bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence‐specific DNA binding activities of nuclear matrix proteins of mammalian lens epithelial cells
Authors: Bagchi M. , Van Wijnen A. , Katar M. , Merriman H. , Lian J. , Stein J. , Stein G. , Maisel H. 
Published: May 2005

AbstractThis study examines matrix and nonmatrix nuclear proteins of the rabbit lens epithelial cells. The nuclear matrix proteins were isolated by modified Penman technique, which requires presence of detergents and nucleases, whereas nonmatrix nuclear proteins were obtained by high salt extraction. The data from these experiments revealed presence of DNA binding activities for SP‐1 and OCT‐1 proteins in both matrix and non‐matrix compartments of rabbit lens epithelial cells. Comparison of the relative abundance of SP‐1 and OCT‐1 binding activities in nuclear matrix and nonmatrix fraction suggest the distribution between these two compartment is cell type specific and possibly related to the control of cell growth. © Wiley‐Liss, Inc.
Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin
Authors: Brown Kathryn J. , Hendry Ian A. , Parish Christopher R. 
Published: May 2005

AbstractHeparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7‐fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10‐fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10‐fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley‐Liss, Inc.
Myoblast and myotude nuclei display similar patterns of heterogneous acetylcholine receptor subunit mRNA expression
Authors: Su Xing , Berman Stephen A. , Sullivan Thomas , Bursztajn Sherry 
Published: May 2005

AbstractMuscle progenitor cells differentiate to myoblasts, and subsequently myotubes, upon expression of muscle specific genes. We and others have previously shown that myotude nuclei, even in the absence of nerve, express AChR α subunit RNA at varying levels, with a small subset (about ten percent) of the nuclei expressing at high levels. These findings raised two important quwstions:(1) is the observed heterogeneneity a unique property of the α subunits, and (2) when does the heterogeneneity begin? In particular, is it induced only at or afer the time of fusion, or does it exist at the myoblast stage? We hasve, therfore, extended our observations to the γ and δ subunits and we also have examined the distributions of AChR α,γ, and δ subunit RNAs in both myoblasts and myotubes. We used intron and intron‐exon probes to detect prespliced trascripts or mature mRNAs in the cells. Because inton‐containing transcripts are not transported out of the nuclei, the distributions of these transcripts can indicate their expression patterns among nuclei in the same myotubes. Our results show that both myotubes and myotubes have distribution of the AChR α,γ, and δ subunit RNAs which differ sharply from that of the U1 RNA or Myo D. Thus, the heterogeneous expression of AChR genes is not only an intrinsic property of muscle cell nuclei (in the sense that it does not require the presence of nerves), but it also exists prior to fusion. Our results suggest that muscle nuclei attain individualized capacities for AChR subunit mRNA production early in their development. Conceptual models consistend with individuality imply an additional level of regulation beyond the known diffusible transcriptional factors. © Wiley‐Liss, Inc.
Intranuclear binding on nucleoplasmin
Authors: Paine Philip L. , Yassin Rihab , Paine Terry Maloney , Vancurva Ivana , George Cathleen , Sahay Neayka 
Published: May 2005

AbstractMany proteins—including not only structural proteins, but also enzymes, hormone receptors, and other transcription factors—accumulate to much higher nuclear than cytoplasmic concentrations. Nuclear Localization sequences or signals (NLSs) within their primary structures entrain specific transport of these proteins through the nuclear pore complexes. Thios transport process is energy‐dependent, but evidence for a true active transport mechanism is not conclusive. An alternative mechanism—facilitated transport of NLS proteins followed by their intranuclear binding—has been implicated by experiments with oil‐isolated nuclei. However, there has been no agreement as to a role for binding in the in vivo nuclear accumulation of NLS‐containing proteins. We Demonstrate herein that a prototypical NLs protein, Nucleoplasmin (NP), binds within the nucleus of the living Xenopus oocyte and that this binding accounts for its nuclear accumulation. © Wiley‐Liss, Inc.
5′ Phosphorylation of DNA in mammalian cells: Identification of a polymin P‐precipitable polynucleotide kinase
Authors: Prinos Panagiotis , Slack Carolyn , Lasko Dana D. 
Published: May 2005

AbstractProteins that catayze 5′ phosphorylation of an oligodeozyribonucleotide substrate can be fractionated by polumin P treatment of whole cell extrats of calf thymus glands. Anion exchange chromatography on Q‐Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, polymin P‐precipitable polynucleotide kinase (PP‐PNK), was futher purified with a 1,500‐fold increase of specific activity compared to the crude polymin fraction. Oligonucleotides, a dephosphorylated 2.9‐kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3′monophosphate was not a substrate. PP‐PNk preparations exhibited an apparent KM of 52 μM for ATP and 8 μM for oligo dT25. The enzyme preparation displayed no detectable 3′ phosphatase or cyclic 2′,3′ phosphohydrolase activities. The sedimentation coefficient of the PP‐Pnk activity was 3.85 as determined by sucrose density gradient analysis; the stokes radius was 45 Å, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimun in the neutral to alkaline range in several buffer systems and is distinct from the DNA Kinase with an acidic pH optimum previously described in calf thymus. © Wiley‐Liss, Inc.
Cells en route to apoptosis are characterized by the upregulation of c‐fos, c‐myc, c‐jun, cdc2, and RB phosphorylation, resembling events of early cell‐cycle traverse
Authors: Pandey Siyaram , Wang Eugenia 
Published: May 2005

