bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the growth factor requirements for stimulation of WI‐38 cells after extended periods of density‐dependent growth arrest
Authors: Owen Thomas A. , Soprano Dianne Robert , Soprano Kenneth J 
Published: February 2005

AbstractWhen cultures of WI‐38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI‐38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long‐term quiescent WI‐38 cells (stimulated 26 days after plating) and short‐term quiescent WI‐38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short‐ and long‐term quiescent WI‐38 cells, in that the same defined medium supplemented with epidermal growth factor [EGF], recombinant human insulin‐like growth factor 1 [IGF‐1], and dexamethasone [DEX] stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long‐term quiescent WI‐38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long‐term density‐arrested WI‐38 cells.
Binding modes of IgG from Pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs
Authors: Milner Yoram , Sagi Ephraim , Timberg Rina , Michel Beno , Me'te'zeau Phlllppe , Goldberg Michel 
Published: February 2005

AbstractPemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P‐IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P‐IgG onto living keratinocytes only. This was shown with several Pernphigus sera or purified P‐IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and “Km” values for P‐IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]‐P‐lgC onto Percollseparated (living) keratinocytes showed the existence of two classes of sites: 2 × l06 sites/cell high‐affinity sites (Kd = 1.5 × 10 −6 M total IgG) and 25 × l06 sites/cell low‐affinity sites (Kd = 6 × 10−5 M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light‐scattering signal, the RNA content, the size and morphology, and the P‐IgG binding to the cells. The results indicated that P‐IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell‐sorter analysis of cells with membrane‐bound P‐IgC, coupled to direct determination of P‐IgC released in the medium, revealed the fate of bound P‐IgG: 40–60% of the P‐IgGs were released in the medium within 30 minutes at 37°C. This was accompanied and followed by a much slower, metabolic energydependent, internalization process of the membrane‐bound P‐IgG. The internalization has been confirmed by electron microscopy of bound P‐lgC labeled with protein A‐gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit “antisurface” antibodies.
Actions of insulin‐like growth factor‐I on the B104 neuronal cell line: Effects on cell replication, receptor characteristics, and influence of secreted binding protein on ligand binding
Authors: Orlowski Craig C. , Chernausek Steven D. , Akeson Richard 
Published: February 2005

AbstractSeveral peptide growth factors influence the growth and differentiation of neural cells. To investigate further the growth‐promoting effects of the somatomedins on cells of neural origin, the authors characterized the binding and mitogenic effects of insulin‐like growth factor‐1 (IGF‐1) on a functionally differentiated rat neuronal cell line (B104). Specific, high‐affinity (Kd ≅ 10−9 M) receptors for IGF‐1 were abundant (approximately 124,000 binding sites/B104 cell). These IGF‐1 receptors were similar to those of non‐neural tissue in that they contained 135,000 dalton binding subunits (demonstrated by affinity labeling and autoradiography) and recognized insulin at high concentrations. IGF‐1 was more potent than insulin at stimulating B 104 cell replication in serum‐free medium and, at an initial concentration of 100 ng/ml, was the only exogenous growth factor needed to maintain growth through several cell divisions. Furthermore, cells of later passage were found to secrete specific IGF binding proteins that produced an unusual, biphasic binding curve in radioligand displacement studies. These binding proteins apparently sequester IGF‐1, limiting its access to the cell. Experiments with B 104 cells may provide useful information about the role of IGFs and their binding proteins as potential regulators of growth and differentiation of the primitive neuroblast.
Differential inhibitory effects of TGF‐β on EGF‐, PDGF‐, and HBGF‐1‐stimulated MG63 human osteosarcoma cell growth: Possible involvement of growth factor interactions at the receptor and postreceptor levels
Authors: Mioh Hiroyuki , Chen Jan‐Kan 
Published: February 2005

