bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties of single potassium channels in guinea pig hepatocytes
Authors: Takanashi Hideo , Sawanobori Tohru , Kamisaka Kazuaki , Maezawa Hidenori , Hiraoka Masayasu 
Published: February 2005

AbstractThe patch‐clamp technique of cell‐attached and inside‐out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K+ concentrations. A linear single‐channel current‐voltage relationship was obtained at the voltage range of ‐80 to ‐20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K+ concentration of 148 mM in the cell‐attached patch membrane, the I‐V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K+]0) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K+]0, indicating that this channel was highly selective for K+. Open probabilities (P0) of the channel were 73‐93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K+ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34‐36°C. P0 in inside‐out patches was not changed by application of 1 μM Ca2+ nor 1 mM Mg2+ to the internal side of patch membranes. It is concluded that a novel type of the K+ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca2+]i, from those in other species. © 1994 Wiley‐Liss, Inc.
Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances functional activities of mature osteoblasts in cultured rat calvaria cells
Authors: Ohishi Keiji , Ishida Hiroshi , Nagata Toshihiko , Yamauchi Noriyuki , Tsurumi Chizuko , Nishikawa Seiji , Wakano Yoichi 
Published: February 2005

AbstractThe effects of thyroid hormone on osteoblastic differentiation and activity were studied in fetal rat calvaria (RC) cells cultured for up to 30 days in medium supplemented with thyroid hormone‐depleted serum. In this condition, the cells proliferated and differentiated to form mineralized bone nodules (BN) and expressed osteoblastic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). The continuous presence of triiodothyronine (T3) at 10‐9‐10‐8 M in the medium inhibited the osteoblastic differentiation: 34% decrease in ALP activity on day 12 and 60% decrease in BN formation on day 15 at 10‐8 M. T3 at these doses had no effect on the DNA content of RC cells at confluence (day 6). Short‐term (48‐h) exposure of T3 at 10‐9 M or higher decreased ALP activity when RC cells were differentiating (days 7–11). However, when BN formation by the cells had already reached a plateau (day 28), the activity was increased by treatment with T3 at 10‐7‐10‐6 M. OCN production was increased dose dependently by this treatment with T3 (2.1‐fold and 1.3‐fold of control at 10‐8 M on days 11 and 28, respectively). Similar increases were observed in the levels of OCN mRNA. In addition, increases in phosphorylated OPN in the medium (day 11) and mineralized matrix (day 28) were observed (1.5‐fold at 10‐8‐10‐6 M), while OPN synthesis and the level of its mRNA were depressed by T3 (60‐70% of control at 10‐8 M). These results suggest that T3 regulates osteoblastic differentiation and activity depending on the state of cell differentiation: T3 suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances the functional activity of mature osteoblasts. © 1994 Wiley‐Liss, Inc.
Evidence that modulation of glucose transporter intrinsic activity is the mechanism involved in the allose‐mediated depression of hexose transport in mammalian cells
Authors: Pratt Susan E. , Colby‐Germinario Susan , Manuel Susannia , Germinario Ralph J. 
Published: February 2005

AbstractIn serum starved V79 Chinese hamster lung fibroblast cells, replacement of D‐glucose with D‐allose resulted in a significant 38 ± 18% (P < 0.05) reduction of 2‐deoxy‐D‐glucose (2‐DG) transport. Similarly, in a respiration‐deficient mutant cell line (V79‐G14), which has elevated 2‐DG transport activity, D‐allose reduced 2‐DG transport by 59 ± 18% (P < 0.05). [3H]D‐allose uptake by V79 cells occurred slowly and was not inhibited by cytochalasin B, suggesting diffusion as the mode of D‐allose entry. Western blot analysis using a rabbit polyclonal antibody to the human erythrocyte glucose transporter (GT) demonstrated that, in both cell lines, GT content and GT subcellular distribution were not significantly different in D‐glucose vs. D‐allose‐treated cells. δ‐Antibody, which has been shown to bind to exofacial epitopes of the GT (Harrison et al., 1990, J. Biol. Chem., 265:5793‐‐5801), did not demonstrate any differences in surface binding to D‐glucose vs. D‐allose‐treated intact V79 cells. D‐allose treatment of 3T3 fibroblasts resulted in a similar decrease (72%) of 2‐DG transport, however D‐allose had no apparent effect on basal sugar transport in 3T3 adipocytes. These results suggest that D‐allose reduces sugar transport through a modulation of the intrinsic activity of the GT, and that D‐allose may act in a tissue‐specific manner. © 1994 Wiley‐Liss, Inc.
Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B
Authors: Pardes Jeffrey B. , Takagi Hajime , Martin Theresa A. , Ochoa M. Sofia , Falanga Vincent 
Published: February 2005

