bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: Evidence for two compartments in a nondegradative pathway
Authors: Raub Thomas J. , Koroly Mary Jo , Roberts R. Michael 
Published: February 2005

AbstractThe internalization and recycling of CHO cell plasma membrane components have been quantified by using iodinated wheat germ agglutinin (WGA) as an adsorptive tracer. Most of these binding sites are thought to be composed of a subpopulation of plasma membrane proteins called high‐molecular‐weight acidic glycoproteins (HMWAG). Greater than 90% of the WGA bound on the cell surface can be removed by brief treatment with N‐acetylglucosamine (GlcNAc). At 37°C, endocytosis of WGA that had been allowed to bind to the surface at 4°C is curvilinear with an initial rapid phase occurring with a t1/2 of 6–8 min. Within 20 min, accumulation has slowed gradually to steady‐state with 65% of the cell‐associated WGA located intracellularly or resistant to remova by GlcNAc. These portions are unaffected by increasing the extracellular concentration of WGA from 0.003 μM. By using pulse‐chase experiments, the observed decrease in rate of endocytosis is shown to be due to return of the WGA‐HMWAG complexes to the cell surface. More than 60% of the WGA that had been internalized is recycled within 30 min, with a mean t1/12 of 17 min. Recycling involved at least two intracellular populations where a significant fraction ( < 30%) of the WGA‐HMWAG complexes are lost gradually from the rapidly recycling pool. Most of the WGA‐HMWAG complexes that had internalized are not delivered to the lysosome. These results demonstrate the magnitude of rapid and continuous recycling of WGA binding sites between the cell surface and endosomes in fibroblasts.
Participation of transient‐type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells
Authors: Suzuki Nobuyuki , Kano Masaakira , Tanakadate Akihiro , Kudo Yoshihisa , Takagi Hiroshi , Yoshioka Tohru 
Published: February 2005

AbstractParticipation of two types of Ca2+ channels (T‐ and L‐types) in the sustained increase of cytosolic‐free Ca2+ concentration ([Ca2+]i) was studied in thyrotropin‐releasing hormone (TRH)‐stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole‐cell voltage‐clamp and Fura‐2 fluorometry, respectively. Phenytoin (100 μM) and Ni2+ (100 μM) selectively blocked T‐type Ca2+ channels and suppressed the TRH‐induced sustained [Ca2+]i increase in single cells. Synthetic ω‐conotoxin (ω‐CgTX, 2 μM) preferentially blocked L‐type Ca2+ channels, but it did not suppress the TRH‐induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T‐type Ca2+ channels in GH3 cells.
Antigen‐Dependent transition of IgE to a detergent‐insoluble form is associated with reduced IgE receptor‐dependent secretion from RBL‐2H3 mast cells
Authors: Seagrave Jeanclare , Oliver Janet M. 
Published: February 2005

AbstractIn mast cells, basophils, and the RBL‐2H3 tumor mast cell model, crosslinking cell surface IgE‐receptor complexes by multivalent ligands activates a signal transduction pathway that leads to the secretion of histamine, serotonin, and other inflammatory mediators. Receptor crosslinking in RBL‐2H3 cells also changes cell surface morphology and increases F‐actin assembly. Previously, Robertson et al. (J. Immunol., 135: 4565–4572, 1986) demonstrated that crosslinked IgE‐receptor complexes become associated with the Triton X‐100‐insoluble fraction (the “cytoskeleton”) of RBL‐2H3 cells and raised the possibility that receptor‐cytoskeletal association may be a required step in the stimulation of secretion. The studies reported here confirm by flow cytometry that crosslinking cell surface IgE by antigen induces the association of the crosslinked complexes with the detergent‐insoluble fraction. Dose‐response studies, also reported here, indicate that the detergent insolubility of the complexes does not correlate with secretion. Thus, secretion increases with antigen concentration to a maximum beyond which more antigen causes less, not more, secretion. There is little residual detergent‐insoluble IgE at the concentrations of antigen that promote optimal secretion, whereas the association of IgE with the detergent‐insoluble fraction is maximal at the high antigen concentrations that result in reduced secretion. The addition of monovalent hapten to reduce the amount of crosslinking caused by high concentrations of antigen increases secretion and simultaneously reduces the association of IgE with the detergent‐insoluble fraction. Dihydrocytochalasin B, an inhibitor of antigen‐stimulated actin polymerization, also increases the rate and extent of secretion and simultaneously delays the association of crosslinked IgE‐receptor complexes with the detergent‐insoluble fraction. From these data, we propose that the association of crosslinked IgE receptors with the detergent‐insoluble fraction of RBL‐2H3 cells increases with increased receptor crosslinking, is enhanced by antigen‐induced actin polymerization, and is more likely related to the termination than the stimulation of secretion. The ligand‐induced conversion of receptors to a detergent‐insoluble form is not restricted to mast cells but occurs in a variety of cell types. Its general function may be to limit the generation or transmission of transmembrane signals.
Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts
Authors: Mikkelsen Ross B. , Asher Craig R. 
Published: February 2005