AbstractThe growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or heparin‐binding growth factor‐1 (HBGF‐1). The mitogenic effect of EGF and PDGF is completely blocked by TFG‐β at 1 ng per ml and the effect of HBGF‐1 is attenuated by 75–80%. Treatment of MG63 cells with TGF‐β reduces HBGF‐1 receptor binding affinity from 1.24 × 10–11 M to 3.51 × 10–11 M with no change on the receptor number (1.1. × 103 per cell). The receptor‐binding affinity of EGF and PDGF is not altered by TGF‐β treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF‐β pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF‐1‐stimulated cells with and without TGF‐β pretreatment. These data suggest that TGF‐β inhibits EGF and PDGF mitogenicity by blocking EGF‐ and PDGF‐stimulated tyrosine kinase activity and attenuates HBGF‐1 mitogenicity by decreasing its receptor affinity.
Electrophysiological properties of isolated rat liver cells
Authors: Sawanobori Tohru , Takanashi Hideo , Hiraoka Masayasu , Iida Yoshitaka , Kamisaka Kazuaki , Maezawa Hidenori 
Published: February 2005

AbstractThe electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole‐cell configuration. The membrane potential in isolated hepatocytes was −42 ± 17 mV (n = 20). The input resistance (Rin) and the time constant (τm) were 51 ± 17 M (the range of 34 to 180 MΩ) (n = 20) and 4.2 ± 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 μF/cm2, the membrane resistance and membrane capacitance were 42. ± 9.0 KΩ cm2 and 87 ± 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current‐voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage‐sensitive Ca2+ channel in the isolated hepatocytes.
Regulation of beta‐adrenergic receptor expression in rat liver
Authors: Schleifer Leonard S. , Black Ira B. , Reid Lola M. 
Published: February 2005

AbstractTo begin defining the factors regulating neurotransmitter receptor expression, we examined beta‐adrenergic receptors in rat liver in vivo and in primary liver cultures under defined hormonal conditions. β‐receptors described a remarkable developmental profile in vivo, increasing fivefold between embryonic days 16 and 20, and decreasing tenfold by early adulthood. The developmental decrease reflected reduced receptor number without a change in receptor properties. The ontogenetic decrease appeared to be specific for β‐receptors α‐receptors developed in a hyperbolic fashion, reaching high plateau values by the third postnatal week.Adult rat liver cells plated into culture re‐expressed high β‐receptor levels, exhibiting a 4‐8‐fold increase. A similar pattern of expression of the β‐receptors, having similar pharmacological properties, was observed in primary liver cultures maintained in serum‐free medium, in a serum‐supplemented medium or in several variations of a serum‐free, hormonally defined medium designed for primary liver cultures. Thus, the degree of expression of the β‐receptors was not found affected by various hormones, by serum, or by any medium condition. By contrast, the degree of expression of the β‐receptors was markedly sensitive to cell density. High expression of the β‐receptors was observed at low cell densities (1‐3 × 106 cells/150 mm dish), and low expression or no expression was observed in confluent cultures (10‐20 × 106 cells/150 mm dish). Our experiments suggest that β‐receptor expression does not follow an immutable program, but may be regulated by density‐dependent cell‐cell interactions.
Platelet‐derived growth factor, epidermal growth factor, and insulin‐like growth factor I regulate specific cell‐cycle parameters of human diploid fibroblasts in serum‐free culture
Authors: Chen Yuhchyau , Rabinovitch Peter S. 
Published: February 2005

AbstractThe growth regulation of human diploid fibroblasts by platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), insulin‐like growth factor I (IGF‐I) (somatomedin C), dexamethasone, and transferrin was investigated in a serumfree, chemically defined culture system. Cell‐cycle kinetic parameters were determined using 5′‐bromodeoxyuridine (BrdU) incorporation and flow cytometric analysis with the DNA‐specific dye Hoechst 33258. We found that PDGF and EGF regulate the proportion of cells capable of entering the cell cycle from the quiescent state, with smaller effects upon the rate of cell transition from G1 into S phase. IGF‐I, on the other hand, regulates the rate of cell exit from G1 without affecting the cycling fraction. Transferrin and dexamethasone showed less effect upon the cell‐cycle kinetics under these culture conditions. The data provide functional evidence that PDGF and EGF regulate similar cell‐kinetic parameters in human fibroblast cultures. IGF‐I is functionally distinct from both PDGF and EGF in its role of regulating G1 exit rate without affecting the cycling fraction. These observations made by BrdU‐Hoechst flow cytometric techniques provide a novel perspective on the regulatory effects exerted by different classes of growth factors, and suggest a mode of interdependence of these mitogens in regulating the net growth rate which could be a feature of growth regulation in vivo. These data also provide a different perspective on the regulation of the growth of fibroblast‐like cells than that of the “competence/progression” cell‐cycle model.
Effect of Na+ flux inhibitors on induction of c‐fos, c‐myc, and ODC genes during cell cycle
Authors: Panet Rivka , Amir Iris , Snyder David , Zonenshein Leora , Atlan Henri , Laskov Reuven , Panet Amos 
Published: February 2005