AbstractWe have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase‐generated fibrin (retention of fibrinopeptide B) and thrombin‐generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)‐beta 1. Fibroblasts seeded on reptilase‐generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin‐generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t‐PA) increased procollagen mRNA and TGF‐beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down‐regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley‐Liss, Inc.
Epidermal growth factor rapidly impairs activation of p34cdc2 protein kinase in HeLa cells at the G2‐M boundary
Authors: Barth Holger , Kinzel Volker 
Published: February 2005

AbstractEpidermal growth factor (EGF) has been shown rapidly to inhibit the transition from G2 phase to mitosis; beyond this transition point the cells are refractory to EGF (Kinzel et al., 1990, Cancer Res., 50:7932–7936). Using synchronized HeLa cells, EGF has now been shown to induce an overall decrease of the histone H1 kinase activity of p34cdc2 after 20 min of treatment, a time course which correlates with the number of cells in metaphase. The kinase level of actively mitotic cells is not altered by EGF. Neither the amount of p34cdc2 protein present nor that of Cyclin B is influenced by EGF, and the formation of the p34cdc2/Cyclin B complex is also unaffected. The use of antiphosphotyrosine antibodies, however, showed that p34cdc2 from cultures treated with EGF was more intensely stained than that of control cells, indicating that EGF treatment prevents the tyrosine dephosphorylation which is required for expression of the protein kinase activity of the complex. Taken together, the results show that EGF in HeLa cells very rapidly prevents the p34cdc2/Cyclin B complex from expressing kinase activity at the G2‐M boundary, which appears to be the cause for the inhibition in G2 phase. © 1995 Wiley‐Liss, Inc.
Effects of mutations in cAMP‐dependent protein kinase on chloride efflux in Caco‐2 human colonic carcinoma cells
Authors: Krolczyk Arkadiusz J. , Bear Christine E. , Lai Patrick F. H. , Schimmer Bernard P. 
Published: February 2005

AbstractIn order to evaluate the importance of cAMP and cAMP‐dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco‐2, human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of regulator subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn2+‐inducible manner and exhibited Zn2+‐inducible inhibition of cAMPdPK activity. The parental and transformed Caco‐2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide‐efflux assay. In the transformants, induction of the protein kinase mutation with ZnSo4 markedly decreased chloride efflux in response to forskolin, the 8‐(4‐chlorophenylthio) analog of cAMP, vasoactive intestial polypeptide, prostaglandin E2 and isoproterenol, whereas Zn2+‐treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue‐regulated chloride channel activity in the Caco‐2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase‐defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation. © 1995 Wiley‐Liss, Inc.
Analysis of EPC‐1 growth state–dependent expression, specificity, and conservation of related sequences
Authors: Pignolo Robert J. , Rotenberg Mitch O. , Cristofalo Vincent J. 
Published: February 2005

AbstractThe transcipt for EPC‐1 (early population doubling level (PDL) cDNA‐1) is induced under conditions of growth arrest due to density‐dependent contact inhibition and/or serum deprivation in early‐passage but not in senescent WI‐38 fibroblasts. We have characterized the EPC‐1 transcript with respect to its cell‐cycle regulation, tissue specificity, and interspecies conservation of related genomic sequences. In low density, quiescent (serum‐deprived), early‐passage fibroblasts that are stimulated to proliferate with fresh serum, steady‐state EPC‐1 transcript levels are steadily reduced until they reach a basal level approximately 24 h after stimulation. However, when early‐passage fibroblasts are made quiescent by both serum deprivation and density‐dependent contact inhibition and then stimulated with serum, steady‐state EPC‐1 transcript levels remain relatively constant throughout a 36 h period following serum stimulation. Senescent WI‐38 cells (>95% life span completed) do not express EPC‐1 under these conditions. We show that differences in the regulation of EPC‐1 transcript levels in early‐passage cells are due to differences in growth state rather than changes in cell densityor contact. We also show that expression of the EPC‐1 transcript is limited to specific cell types and that related genomic sequences are found in all mammalian species examined as well as in the chicken. © 1995 Wiley‐Liss, Inc.
Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells
Authors: Ortiz Marcela B. , Goin Mercedes , de Alzaga Maria B. Gomez , Hammarstrom Swen , de Asua Luis Jimenez 
Published: February 2005

AbstractLovastatin (LOV), a hydroxy‐methylglutaryl‐coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F2α (PGF2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV‐induced blockage of PGF2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF‐ or PGF2α‐stimulated cells, LOV did not inhibit [3H]leucine or [3H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley‐Liss, Inc.
Expression of tissue‐type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on matrigel
Authors: Ito Ken‐Ichi , Ryuto Masahiro , Ushiro Shin , Ono Mayumi , Sugenoya Akira , Kuraoka Akio , Shibata Yosaburo , Kuwano Michihiko 
Published: February 2005