AbstractThe effects of hyperthermia (41–43°C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41°C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37°C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+: H+ exchange. Shifting temperature to 41°C increased ouabain‐sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43°C for 60 min, the membrane potential of log phase cells depolarized (20–35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41°C only in the presence of ouabain. At 43°C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43°C, 0.25 mM amiloride inhibited heat‐stimulated Na+ uptake. Na+ efflux was enhanced at 41°C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41°C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures ≥43°C, the Na+ pump is inhibited.
Insulin‐like growth factor (IGF) binding to human fibroblast and glioblastoma cells: The modulating effect of cell released IGF binding proteins (IGFBPs)
Authors: McCusker R. H. , Camacho‐Hubner C. , Clemmons D. R. , Bayne M. L. , Cascieri M. A. 
Published: February 2005

AbstractThe cell surface of human fibroblasts contains not only type IIGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF‐I‐cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125l]‐IGF‐l binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]‐IGF‐I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]‐IGF‐I added) since they have a higher affinity than type IIGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF‐I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]‐IGF‐I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]‐IGF‐I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125 I]‐IGF‐l that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]‐IGF‐l (IQAYLj‐IGF‐I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF‐I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF‐I and the [QAYL]‐IGF‐I compete equally with the [125I]‐[QAYL]‐IGF‐I for binding to cell surfaces, whereas unlabeled [QAYL]‐IGF‐I is > 25‐fold less potent compared to IGF‐I in competing with [125I]‐IGF‐I for cell surface binding. Specific binding of [125I]‐[QAYL]‐IGF‐I to GM10 and 1690 cell surfaces is < 20% of [125I]‐IGF‐I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]‐IGF‐I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.
Stimulating effects of insulin and low‐density lipoprotein on cell growth and macromolecular syntheses of HeLa cells cultured in K+‐depleted medium
Authors: Hosokawa Keiko , Yamaguchi Hisao , Ikehara Toshitaka , Takahashi Akira , Miyamoto Hiroshi 
Published: February 2005

AbstractThe influence of the intracellular K+ concentration on the effects of growth factors (insulin, EGF, hydrocortisone, and transferrin) and LDL on growth of HeLa cells was investigated. Upon replacenient of K+ in a chemically defined medium (K+‐CDM) by Rb+ (Rb+‐CDM), about 80% of the intracellular K+ was replaced by Rb+ within 24 h, but showed no further change in the next 24 h, irrespective of addition of dialyzed calf serum (5%) or growth factors to the medium. In Rb+‐CDM, cell growth and DNA synthesis were greatly suppressed, although cell viability was not significantly altered for 72 h. The suppression of cell growth was partially restored by addition of serum, insulin (5 μg/ml), or LDL (2.5 mg/ml) to Rb+ ‐CDM. A combination of serum and insulin or insulin and LDL stimulated cell growth to approximately the level in K+‐CDM without any addition, but a combination of serum and LDL did not have more effect than that of serum alone. Unexpectedly, other factors were ineffective in stimulating growth in Rb+‐CDM. In Rb+‐CDM, the effect of insulin was lost in 24–48 h, whereas that of LDL persisted for at least 96 h. Insulin and LDL also enhanced growth in K+‐CDM. After cessation of cell growth in Rb+‐CDM for 24 h, addition of insulin and/or LDL markedly restored cell growth and DNA synthesis. Therefore, insulin and LDL may stimulate certain mechanisms required for cell growth that can operate in K+‐deficient conditions.
Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: Protease complex receptors increase during in vitro myogenesis
Authors: Festoff Barry W. , Rao Jasti S. , Rayford Alan , Hantaï Daniel 
Published: February 2005

