bluuebunny/crossref_metadata_2025_split huggingface dataset:
Global change of gene expression at late G1/S boundary may occur in human IMR‐90 diploid fibroblasts during senescence
Authors: Pang Jong Hwei , Chen Kuang Yu 
Published: February 2005

AbstractThe hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR‐90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263: 11431–11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR‐90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR‐90 cells but not in old IMR‐90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age‐dependent attenuation in activities. In contrast, expression of growth‐related genes such as elF‐5A, c‐Ha‐ras, and β‐actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp‐1 binding site as the most common cis‐element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled. © 1994 Wiley‐Liss, Inc.
Autocrine downregulation of fibroblast growth factor receptors in F9 teratocarcinoma cells
Authors: Moscatelli David 
Published: February 2005

AbstractThe regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind 125I‐basic FGF (FGF‐2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of 125I‐FGF‐2 was due to an increase in the number of receptors with no change in their affinity. When 125I‐FGF‐2 was cross‐linked to cell surface receptors, an increase in FGF‐2‐receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF‐4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF‐receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased 125I‐FGF‐2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF‐4 increased the 125I‐FGF‐2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley‐Liss, Inc.
Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands
Authors: Métioui Mourad , Grosfils Katrina , Dehaye Jean‐Paul 
Published: February 2005

AbstractRat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura‐2.In the absence of extracellular magnesium and calcium ([Ca2+]o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca2+]o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura‐2; 10 m̈M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca2+]in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca2+‐free/Ca2+ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca2+]in and the calcium uptake but potentiated the response to ATP.These results suggest that, after binding to purinergic receptors, extracellular ATP4‐ increases [Ca2+]in. ATP4‐ does not mobilize thapsigargin‐sensitive intracellular calcium pools (among which is the IP3‐sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley‐Liss, Inc.
Signalling the molecular stress response to nephrotoxic and mutagenic cysteine conjugates: Differential roles for protein synthesis and calcium in the induction of c‐fos and c‐myc mRNA in LLC‐PK1 cells
Authors: Yu Kefu , Chen Qin , Liu Hong , Zhan Yi , Stevens James L. 
Published: February 2005

AbstractNephrotoxic and mutagenic cysteine conjugates (NCC) are activated by the enzyme cysteine conjugate, β‐lyase, to reactive acylating species which bind covalently to cellular macromolecules. We now show that an early event after treatment of LLC‐PK1 cells with NCC is the induction of mRNA for both c‐fos and c‐myc. Treatment with S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC) induced c‐fos (53‐fold) and c‐myc mRNA (20‐fold) and increased transcription about 3‐fold for both genes. Covalent binding was required for induction of both mRNAs. Dithiothreitol partially prevented induction of both c‐fos and c‐myc RNA. Buffering the DCVC‐induced increase in cytosolic free calcium had no effect on c‐fos mRNA but partially blocked c‐myc mRNA induction. Cycloheximide blocked the induction of c‐myc mRNA in the absence of an effect on c‐fos induction. The data suggest that the increase in c‐fos mRNA is a primary response to DCVC toxicity and occurs without a requirement for protein synthesis or an increase in intracellular free calcium. In contrast, c‐myc induction requires protein synthesis, suggesting that the presence of another primary response factor may regulate induction either transcriptionally or posttranscriptionally. The data suggest that different signalling pathways regulate induction of c‐fos and c‐myc mRNA in response to stress caused by reactive acylating species. © 1994 Wiley‐Liss, Inc.
Native TIMP‐free 70 kDa progelatinase (MMP‐2) secreted at elevated levels by RSV transformed fibroblasts
Authors: Stefansson Steingrimur , Aimes Ronald T. , Seward Nancy B. , Alexander Daniela S. , Quigley James P. 
Published: February 2005

AbstractRous sarcoma virus‐transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase‐2 (MMP‐2). Affinity‐purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP‐2 and a native 70 kDa progelatinase free of any detectable TIMP‐2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS‐PAGE and nondenaturing, native gel electrophoresis. The purified TIMP‐free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP‐free progelatinase with the organomercurial, p‐aminophenylmercuric acetate (APMA), results in rapid (5‐60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP‐free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP‐complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6‐24 hours and the specific enzyme activity of the preparation is 8 to 18‐fold lower. Purified avian TIMP‐2 added to the TIMP‐free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP‐free progelatinase than the normal CEF cultures which produce predominantly TIMP‐complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP‐free progelatinase, the suppression of its rapid activation by TIMP‐2, and the potential effect of the altered balance between TIMP‐free and TIMP‐complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed. © 1994 Wiley‐Liss, Inc.
Insulin‐like growth factor (IGF) binding to a murine mammary epithelial cell line
Authors: Hadsell Darryl L. , Gibson C. Anne , Baumrucker Craig R. 
Published: February 2005

