechanisms of ATP‐induced hyperpolarization in renal epitheloid MDCK‐cells
Authors: Paulmichl M. , Pfeilschifter J. , Wöll E. , Lang F. 
Published: February 2005

AbstractPrevious studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)‐cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12‐O‐tetradecanoyl phorbol 13‐acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5‐tris‐phosphate (IP3), inositol 1,3,4,5‐tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.
Growth and differentiation of primary tracheal epithelial cells in culture: Regulation by extracellular calcium
Authors: Martin Walter R. , Brown Chris , Zhang Yao Juan , Wu Reen 
Published: February 2005

AbstractGrowth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum‐free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40‐60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (≥ 0.3 mM). High calcium medium also increased cell‐cell interactions, formation of desmosomes, and multi‐cell layering. The relative content of mucous cells, which were identified by a mucin‐specific monoclonal antibody and the presence of mucus‐secreting granules at the ultrastructural level, was greater in the high‐calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H‐glucosamine into mucous glycoprotein fractions, was also increased more than 5‐fold in media containing high calcium content ( ≥ 0.6 mM). In contrast, MTE cells cultured in low calcium medium ( < 0.15 mM) were squamous‐like with prominent tonofiiaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.
Brefeldin A induces endoplasmic reticulum‐associated O‐glycosylation of galactosyltransferase
Authors: Bosshart Herbert , Straehl Peter , Berger Bea , Berger Eric G. 
Published: February 2005

AbstractRecent data from several laboratories show that Brefeldin A (BFA) induces a microtubule‐dependent back‐flow of Golgi components to the endoplasmic reticulum (ER) thereby causing disassembly of the Golgi apparatus and its fusion with ER membranes. In order to delineate the effect of BFA on resident Golgi proteins, we investigated its effect on biosynthesis, maturation and intracellular transport of galactosyltransferase (gal‐T), an established trans‐Golgi enzyme. Using a protocol of metabolic labeling/immunoprecipitation followed by electrophoretic/fluorographic analysis, we show that in the presence of BFA, gal‐T matures to a molecular form of 48.5 kD, a size intermediate between the 2 precursor forms of 44 and 47 and the mature form of 54 kD (Strous and Berger: j. Biol. Chem., 257:7623–28, 1982). Little mature form was detectable in the presence of BFA even after prolonged chase times of up to 28 hr. The intermediate form was sensitive to O‐glycanase and endoglycosidase H, indicating early O‐glycosylation without sialylation and lack of complex N‐glycosylation, respectively. In order to define the compartment responsible for O‐glycosylation in the presence of BFA, a temperature block of 25°C was applied which inhibited recovery of Golgi elements from BFA‐induced fusion with ER. At this temperature and in absence of BFA, biosynthesis of gal‐T was not appreciably affected, while maturation was completely inhibited as indicated by the presence of unmodified precursor forms of gal‐T. After 60 min preincubation with BFA, a time period sufficient to demonstrate complete fusion of Golgi with ER, subsequent biosynthesis of gal‐T at 25°C in absence of BFA led to the intermediate form, while precursor forms were not detectable. These data provide direct evidence for BFA‐induced redistribution to the ER of Golgi enzymes involved in O‐glycosylation and their early functional involvement in biosynthesis of newly synthesized gal‐T.
Restoration of adhesive potentials of Ehrlich ascites carcinoma cells by modification of plasma membrane
Authors: Bogdanov Alexei A. , Gordeeva Larisa V. , Baibakov Boris A. , Margolis Leonid B. , Torchilin Vladimir P. 
Published: February 2005

AbstractA novel technique for modulating the spreading of ascites cells has been developed. Plasma membranes of Ehrlich ascites carcinoma cells were modified in two different ways: 1) biotin residues were covalently coupled to membrane components; 2) biotinylated lipid was introduced into plasma membranes. Adhesion and spreading of modified cells on avidin‐coated substrates were studied and compared to those of non‐modified cells. Both types of membrane alteration were shown to induce specific (biotin‐dependent) interaction with immobilized avidin with resultant cell spreading. Spread cells attained epithelioid‐like morphology with the formation of wide thin lamellae, focal contacts with substrate, and circular actin bundles. The process of spreading was shown to be energy‐dependent: it could be blocked by metabolic inhibitors and by low temperature. Formation of extended lamellae was prevented by preincubation of cells in the presence of cytochalasin B. The effects of metabolic poisons, low temperature, and microfilament–disruptive drugs were reversible and after the restoration of physiological conditions the cells resumed the spreading process. Immunoprecipitation of biotinylated cell lysates with antiserum to cytoplasmic domain of 3,‐integrin subunit revealed a major 110 kD avidin‐binding component. We conclude that lack of spreading of ascites carcinoma cells may be explained by the lack of functionally active adhesion‐ and spreading‐competent cell‐surface receptors, but may not be attributed to the defects in intracellular function or organization. Intracellular machinery of cell spreading is preserved in these ascites cells and could be turned on by cell attachment to the substrate via artificial adhesive site incorporated into plasma membrane.
Adhesion of mouse mast cells to fibroblasts: Adverse effects of steel (SI) mutation
Authors: Kaneko Yoshiyuki , Takenawa Jun , Yoshida Osamu , Fujita Kohichi , Sugimoto Kenkichi , Nakayama Hiroki , Fujita Jun 
Published: February 2005

