bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acidic fibroblast growth factor (HBGF‐1) stimulates DNA synthesis in primary rat hepatocyte cultures
Authors: Houck Keith A. , Zarnegar Reza , Muga Stephanie J. , Michalopoulos George K. 
Published: February 2005

AbstractAcidic fibroblast growth factor (aFGF) stimulated DNA synthesis in primary rat hepatocyte cultures in a dose‐dependent manner with maximal effect at 10–50 ng ml−1. This activity was dependent on the presence of heparin at a concentration of 10–50 μ·ml−1. Insulin interacted synergistically with aFGF, as it did with epidermal growth factor (EGF). The response to aFGF was only 50% that found with EGF. The disparity was not due to different kinetics of DNA synthesis, since the peak response for both growth factors occurred at 36–72 hr after plating of the hepatocytes. The potential relevance of this novel hepatocyte mitogen to normal and pathological liver growth is discussed.
Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes
Authors: Gladhaug Ivar P. , Christoffersen Thoralf 
Published: February 2005

AbstractIn previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature‐ and time‐dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Glad‐haug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride‐treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride‐treated hepatocytes the phorbol ester TPA strongly inhibited high‐affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand‐independent EGF receptor cycling pathway in hepatocytes.
Transcriptional regulation of DF3 gene expression in human MCF‐7 breast carcinoma cells
Authors: Abe Miyako , Kufe Donald 
Published: February 2005

AbstractThe DF3 gene codes for a high molecular weight human breast tumor‐associated glycoprotein. The detection of this antigen in human milk has also suggested that its expression represents a differentiated function of mammary epithelium. The present studies have examined the regulation of DF3 gene expression in human MCF‐7 breast carcinoma cells. These cells express two DF3 transcripts of 4.5 and 7.0 kb. Treatment of MCF‐7 cells with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) was associated with increases in levels of both DF3 mRNAs. When nuclear run‐on assays were used, DF3 gene transcription was at low to undetectable levels in untreated MCF‐7 cells and was increased after TPA exposure. TPA‐induced increases in DF3 mRNA levels were also inhibited by actinomycin D (ACT). MCF‐7 cells exposed to ACT further demonstrated that the half‐lives of the 4.5 and 7.0 kb transcripts are 26 and 11 h, respectively. The results also demonstrate that the protein synthesis inhibitor, cycloheximide (CHX), increases DF3 mRNA levels in MCF‐7 cells. These effects of CHX were sensitive to actinomycin D and not associated with stabilization of the DF3 transcripts. Taken together, these findings indicate that DF3 gene expression is controlled at a transcriptional level in TPA‐ and CHX‐treated MCF‐7 cells.
Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild‐type and mutant S49 T lymphoma cells
Authors: Albert Daniel A. , Nodzenski Edwardine , Yim Gloria , Kowalski Janice 
Published: February 2005

AbstractRibonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and G2/M phase with low activity and low concentrations of the M2 subunit in G1 phase. Pharmacologic concentrations of cyclic AMP arrest S49 cells in the G1 phase of the cell cycle. We investigated the effect of cyclic AMP on M2 messenger RNA concentrations using RNA from exponentially growing and elu‐triated, cell cycle‐enriched populations. To discern whether cyclic AMP‐induced G1 arrest was associated with low concentrations of M2‐specific messenger RNA, we probed blots with a full‐length cDNA for M2. Cell cycle variation in M2 messenger RNA concentrations was similar in wild‐type, hydroxyurea‐resistant cells with amplified M2 activity, and cyclic AMP‐dependent protein kinase‐deficient cell lines. All lines had low amounts of M2‐specific mRNA in early G1, an increase at the late G1/early S phase interface, a decrease in mid S phase, and another increase in late S phase that continued through G2/M. These concentrations did not directly correlate with enzyme activity, suggesting other regulatory effects might participate in determining ribonucleotide rectase activity. Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2‐specific messenger RNA concentration. This effect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP‐dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional activation of the M2 gene.
Clonal segregation of multiple and overlapping matrix adhesive responses in dorsal root neuronal derivative cells
Authors: Barletta Emanuela , Culp Lloyd A. 
Published: February 2005

AbstractClones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence–;plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1‐binding substratum, and platelet factor‐4 (PF4) as a model proteoglycan‐binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum‐specific pattern and varied from 0 to >60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4‐ and CTB‐directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibitior, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neurites for all clones, indicating a widelyused negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of these processes.
K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation
Authors: Järvinen Maarit , Andersson Leif C. , Virtanen Ismo 
Published: February 2005

AbstractThe effects of differentiation‐modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki‐67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF‐7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG‐1) did not contain cytokeratin‐ or EMA‐immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.
Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes
Authors: Engelmann Bernd , Schumacher Udo , Duhm Jochen 
Published: February 2005

AbstractThe uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22°C proceeded with a t½ of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5–6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20–500 μM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one‐fourth. Fluorescence microscopy of red cells incubated with 100 μM CTC at 22°C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter >0.2 <1μm) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0°C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.
Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts
Authors: Varani James , Chakrabarty Subhas 
Published: February 2005

AbstractIn previous studies it was shown that transformation of AKR fibroblasts with 3‐methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N‐dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR‐2B) and their transformed counterparts (AKR‐MCA) bound 125I‐fibronectin in a receptor‐like fashion, but the AKR‐MCA cells had only 20% of the receptors found on the AKR‐2B cells. Whole cell extracts prepared from the AKR‐2B cells and separated by sodium dodecyl sufate‐polyacrylamide gel electrophoresis under reducing conditions were examined for 125I‐fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR‐MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I‐fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF‐sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I‐fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the suosequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell‐substrate adhesion. The AKR‐2B cells rapidly attached and spread on bovine serum albumin‐coated dishes and on fibronectin‐coated dishes, whereas the AKR‐MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell‐substrate adhesion.
Effect of epidermal growth factor on rat pleural mesothelial cell growth
Authors: Van Der Meeren A. , Levy F. , Renier A. , Jaurand M. C. , Katz A. 
Published: February 2005

