bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and functional characterization of proteoglycans produced by SI/SId murine bone marrow stromal cell lines
Authors: Bentley Stuart A. , Kirby Suzanne L. , Anklesaria Pervin , Greenberger Joel S. 
Published: February 2005

AbstractThe Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (SI/SId) animals and two control stromal cell lines, one (+/+2.4) from a non‐anemic littermate, and one (GBI/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCI density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co‐culture of an IL‐3‐dependent cell line (FDC‐P1) with cell‐free ECM preparations from an SI/SId and a control (GBI/6) stromal cell line, with and without pre‐incubation with IL‐3. Cell‐free ECM preparations from SI/SId and control cell lines supported FDC‐P1 growth to an approximately equal extent after pre‐incubation with II‐3. FDC‐P1 growth support by ECM preparations from both cell lines was also observed without IL‐3 pre‐incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.
Basic fibroblast growth factor accumulates in the nuclei of various bFGF‐producing cell types
Authors: Tessler Shoshanna , Neufeld Gera 
Published: February 2005

AbstractThe intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK‐21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus: bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei ofuntransfeted cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti‐bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin‐sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK‐21 cells. The mitogenic cactivity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear, these findings suggest that some biologicaL activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF‐ to DNA.
Transforming growth factor β1 enhances expression of 50 kDa protein related to 2′‐5′ oligoadenylate synthetase in human sperm cells
Authors: Naz Rajesh K. , Kumar Rakesh 
Published: February 2005

AbstractHuman cellular polypeptide factors, namely interferon‐α, interferon‐γ, transforming growth factor (TGF)‐α, and TGF‐β, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2′‐5′ oligoadenylate [2–5(A)]synthetase activity. TGF‐α affected neither motility nor the levels of 2–5(A) synthetase in sperm cells. TGF‐β, had no affect on sperm motility, yet it caused an induction of 2–5(A)synthetase activity. Western immunoblot analysis of TGF‐β1‐treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30–92 kDa; the level of 50 kDa protein increased after treatment with TGF‐β1. The treatment of sperm cells with TGF‐β1 did not affect their penetration in zona‐free hamster eggs (SPA). These results indicate that TGF‐β1 enhances expression of a 50 kDa protein related to 2–5(A)synthetase in human sperm cells along with other minor proteins, and this increase does not affect sperm motility and SPA.
Decline of poly(ADP‐ribosyl)ation during in vitro senescence in human diploid fibroblasts
Authors: Dell'orco R. T. , Anderson L. E. 
Published: February 2005

AbstractPoly(ADP‐ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast‐like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP‐ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30–60% as the cells moved through their in vitro life spans. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP‐ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.
Growth factor‐independent proliferation of normal human neonatal keratinocytes: Production of autocrine‐ and paracrine‐acting mitogenic factors
Authors: Cook Paul W. , Pittelkow Mark R. , Shipley Gary D. 
Published: February 2005

AbstractWhen normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 × 103/cells cm2, the cells proliferated continuously and the addition of IGF‐I, EGF, TGFα, or aFGF did not significantly alter growth rate. Heparin sulfate, TGFβ, or suramin inhibited keratinocyte growth factor‐independent proliferation. The addition of EGF, TGFα, or aFGF reversed heparin‐induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte‐derived conditioned medium (CM) indicated the presence of TGFα peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte‐derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti‐EGF receptor mAb inhibited CM‐induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte‐derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR‐2B cells, and EGF‐receptorless mouse NR6 cells. CM‐stimulated [3H] thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti‐EGF receptor mAb. Heparin sulfate partially inhibited CM‐induced keratinocyte clonal growth and [3H] thymidine incorporation into quiescent AKR‐2B cells. We hypothesize from these data that autocrine and paracrine‐acting factors produced by kerati‐nocytes modiated their effect through the activation of both EGF receptor‐dependent and EGF receptor‐independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.
Induction of cell cycle‐dependent genes during cell cycle progression of arterial smooth muscle cells in culture
Authors: Gadeau Alain‐Pierre , Campan Michel , Desgranges Claude 
Published: February 2005

AbstractSerum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle‐dependent in various cell types also demonstrate a cell cycle‐dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: “immediate early” genes (c‐fos, c‐myc,…), “delayed early” genes (2F1,…), and “late‐G1” genes (proliferating cell nuclear antigen, thymidine kinase,…). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycledependent: A21, the rat JE gene, and L51 can be classified as “immediate early” genes, while M11 represents a new member of the “delayed early” gene family.
Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non‐ionic detergent triton X‐100
Authors: Miseta A. , Kellermayer M. , Ludany A. , Cameron I. L. , Hazlewood C. F. 
Published: February 2005

AbstractThe release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X‐100 (a non‐ionic detergent). This Triton X‐100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X‐100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein‐water‐ion interactions within the lens is the rate‐limiting step of the observed equilibration of monovalent cations and sucrose molecules.
Structural/functional relationships between internal and external MSH receptors: Modulation of expression in Cloudman melanoma cells by UVB radiation
Authors: Chakraborty Ashok K. , Orlow Seth J. , Bolognia Jean L. , Pawelek John M. 
Published: February 2005

AbstractExpression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142: 129–136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50–53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co‐purified with a sub‐cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bologniaet al: J. Invest. Derm., 92:651–656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomittant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.
The basic fibroblast growth factor‐saporin mitotoxin acts through the basic fibroblast growth factor receptor
Authors: Lappi Douglas A. , Maher Pamela A. , Martineau Darlene , Baird Andrew 
Published: February 2005