AbstractMouse bone marrow‐derived cultured mast cells proliferate on +/+ mouse embryo‐derived 3T3 fibroblasts, but not on SI/SId mouse embryo‐derived 3T3 fibroblasts, in the absence of IL‐3 and IL‐4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888–2891, 1989). To further characterize the mast cell‐fibrpblast interactions and the effects cf SI mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 103 to 5 × 105 cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited byan RGD‐containingpeptide, an anti‐LFA‐1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from SI/SId mouse embryos. Adhesion to mouse spleen‐derived fibroblasts lacking mast cell‐supporting activitywas comparable to that to SI/SId/3T3 cells. The failure of mast cells to adhere to fibroblasts with the SI mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type SI gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.
Lateral diffusion of CD4 on the surface of a human neoplastic T‐cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV‐1)
Authors: Pal Ranajit , Nair B. C. , Hoke George M. , Sarngadharan M. G. , Edidin Michael 
Published: February 2005

AbstractThe envelope glycoprotein (gp120) of HIV‐1 was labeled with fluorescein by using 6‐[4,6‐dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4‐positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein‐labeled gp120 with CD4 was observed in a solid‐phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV‐1‐infected cells on CEM cells was significantly inhibited in the presence of fluorescein‐labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein‐labeled gp120 was approximately 5 × 10−10 cm2sec−1, with nearly 61 % of the receptor molecules being mobile. Binding of anti‐gp120 monoclonal antibody to the CD4‐gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.
Culture‐induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells
Authors: Speir Edith , Sasse Joachim , Shrivastav Shashi , Casscells Ward 
Published: February 2005

AbstractThe activity of acidic and basic fibroblast growth factor‐like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4‐fold more bFGF‐like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10‐fold more bFGF‐like activity than the fresh cell lysates. We consistently find aFGF‐like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3‐fold more aFGF‐like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF‐like activity in cell and ECM extracts was 8‐fold higher than in fresh cell extracts.The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin‐sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti‐aFGF (1.1 M), or anti‐bFGF (1.5M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF‐like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125laFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.
Analysis by cell hybridization of mechanisms that regulate β‐Adrenergic responses in reticulocytes and in differentiating erythroid cells
Authors: Porzig Hartmut , Moudry Radmila , Montandon Jean‐Blaise 
Published: February 2005

AbstractIn intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t½ ≈ 2 h) during which the initial activity was reduced by 40–50% was followed by a slow phase with t½ close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (‐)isoprenaline‐ or forskolin‐stimulated activities behaved similarly and bacterial toxin‐monitored Gs and Gi proteins remained stable. The mechanism of the initial decrease in hormonal responsive ness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte‐derived β‐adrenoceptors and MEL cell‐derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide‐induced differentiation of MEL cells showed stable or increasing rates of receptor‐coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gs protein in these cells. A cyclase‐stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short‐term regulation of hormonal responsiveness. We conclude that the strength of β‐adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein‐mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably via a cytosolic proteolytic pathway.
GM‐CSF and phorbol esters modulate GM‐CSF receptor expression by independent mechanisms
Authors: Brizzi Maria F. , Arduino Carlo , Carlo Avanzi G. , Bussolino Federico , Pegoraro Luigi 
Published: February 2005

AbstractHuman granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) (0.1 nM) down‐modulates its receptor in IL‐3/GM‐CSF dependent M‐07e cells, in KG‐1 cells and normal granulocytes, whereas phorbol esters 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM) down‐modulates the GM‐CSF receptor in M‐07e cells and granulocytes but not in KG‐1 cells. As data analysis shows by nonlinear regression, the decreased binding ability depends on a reduction of the binding sites with no significant change of their dissociation constant. To gain insight into the mechanisms involved in the GM‐CSF receptor regulation, we investigated the role of protein kinase C (PKC). GM‐CSF, unlike TPA, was unable to activate PKC in all the cells studied. Moreover, unlike TPA, GM‐CSF was still able to down‐modulate its receptor in cells where PKC was inhibited by 1‐(5‐isoquinolonesulphonyl)‐2‐methylpiperazine (H7) and staurosporine or in cells where PKC was exhausted by prolonged incubation with 1 μM TPA. Finally, the receptor re‐expression rate was accelerated by protein kinases inhibitors. These results, taken together, indicate the presence of a PKC‐dependent and ‐independent down‐modulation mechanism and a negative role of the endogeneous protein kinases in GM‐CSF receptor re‐expression.
Modulation of Na+/K+ exchange potentiates lipopolysaccharide‐induced gene expression in murine peritoneal macrophages
Authors: Ohmori Yoshihiro , Reynolds Elwood , Hamilton Thomas A. 
Published: February 2005