AbstractThe goal of this study was to relate insulin‐like growth factor (IGF) binding with IGF‐stimulated growth in a murine mammary epithelial (COMMA‐D/MME) cell line. Affinity crosslinking with [125I]IGF‐I showed major bands at 224,000 and 148,000 Mr that were ablated by the inclusion of IGF‐I or ‐II at 130 nM. Scatchard‐transformed [125I]IGF‐I binding data was best fit by a two‐site model, with 24,000 sites possessing a Kd of 0.33 nM and 1,000 sites possessing a Kd of 8.09 nM per cell. Competition analysis showed ED50 values for IGF‐I, ‐II, and insulin to be 0.90 ± 0.03, 7.15 ± 4.27, and 335 ± 104 nM, respectively. Affinity crosslinking of [125I]IGF‐II showed three major bands of 230,000, 148,000, and 61,000 to 58,000 Mr. Unlabeled IGF‐II ablated all bands, while IGF‐I and insulin ablated only the 148,000 Mr band. Competition analysis showed ED50 values for unlabeled IGF‐I and ‐II to be 0.10 ± 0.01 and 5.31 ± 2.04 nM, respectively. In spite of the receptor affinity differences, no significant difference was noted in IGF‐I and ‐II in capacity to stimulate cell growth. These data indicate that COMMA‐D/MME cells express IGF receptors and that both IGFs are mitogenic. © 1994 Wiley‐Liss, Inc.
Differential effects of superoxide, hydrogen peroxide, and hydroxyl radical on intracellular calcium in human endothelial cells
Authors: Dreher D. , Junod A. F. 
Published: February 2005

AbstractChanges in intracellular Ca2+ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine‐xanthine oxidase system (X‐XO) mobilizes Ca2+ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O2 species involved in the disruption of calcium homeostasis by X‐XO, we studied the effect of X‐XO on [Ca2+]i by spectrofluorimetry with fura‐2 in human umbilical vein endothelial cells (HUVEC). The [Ca2+]i response to X‐XO was essentially diminished by superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (200 U/ml), which scavenge the superoxide anion, O2−, or H2O2, respectively. The [Ca2+]i increase stimulated by 10 nmol H2O2/ml/min, generated from the glucose‐glucose oxidase system, or 10 μM H2O2, given as bolus, was about a third of that induced by X‐XO (10 nmol O2−/ml/min) but was comparable to that induced by X‐XO in the presence of SOD. The X‐XO—stimulated [Ca2+]i increase was significantly reduced by 100 μM o‐phenanthroline, which inhibits the iron‐catalysed formation of the hydroxyl radical. On the other hand, the [Ca2+]i response to low dose X‐XO (1 nmol O2−/ml/min) was markedly enhanced in the presence of 1 μM H2O2, which itself had no effect on [Ca2+]i. More than 50% of this synergistic effect was prevented by o‐phenanthroline. These results indicate that the effect of X‐XO on calcium homeostasis appears to result from an interaction of O2− and H2O2, which could be explained by the formation of the hydroxyl radical. © 1995 Wiley‐Liss, Inc.
Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells
Authors: Graham Martin F. , Willey Amy , Adams John , Yager Dorne , Diegelmann Robert F. 
Published: February 2005

AbstractThe role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L‐[5‐3H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate‐replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady‐state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two‐ to fourfold. L‐ascorbate phosphate did not increase the activity of L‐ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady‐state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L‐ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L‐ascorbate‐phosphate is not an adequate substitute for L‐ascorbate in this cell line. © 1995 Wiley‐Liss, Inc.
Activation of protein kinase C inhibits ATP‐induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways
Authors: Gallinaro Brian J. , Reimer Wesley J. , Dixon S. Jeffrey 
Published: February 2005

AbstractExtracellular ATP elicits transient elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in osteoblasts through interaction with more than one subtype of cell surface P2‐purinoceptor. Elevation of [Ca2+]i arises, at least in part, by release of Ca2+ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca2+]i of indo‐1‐loaded UMR‐106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca2+, ATP (100 μM) induced transient elevation of [Ca2+]i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12‐O‐tetradecanoyl‐β‐phorbol 13‐acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca2+]i. Inhibition was half‐maximal at approximately 1 nM TPA. The Ca2+ response to ATP was also inhibited by the PKC activators 1,2‐dioctanoyl‐sn‐glycerol or 4β‐phorbol 12, 13‐dibutyrate, but not by the control compounds 4α‐phorbol or 4α‐phorbol 12, 13‐didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H‐7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca2+]i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P2Y‐receptor agonists, ADP and 2‐methylthioATP, than the response to the P2U‐receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley‐Liss, Inc.
Quantitative export of FGF‐2 occurs through an alternative, energy‐dependent, non‐ER/Golgi pathway
Authors: Florkiewicz Robert Z. , Majack Richard A. , Buechler Robbie D. , Florkiewicz Elin 
Published: February 2005