AbstractIn the course of studies on the regulation of plasminogen activator‐mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myo‐blast medium was compared to that of human PNI purified from human fibro‐blasls. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of (a) increased PNI proteolytic inhibitory activity expression and (b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle suface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.
Cellular distribution and biological activity of epidermal growth factor receptors in A431 cells are influenced by cell‐cell contact
Authors: Lichtner Rosemarie B. , Schirrmacher Volker 
Published: February 2005

AbstractThe potential significance of cell‐cell interactions on EGF receptor (EGFR) activity was investigated in cultured adherent A431 cells seeded as single‐cell suspensions with different initial cell densities. In dense cultures, EGFRs were mainly localised at cell boundaries and in microvilli as shown by immunofluorescence analysis with an EGFR‐specific antibody while in sparse cultures the distribution of EGFRs was more diffuse. Scatchard analysis showed that as cell density decreased the number of high‐affinity receptors increased considerably. Upon treatment of adherent intact cells with EGF all cells in sparse cultures contained activated EGFRs as demonstrated by immunofluorescence analysis with a phosphotyrosine‐specific antibody, while in dense cultures mainly cells at the periphery of a cluster and especially at their expanding borders exhibited activated EGFRs. EGF‐induced phosphorylation in intact cells was greatly enhanced in sparse compared with dense cultures as demonstrated by immunoprecipitation with a phosphotyrosine‐specific antibody. In contrast to intact cells, in cytoskeleton preparations, obtained after mild detergent treatment of adherent cells, EGFRs were able to undergo EGF‐independent phosphorylation. Pretreatment of cells with EGF led to enhanced tyrosine phosphorylation of cytoskeletal‐associated proteins. Our observations suggest that cell density has a considerable effect on the subcellular localisation as well as biological activity of the EGFR. Thus, in intact A431 cells growing with extensive cell‐cell interactions some negative control mechanisms preventing EGFR activation may be exerted by adjacent cells.
Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: Differentiation response distinguishes KGF from EGF family
Authors: Marchese C. , Messina A. , Faggioni A. , Torrisi M. R. , Frati L. , Ron D. , Rubin J. , Aaronson S. A. 
Published: February 2005

AbstractHuman keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF‐ or EGF‐stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGFα, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.
Human recombinant interleukin‐1β‐ and tumor necrosis factor α‐mediated suppression of heparin‐like compounds on cultured porcine aortic endothelial cells
Authors: Shimada Kazuyuki , Ozawa Toshio , Kobayashi Masashi 
Published: February 2005

AbstractCytokines are known to tip the balance of the coagulant‐anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface‐associated heparin‐like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin‐1β (rIL‐1β) or tumor necrosis factor α (rTNFα) in a dose‐ and time‐dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rlL‐1β or 5 ng/ml of rTNFα for 12–24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S‐heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I‐Labeled antithrombin III to the endothelial cell surface was reduced to 40–60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin‐like) activity was partially diminished in cytokine‐treated endothelial cells. Thus, cytokine‐mediated suppression of heparin‐like substance on endothelial cells appears to be another cytokine‐inducible endothelial effects affecting coagulation.
Role of extracellular signaling on endothelial cell proliferation and protein N‐glycosylation
Authors: Oliveira Caroline M. , Banerjee Dipak K. 
Published: February 2005

AbstractIn vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine‐linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in Go/G1 phase. The cellular response to protein N‐glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS‐PAGE indicated that glycosylation of 80–120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry “high mannose” and “complex” type oligosaccharide chains attached to their protein core.
Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer
Authors: Milton Shirlette G. , Knutson Victoria P. 
Published: February 2005

AbstractIn cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin section electron microscopy. Although structurally similar, the tight junctional complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3‐C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer‐filter systems were also compared. The permeability coefficient of albumin across the endotnelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of en‐dothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.