AbstractThe role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride‐sensitive Na+/H + antiport and the bumetanide‐sensitive Na + /K + /CI‐ cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain‐sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na + /K + pump stimulation was obtained.Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S‐phase was completely inhibited. To investigate the mode of cell‐cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes‐c‐fos, c‐myc, and ornithin decarboxylase (ODC)‐was measured during cell transition through the G1‐phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c‐fos, c‐myc, and ODC mRNAs. These results suggest that stimulation of Na+ fluxes by serum growth factors is essential for cell transition into the S‐phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c‐fos, c‐myc, and ODC genes induction.
Regulation of hepatocyte growth: Alpha‐1 adrenergic receptor and ras p21 changes in liver regeneration
Authors: Cruise Jennifer L. , Muga Stephanie J. , Lee Yi‐Sheng , Michalopoulos George K. 
Published: February 2005

AbstractCatecholamines, acting via the alpha‐1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha‐1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]‐prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]‐myo‐inositol was transiently reduced between 8 and 16 hr, when alpha‐1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha‐1 receptors and ras protein in the early events of liver regeneration.
Control of protein synthesis by extracellular Na+ in cultured fibroblasts
Authors: Petronini Pier Giorgio , Tramacere Mariarosaria , Mazzini Alberto , Kay John E. , Borghetti Angelo F. 
Published: February 2005

AbstractIn chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum‐dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate‐containing media, but at lower or higher than physiological pH. Analysis by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.
Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes
Authors: Schmidt Rainer , Cathelineau Christine , Cavey Marie T. , Dionisius Veronique , Michel Serge , Shroot Braham , Reichert Uwe 
Published: February 2005

AbstractSodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross‐linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus‐40 (SV‐40)‐transformed human foreskin keratinocyte line SV‐K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane‐associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane‐associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.
Epidermal growth factor: Modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport
Authors: Gawel‐Thompson Kathleen J. , Greene Robert M. 
Published: February 2005

AbstractPolyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose‐ and time‐dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4‐6 hours. EGF also stimulated the initial rate of putrescine transport in dose‐ and time‐dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 μM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 μM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF‐treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 μg/ml) and actinomycin D (50 μg/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca+ + while α‐aminoisobutryic acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO‐treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.
Protein kinase C activation and down‐regulation in relation to phorbol ester‐induced differentiation of SH‐SY5Y human neuroblastoma cells
Authors: Heikkilä Jari E. , Åkerlind Göran , Åkerman Karl E. O. 
Published: February 2005

AbstractThe role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH‐SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane‐associated protein kinase C (PK‐C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding‐up of cells. A subsequent decrease in particulate PK‐C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron‐specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1‐(5‐lsoquinolinesulphonyl)‐2‐methylpiperazine (H‐7), an inhibitor of PK‐C and cyclic nucleotide‐dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N‐(2‐guanidinoethyl)‐5‐isoquinolinesulphonamide (HA‐1004), which primarily inhibits cyclic nucleotide‐dependent protein kinases, had no effect on the TPA‐induced phenotypic differentiation. H‐7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron‐specific enolase level. Both H‐7 and HA‐1004 inhibited the TPA‐induced increase in noradrenaline content.