AbstractAlthough basic fibroblast growth factor (bFGF/FGF‐2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS‐1 cell expression system, we have identified a novel membrane‐associated transport pathway that mediates export of FGF‐2. This export pathway is specific for the 18‐kD isoform of FGF‐2, is resistant to the anti‐Golgi effects of Brefeldin A, and is energy‐dependent. In FGF‐2‐transfected COS‐1 cells, this ER/Golgi‐independent pathway appears to be constitutively active and functions to quantitatively export metabolically‐labeled 18‐kD FGF‐2. Co‐transfection and co‐immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF‐2. When neomycin phosphotransferase was appended to the COOH‐terminus of 18‐kD FGF‐2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF‐2 domain extracellular and remained cell‐associated following alkaline carbonate extraction. Taken together, the data suggest that FGF‐2 is “exported” from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide‐mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF‐2 export. Such an intervention may be of considerable use for the treatment of angiogenesis‐dependent diseases involving FGF‐2. © 1995 Wiley‐Liss, Inc.
Effect of phosphotyrosyl‐IRS‐1 level and insulin receptor tyrosine kinase activity on insulin‐stimulated phosphatidylinositol 3, MAP, and S6 kinase activities
Authors: Wilden Peter A. , Broadway Dale E. 
Published: February 2005

AbstractThe role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS‐1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS‐1, the insulin receptor, or both IRS‐1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS‐1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin‐stimulated IRS‐1 associated Ptdlns 3′‐kinase activit in these cells was 1‐, 1.5‐, 3‐, and 3‐fold, while insulin sensitivity, as determined by ED50, was 1‐, 2.5‐, 10‐, and 10‐fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl‐IRS‐1; however, the increased level of phosphotyrosyl‐IRS‐1 seen in CHO/IR/IRS‐1 cells did not further increase these responses. Likewise, maximal insulin‐stimulated MAP kinase activity in these cell lines increased in parallel with IRS‐1 tyrosine phosphorylation except in the CHO/IR/IRS‐1 cell lines with activity levels of one‐, five‐, nine‐, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin‐stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl‐IRS‐1, but an increase was observed with insulin‐stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS‐1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS‐1 and other possible cellular substrates. © 1995 Wiley‐Liss, Inc.
Inhibition of heme synthesis induces apoptosis in human erythroid progenitor cells
Authors: Muta Koichiro , Krantz Sanford B. 
Published: February 2005

AbstractHeme synthesis by erythroid progenitor cells is maintained by erythropoietin (EP), insulin‐like growth factor‐I (IGF‐I), and stem cell factor (SCF), and without these growth factors apoptosis (programmed cell death) occurs. To clarify the possible interaction between heme synthesis and programmed cell death of human erythroid progenitor cells, the effect of specific inhibition of heme synthesis on apoptosis of highly purified human erythroid colony forming cells (ECFC) was studied. When the amount of uncleaved DNA was determined as a measure of apoptosis, the heme synthesis inhibitors, succinylacetone (SA) (0.1 mmol/L) or isonicotinic acid hydrazide (INH) (10 mmol/L), significantly decreased the amount of uncleaved DNA (P < 0.01) in the presence of erythropoietin (EP). Addition of recombinant heavy‐chain ferritin (rHF) (10 nmol/L), or deprivation of transferrin from the culture medium, which decreased heme synthesis, also reduced the amount of uncleaved DNA (P < 0.01). The production of apoptosis by diverse inhibitors of heme synthesis was in each case reversed by the addition of hemin (0.1 mmol/L) and did not occur with HL‐60 cells. When the colony‐forming capacity of ECFC was determined by plasma clot assay, SA, INH, or rHF reduced the number of CFU‐E (P < 0.01), and the effect of SA was reversed by hemin. The addition of SA did not alter the c‐myc response of ECFC to EP. These data indicate that inhibition of heme synthesis induces apoptosis of human erythroid progenitor cells, in a manner independent of an early c‐myc response, and suggest that the presence of apoptosis in ineffective erythropoiesis may be secondary to impaired heme synthesis. © 1995 Wiley‐Liss, Inc.