AbstractHuman omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb‐like structures and capillary‐like networks within 18 h. Cross‐sections of the capillary networks show them to be tube‐like structures. Northern blot analysis showed that tissue‐type plasminogen activator (t‐PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t‐PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor‐1 (PAI‐1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI‐1 mRNA level was increased eightfold initially at 4 h over that at O h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP‐2) mRNA were increased only a slightly within 2–4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP‐1 mRNA level increased up to 18 h, reaching around three times the level at O h. However, on collagen‐coated dishes, cellular levels of t‐PA, PAI‐1, 72 kD type IV collagenase, TIMP‐1, and TIMP‐2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t‐PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti‐transforming growth factor‐β (TGF‐β) antibody. In contrast, both PAI‐1 and TIMP‐1 mRNA levels at 18 h were reduced in the presence of anti‐TGF‐β antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti‐t‐PA antibody. Epidermal growth factor (EGF) enhanced t‐PA gene expression and TGF‐β inhibited its expression in HOME cells cultured on collagen‐coated dishes. On the other hand, TGF‐β enhanced cellular expression of the PAI‐1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti‐angiogenic TGF‐β through modulation of PA activity. © 1995 Wiley‐Liss, Inc.
Activation of a transfected FGFR‐1 receptor in Madin‐Darby epithelial cells results in a reversible loss of epithelial properties
Authors: Migdal Michal , Soker Shay , Yarden Yosef , Neufeld Gera 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine‐kinase family of receptors. We have found that Madin‐Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse‐derived cDNA encoding FGF receptor‐1 (FGFR‐1) in MDCK cells resulted in the acquisition of a fibroblast‐like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF‐induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR‐1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR‐1 ‐expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR‐1‐transfected cells with bFGF resulted in the loss of trans‐epithelial resitance. Monolayers of parental MDCK cells did not lose their trans‐epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans‐epithelial resistance, indicating that the effects on the trans‐epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans‐epithelial resistance is separable from the morphological transition. © 1995 Wiley‐Liss, Inc.
Receptor binding of PDGF‐AA and PDGF‐BB, and the modulation of PDGF receptors by TGF‐β, in human periodontal ligament cells
Authors: Oates T. W. , Kose K. N. , Xie J. F. , Graves D. T. , Collins J. M. , Cochran D. L. 
Published: February 2005

AbstractThe growth factors PDGF‐AA and PDGF‐BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF‐AA has been shown to be specifically inhibited by TGF‐β. The purpose of the present investigation was to examine the binding of PDGF‐AA and PDGF‐BB, and the modulation of PDGF binding by TGF‐β, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF‐AA high‐affinity binding sites per cell with a dissociation constant (Kd) of 0.66 nM and an average of 36,000 PDGF‐BB binding sites per cell with a dissociation constant (kd) of 0.44 nM. After treatment with TGF‐β, the receptor number for PDGF‐AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of α receptor subunits. Northern blot analysis identified the TGF‐β‐mediated decrease in PDGF α receptor subunit mRNA levels. PDGF‐BB showed little change in the number of binding sites or in the binding affinity with TGF‐β treatment, and the data were consistent with an increase in the number of β receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF‐AA and PDGF‐BB in hPDL cells. Also, modulation of PDGF binding, by TFG‐β, was shown to result in a reduced number of α receptor subunits with an increase in the number of β receptor subunits. © 1995 Wiley‐Liss, Inc.
Zinc inhibits turnover of labile mRNAs in intact cells
Authors: Taylor Gregory A. , Blackshear Perry J. 
Published: February 2005

AbstractFor immediate early genes such as the c‐fos proto‐oncogene, mRNA breakdown is very rapid and is largely responsible for the transient nature of mRNA accumulation after transcription is stimulated. We found that in several types of cultured cells and in mice, Zn++ caused marked accumulation of c‐fos mRNA and that of another labile mRNA, that encoding the tristetraprolin (TTP) protein. Exposure of TK‐L cells to 100 μM ZnSO4 caused an increase of c‐fos and TTP mRNA levels within 1 h that reached peak levels in 4–8 h and remained constant to 12 h. Increases in fos protein accumulation were also noted. When the cells were exposed to Zn++ for 4 h and then exposed to actinomycin D, both c‐fos and TTP mRNA levels remained constant for up to 10 h, indicating that Zn++ was preventing the breakdown of both c‐fos and TTP mRNA. Also, 100 μM ZnSO4 inhibited protein synthesis in TK‐L cells, suggesting that the effect on mRNA accumulation © 1995 Wiley‐Liss, Inc.
Cell density—dependent regulation of PLCγ1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC‐6)
Authors: Polk D. Brent , McCollum Gary W. , Carpenter Graham 
Published: February 2005

AbstractAdministration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC‐6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC‐6 cells with EGF provoked a significant increase in the hydrolysis of Ptdlns 4,5‐P2 by immunoisolated PLCγ1. The tyrosine phosphorlation state of PLCγ1 and the association of PLCγ1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2‐containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLCγ1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLCγ1, therefore, is regulated by physiological factors dependent on cell density in IEC‐6 cells. © 1995 Wiley‐Liss, Inc.