AbstractDensity‐arrested quiescent murine Balb/c‐3T3 cells are dependent upon growth factors for their survival. Withdrawal of serum from their medium induces rapid cell death, the mechanism of which is not yet fully understood. We have studied the effect of serum deprivation on density‐inhibited quiescent Swiss 3T3 cells and found that they undergo rapid cell death upon total withdrawal of serum. The nature of this cell death is similar to apoptosis, as shown by cellular and nulear morphology and DNA fragmentation into oligonucleosomal fragments. Investigating the regulation of early cell‐cycle genes during this process, we found that c‐myc, c‐jun, c‐fos, and cdc2, protein presence is induced after serum deprivation, when the phosphorylated form of the RB protein also appears. The upregulation of these genes' protein products is coupled with the appearance of PCNA, a proliferation‐specific nuclear antigen, as well as significant incorporation of BrdU, which may reflect DNA repair activity; in situ analysis shows that BrdU‐positive cells are also positive for DNA fragmentation. These results suggest that en route to apoptosis, cells undergo events typical of early cell‐cycle traverse by expressing early G1 genes and may even experience the late G1/S phase boundary, as shown by the presence of PCNA. However, the demonstrated ability of these cells to traverse the G1 phase of the cell cycle seems to be an abortive event, since they die shortly afterwards.
Suramin inhibits C6 glioma‐induced angiogenesis in vitro
Authors: Coomber Brenda L. 
Published: May 2005

AbstractAspects of tumor‐induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase‐contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1‐μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8‐μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor‐derived growth factor release and/or endothelium‐mediated matrix proteolysis.
Collagen binding activity of recombinant and N‐terminally modified annexin V (anchorin CII)
Authors: Turnay Javier , Pfannmüller Eva , Lizarbe María Antonia , Bertling Wolf M. , von der Mark Klaus 
Published: May 2005

AbstractWe have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N‐terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio‐β‐D‐galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion‐exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium‐independent manner. Heat denaturing of collagen decreases this interaction while pepsin‐treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N‐terminal tail of annexin V is not essential for collagen binding.
Effects of c‐Jun and a negative dominant mutation of c‐Jun on differentiation and gene expression in lens epithelial cells
Authors: Rinaudo Jo Ann , Vacchiano Emanuel , Zelenka Peggy S. 
Published: May 2005

AbstractWe have used a retroviral vector (RCAS) to overexpress wild‐type chicken c‐Jun or a deletion mutant of chicken c‐Jun (JunΔ7) lacking the DNA binding region to investigate the possible role of c‐Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c‐Jun increased the rate of cell proliferation and greatly delayed the appearance of “lentoid bodies,” structures which contain differentiated cells expressing fiber cell markers. Excess c‐Jun expression also significantly decreased the level of βA3/A1‐crystallin mRNA, without affecting αA‐crystallin mRNA. In contrast, the mutated protein, JunΔ7, had no effect no proliferation or differentiation but markedly increased the level of αA‐crystallin mRNA in proliferating cell cultures. These results suggest that c‐Jun or Jun‐related proteins may be negative regulators of αA‐ and βA3/A1‐crystallin genes in proliferating lens cells.
Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS‐1 leading to IRS‐1 association with the phosphatidylinositol 3‐kinase
Authors: Wilden Peter A. , Broadway Dale 
Published: May 2005

AbstractTo analyze the mechanism of action of the insulinomimetic agents H2O2, vanadate, and pervanadate (H2O2 and vanadate), CHO cells or CHO cells that overexpress wild‐type or mutant insulin receptor and/or the insulin receptor substrate (IRS‐1) were used. H2O2 or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pevanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS‐1. However, the insulin receptor and IRS‐1 coimmunoprecipitate from insulin‐treated but not from pervanadate‐treated cells. Pervanadate‐induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS‐1 in vivo and IRS‐1 peptides in vitro equal to that induced by insulin treatment. Pervanadate‐enhanced phosphorylation of IRS‐1 led to a fifteenfold increase in IRS‐1–associated phosphatidylinositol (Ptdlns) 3‐kinase activity. However, insulin receptor–associated Ptdlns 3‐kinase activity from pervanadate‐treated cells was not detectable, while insulin receptor–associated Ptdlns 3‐kinase activity from insulin‐treated cells was 20% of the IRS‐1‐associated activity. Thus, pervanadate but not H2O2 or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS‐1 association.
U2‐SnRNP B″ protein gene is an early growth‐inducible gene
Authors: Laitinen Jens , Saris Per , Hölttä Erkki , Pettersson Ingvar 
Published: May 2005