AbstractThe role of Na+/K+ exchange in regulating lipopolysaccharide (LPS)‐mediated induction of cytokine gene expression has been examined in murine peritoneal macrophages. Depletion of K+ from the culture medium resulted in a three‐ to five‐fold potentiation of tumor necrosis factor‐α (TNFα), KC (gro), and IP‐10 mRNA expression in LPS‐treated macrophages. The potentiating effect was apparently the result of inhibition of Na+/K+ exchange through the Na+/K+‐adenosine triphosphatase (ATPase) because ouabain‐mediated inhibition of Na+/K+‐ATPase was also able to potentiate cytokine mRNA expression as much or more than did K+ depletion. The effects of K+ depletion or ouabain treatment were not caused by depolarization of the macrophage membrane because depolarization mediated by elevating extracellular K+ levels was inhibitory to cytokine mRNA expression. Depletion of Na+ by substitution with cho ine in the culture medium also markedly potentiated LPS‐induced gene expression. The Na+/H+ antiporter was not, however, involved in potentiating cytokine expression because treatment of macrophages with amiloride either had no effect on or was inhibitory to the LPS‐induced changes in mRNA levels. The potentiation of gene expression was selective and was at least partially the result of increased transcriptional activity of each gene. Whereas Na+ depletion and ouabain both inhibited 86Rb+ uptake by macrophages, treatment with LPS had no effect either on Rb+ uptake or on efflux. Thus altered Na+/K+ exchange is not a component of the primary signalling pathway(s) mediating response to LPS. Nevertheless, modulation of macrophage Na+/K+ exchange by agents encountered during an inflammatory response may be an important determinant of the magnitude and quality of specific gene expression.
Differential regulation of the expression of transforming growth factor‐βs 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK‐49F and A549 cells
Authors: Danielpour David , Kim Kyung Young , Winokur Thomas S. , Sporn Michael B. 
Published: February 2005

AbstractAlthough most biological activities of transforming growth factor‐βs 1 and 2 (TGF‐β1 and TGF‐β2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme‐linked immunosorbent assays for TGF‐β1 and TGF‐β2 to examine the effects of a variety of treatments on expression of these two TGF‐β isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF‐β1 and not TGF‐β2, whereas retinoic acid (RA) induces secretion of TGF‐β2 and not TGF‐β1 in NRK‐49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF‐β2, while RA decreases the levels of TGF‐β1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF‐β1 and TGF‐β2 in A549 cells, while selectively inhibiting TGF‐β1 secretion in NRK‐49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF‐β1 and TGF‐β2, which were studied on NRK‐49F cells, demonstrate that EGF blocks the induction of TGF‐β2 mRNA and peptide by RA, while Dex inhibits the induction of TGF‐β1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF‐βs1 and 2.
Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen‐responsive shionogi carcinoma 115
Authors: Kasayama Soji , Sumitani Satoru , Tanaka Akira , Yamanishi Hiroshi , Nakamura Nobuyoshi , Matsumoto Keishi , Sato Bunzo 
Published: February 2005

AbstractAn androgen‐responsive cloned cell line (SC‐3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)‐like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC‐3 cells in an autocrine mechanism. Since the androgen‐induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC‐3 cells. Heparin was found to exhibit significant inhibition of testosterone‐induced growth in a concentration‐dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 μg/ml. DNA synthesis of SC‐3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC‐3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen‐induced growth factor in the conditioned medium from testosterone‐treated cells. These results indicate that heparin inhibits autocrine loop in SC‐3 cells induced by androgen. Thus the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.
Heparanase activity in cultured endothelial cells
Authors: Godder K. , Vlodavsky I. , Eldor A. , Weksler B. B. , Haimovitz‐Freidman A. , Fuks Z. 
Published: February 2005

AbstractThis study was undertaken to identify a heparan sulfate (HS) degradation endoglycosidase (heparanase) in cultured endothelial cells (EC) and to characterize the requirements for its release and subsequent degradation of HS side chains in the subendothelial extracellular matrix (ECM). Intact EC, EC lysates, or EC conditioned media from different sources were incubated with metabolically Na235SO4‐labeled ECM produced by bovine EC. The released sulfated products were analyzed by gel filtration on Sepharose 6B. Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) lysates expressed heparanase activity as indicated by release of most of the radioactivity from ECM as HS fragments that are one‐fifth to one‐sixth the size of the intact HS side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin. Rabbit coronary microvascular EC and bovine brain capillary EC lysates showed less heparanase activity (30–35%), whereas bovine aortic and corneal EC showed no activity. Intact HUVEC, piated directly on the labeled ECM, expressed low enzyme activity that was not changed when cells were exposed to various agents. Exposure of HUVEC to interleukin‐1, phorbol myristate acetate, tumor necrosis factor, endotoxin, thrombin, calcium ionophore A23187, fibroblast growth factor, or radiation did not induce release of the enzyme to the medium or degradation of HS in the ECM, as long as the cells remained viable. EC differ from various normal and malignant cells that degrade HS by virtue of their inability to release the enzyme. We suggest that heparanase release during vessel wall injury may regulate the growth of EC and smooth muscle by release of HS degradation products in processes such as wound healing, neovascularization, and atherosclerosis.