AbstractWe recently reported that the growth of normal rat pleural mesothelial cells (RPMCs) is inhibited by conditioned media from either in vivo or in vitro transformed RPMCs. In this study we report that the growth of normal RPMCs is inhibited by epidermal growth factor (EGF). This was demonstrated by using three methods of investigation. Two types of studies were carried out with growing cells. First, cell counts indicated that the number of cells was reduced in EGF‐treated cultures when compared with untreated cultures. Second, the percentage of S cells detected by flow cytometry following treatment with EGF was lower than without EGF. In other experiments, incorporation of tritiated thymidine in confluent cells was decreased by EGF treatment, either in the presence or absence of fetal calf serum; these effects were dose dependent and were observed from 2 ng/ml EGF. Lower EGF concentrations did not significantly modify thymidine incorporation when compared with untreated cells. Analysis of 125I EGF binding experiments by the Scatchard method indicated that RPMCs possess EGF receptors (about 105 per cell) with low ligand binding affinity (Kd = 1.7 ± 0.4 nM). These results indicate that EGF might modulate the growth of RPMCs.
Protein disulphide isomerase from human peripheral blood neutrophils
Authors: Bassuk James A. , Capodici Constance , Berg Richard A. 
Published: February 2005

AbstractProtein disulphide isomerase (PDI) is a 56 kDa resident polypeptide of the endoplasmic reticulum of many cell types. We evaluated the ability of human peripheral blood polymorphonuclear neutrophils (PMN) to synthesize both mRNA and proteins. Using in vitro [35S]‐methionine labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized polyclonal and monoclonal antibodies and analysis by gel electrophoresis, PMN were shown to synthesize many proteins, including actin. In contrast, incorporation of [35S]‐methionine into PDI was not detected. Purification of total RNA from PMN and analysis by Northern blots demonstrated the presence in PMN of PDI‐RNA. Western immunoblot evaluations of total PMN protein display an immunoreactive‐PDI of 56 kDa. Indirect immunofluorescence studies suggest an abundance of immunoreactive‐PDI throughout PMN. We therefore conclude that PDI is synthesized in precursor cells of the bone marrow. Phorbol 12‐myristate 13‐aceiate, a reagent known to affect the degranulation of specific granules, causes the release of immunoreactive‐PDI into a post‐centrifugation supernatant. PDI, a ubiquitous endoplasmic reticulum resident protein, is shown here to be associated with specific granules in a cell type which has lost its intracellular membrane network during terminal differentiation.
Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: Evidence for an inhibitor of vincristine binding
Authors: Bowman Laura C. , Houghton Janet A. , Houghton Peter J. 
Published: February 2005

AbstractIn cytosols from human rhabdomyosarcoma xenografts, the formation of vincris‐tine (VCR)‐tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695–700, 1986). We have further examined this modulation to determine whether (a) GTP was protecting the VCR binding site from denaturation, (b) the enhancement of complex formation was guanosine specific, and (c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP‐depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37°C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 μM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three‐fold, while GMP, GMP‐PNP, and ITP increased formation 1.5‐fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization‐depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20–50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.
Collagen‐Binding proteins of mammary epithelial cells are related to Ca2+‐and phospholipid‐binding annexins
Authors: Wirl Gerhard , Schwartz‐Albiez Reinhard 
Published: February 2005

AbstractThree major proteins of 34, 36, and 38 kDa were isolated from mebrane preparations of chemically induced mammary tumors of the rat by collagen type I affinity chromatography and therefore were termed collagen‐binding proteins (CBP). Three proteins in the same molecular weight range isolated from cell extracts by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on hydroxylapatite were demonstrated to be immunologically related to CBP. As shown by immunoblot analysis, an antiserum directed against the cluster of the 34–38 kDa proteins reacted strongly with porcine intestinal protein I, weakly with porcine lipocortin I, and very weakly with porcine intestinal protein II. Antiserum against the 34 kDa protein reacted weakly with protein I but strongly with protein II. All three CEP reacted with protein I/calpactin l‐specific antiserum of immunoblots and immunoprecipitation in experiments. However, antisera directed against CBP failed to show cross‐reaction with collagen‐binding protein anchorin II from chicken chondrocytes. Conversely, antisera against anchorin II did not react with CBP. Antiserum AS/87 immunoprecipitated CBP of 38 kDA that was labeled in a lactoperoxydase‐catalyzed iodination, suggesting that this polypeptide is associated with the cell surface. Further, all three CBP were found to be phosphorylated by incubating mammary cells with 32P‐orthophosphate. CBP bound to epithelial cell membranes in a Ca2+ dependent manner (= Triton × 100 insoluble form). Fractionated extraction and immunofluorescence microscopy also show that another form of CBP (= Triton × 100 soluble form) exists in these cells and is associated with a granular fraction. We therefore conclude that mammary collagen‐binding proteins represent members of a family of Ca2+‐binding membrane proteins. The 38 kDa CBP seems closely related to the pp60src kinase substrate protein I/calpactin I monomer, the 34 kDa CBP seems to be related or equivalent to protein II, while the relationship of the 36 kDa CBP to other defined proteins is still unclear.