AbstractIn this work we isolated mouse U2‐snRNP–specific b″ clones and analysed the expression of the mouse U2‐snRNP–specific b″ and U1‐snRNP–specific 70K genes in NIH‐3T3 fibroblasts. Stimulation of growth‐arrested NIH‐3T3 cells with serum was found to evoke a rapid increase in the amount of cytoplasmic b″ and 70K mRNAs. These increases in mRNA did not require de novo protein synthesis. Moreover, the inhibition of protein synthesis by cycloheximide caused a superinduction in the amounts of the U1‐snRNP–specific 70K transcripts. We also found that c‐Ha‐rasval12 oncogene‐transformed NIH‐3T3 cells have higher of the b″ and 70K mRNAs than the normal 3T3 cells. These data imply that the b″ and 70K are early growth response genes, and their enhanced expression might be of significance in the processing of pre‐mRNAs into mature mRNAs.
G/C element contributes to the cell line–specific expression of the proximal osteocalcin promoter
Authors: Goldberg Daniella , Gardiner Edith , Morrison Nigel A. , Eisman John A. 
Published: May 2005

AbstractSequential activation of cell type–specific genes occurs during osteoblast development. The promoter of one such gene, osteocalcin, has been widely studied, but the DNA sequences that govern osteoblast‐specific expression have not been defined. The proximal osteocalcin promoter linked to pTKCAT directs strong promoter activity in osteoblast‐like ROS17/2.8 cells and comparatively weak promoter activity in nonosteoblastic NIH3T3 cells. To identify sequences important in conferring cell‐specific expression of the osteocalcin gene, a deletion series of the human proximal promoter was constructed and the activities assessed in ROS17/2.8 and NIH3T3 cells. These studies identified a 30 bp sequence within the proximal promoter (osteocalcin repressor element‐1 [ORE‐1]) which is responsible for repressing the transcriptional activity in NIH3T3 cells. In electrophoretic mobility shift assays from both NIH3T3 and ROS17/2.8 cells, a protein complex bound to the ORE‐1 that was related to a complex which binds the G/C‐rich repressor element in the collagen type I (α1) promoter. In addition, there was a second complex from NIH3T3 cells but not ROS17/2.8 cells that bound the ORE‐1 fragment. The presence of this additional factor in NIH3T3 cells parallels the observation that constructs carrying the ORE‐1 sequence have repressed promoter activity relative to the analogous constructs lacking the ORE‐1 when transfected into NIH3T3 and suggests that the NIH3T3‐specific factor is a repressor. These data indicate that the G/C element in the ORE‐1 contributes to the repression of osteocalcin gene transcription in a nonosteoblast cell line. The high homology between the ORE‐1 sequence and a related sequence and a related sequence in the collagen type I (α2) proximal promoter suggests that homologous regions in other osteoblast‐expressed genes may function similarly.
1,25‐Dihydroxyvitamin D3: A novel agent for enhancing wound healing
Authors: Tian Xiao Q. , Chen Tai C. , Holick Michael F. 
Published: May 2005

Abstract1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)2D3 affects both fibroblast and keratinocytes, we evalauated the effect of 1,25(OH)2D3 or wound healing. We investigated the effect of the topically applied 1,25(OH)2D3 or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)2D3 at concentrations between 5 and 50 ng/day caused a dose‐dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)2D3 specifically promoted healing between 1–5 days after wounding as compared with vitamin D (0.5 μg/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)2D3 and its analogues may be a new class of compounds that could be developed to enhance wound healing. © 1995 Wiley‐Liss, Inc.
Regulation of ICAM‐1 mRNA stability by cycloheximide: Role of serine/threonine phosphorylation and protein synthesis
Authors: Ohh Michael , Takei Fumio 
Published: May 2005

AbstractCycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3′UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule‐1 (ICAM‐1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4‐10 (lymphoid endothelial cell), and ICAM‐1‐transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM‐1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stablization of the ICAM‐1 mRNA as indicated by the half‐life analysis. To determine whether this effect is dependent on the 3′UTR containing the AUUUA sequences, L cells were transfected with either the full‐length ICAM‐1 cDNA or a truncated form lacking the AUUUA sequences in the 3′UTR (ICAM‐1Δ3). There was no discernible difference in the effect of cycloheximide on ICAM‐1 mRNA accumulation or half‐life between the two types of transfected cells. The effect of cycloheximide on ICAM‐1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H‐7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM‐1 as well as de novo synthesis of ICAM‐1 in SVEC4‐10 and the ICAM‐1‐transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM‐1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM‐1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect. © 1995 Wiley‐Liss, Inc.
Involvement of protein phsophatase‐1 in cytoskeletal organization of cultured endothelial cells
Authors: Shinoki Nobutoshi , Sakon Masato , Kambayashi Jun‐ichi , Ikeda Masataka , Oiki Eiji , Okuyama Masaki , Fujitani Kazumasa , Yano Yoshiko , Kawasaki Tomio , Monden Morito 
Published: May 2005

AbstractThe phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP‐1) and 2A (PP‐2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F‐actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non‐selective kinase inhibitor, K‐252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP‐1. The catalytic subunit of PP‐1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP‐1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley‐Liss, Inc.