AbstractWe have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross‐linking experiments show that radio‐labeled basic fibroblast growth factor‐saporin (FGF‐SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF‐SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF‐SAP which will result in cytotoxicity occurs very rapidly: 5 minutes of incubation of 10 nM basic FGF‐SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that basic FGF‐SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti‐FGF for therapeutic and research uses in vivo is discussed.
Steady state levels of hepatic α1 and β2‐adrenergic receptors and gene transcripts during development of the male rat
Authors: Rossby S. Paul , Cornett Lawrence E. 
Published: February 2005

AbstractMetabolic events stimulated by epinephrine and norepinephrine in hepatocytes isolated from fetal and early postnatal male rats are largely mediated through the β2‐adrenergic receptor‐/cyclic AMP dependent‐system, whereas the same stimuli are transduced through the α1‐adrenergic receptor‐/phosphatidylinositol dependent‐system in hepatocytes isolated from young adult male rats. This developmental transition was investigated by correlating hepatic α1‐ and β2‐adrenergic receptor gene transcript levels with receptor levels as determined with selective radioligands in livers from late fetal to postnatal day 120 male Sprague‐Dawley rats. β2‐Adrenergic receptor concentration, initially high in membrane preparations isolated from fetal livers (203 ± 21 fmol/mg protein), dropped precipitously n postnatal day 6 livers (14± 2 fmol/mg protein) and remained low throughout development out to postnatal day 90.β‐Adrenergic receptor mRNA levels were highest in fetal livers, were decreased somewhat in postnatal day 6 livers and were uncetectable in livers beyond postnatal day 15. In contrast, hepatic α‐adrenergic receptor concentration was relatively low in fetal livers (86 ± 25 fmol/mg protein) and remained low until postnatal day 18. Thereafter, a steady increase in α1‐adrenergic receptors was observed until adult levels. (270 ± 24 fmol/mg protein) were achieved at postnatal day 27. α1‐Adrenergic receptor mRNA levels increased ∼ 3‐fold, reaching a peak at postnatal day 24. Surprisingly, at postnatal day 30 hepatic α1‐adrenergic receptor mRNA levels dropped to fetal levels; but, gradually increased with continued development. Thus, hepatic α1‐ and β2‐adrenergic receptors appear to be under complex regulatory control which may include transcriptional, as well as post‐transcriptional, mechanisms.
Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts
Authors: Bos M. P. , van Leeuwen J. P. T. M. , Herrmann‐Erlee M. P. M. 
Published: February 2005

AbstractWe studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12‐myristate 13‐acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH‐induced cAMP accumulation was not due to an effect on the PTH‐receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G‐protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)‐induced cAMP accumulation; i.e., at concentrations ⩾ 10−6 M, PMA potentiated the PGE2‐induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 ⩽ 5.10−7 M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2‐induced cAMP accumulation.
Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas
Authors: Bisgaard Hanne Cathrine , Thorgeirsson Snorri S. 
Published: February 2005

AbstractThe appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic‐type tissue in the adult rat liver can be experimentally induced (Reddy et al.: J. Cell Biol., 98:2082–2090, 1984; Rao et al., J. Histochem. Cytochem., 34:197–201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light‐and electron‐microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and β‐tubulin were present in all cell lines. In addition, simple epithelial type II cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type II cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth‐inhibitory effects of TGF‐β1. No expression of tissue‐ or cell‐specific proteins such as α‐fetoprotein, albumin, amylase, elastase, or γ‐glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a “precursor” or “stem” cell compartment in vivo.
Proliferation of smooth muscle cells in the inner and outer layers of the tunica media of arteries: An in vitro study
Authors: Betz E. , Fallier‐Becker P. , Wolburg‐Buchholz K. , Fotev Z. 
Published: February 2005

AbstractDuring the development of atherosclerotic and fibromuscular proliferates/lesions, smooth muscle cells (SMC in the media, particularly near the lumen, are activated to migrate into the intima, where they continue to proliferate to form an intimal thickening. It is to date unclear whether SMCs situated adjacent to the adventitia possess a lower capacity to proliferate because they are a special subpopulation of medial SMCs or because the adventitia excerts an inhibitory effect. We have, therefore, developed an in vitro system whereby we have attempted to clear up this uncertainty. The following observations were made from the in vitro experiments:Media‐explants from rabbit aorta were laid on a polycarbonate filter with pores 5 μm in diameter. The SMCs migrated through the pores and formed a fibromuscular proliferate on the other side of the filter.Endothelial cells were seeded on one side of the filter before media‐explants were laid on the other side of the filter. The confluent endothelium inhibited migration of SMCs through the filter pores.Media‐explants were placed between two polycarbonate filters (pores 5 μm diameter). In this “sandwich” arrangement SMCs migrated through both filters, i.e., in both directions. The quantity of migrating and proliferating cells through both filters was almost identical. This suggests that there is no difference in the migratory and proliferate capacity of SMCs in the inner and outer layers in the media of arteries.To investigate the influence of the adventitia on medial SMCs, media‐explants were placed between a lower (5 μm) and an upper (0.2 μm) filter adventitia‐explants were laid above the media‐explants. The 0.2 μm filter prevented migration of SMCs from the media‐explant into the adventitia and migration of fibroblasts from the adventitia into the media. Interestingly, the adventitial tissue inhibited proliferation of SMCs at the abluminal and migration and proliferation at the luminal side of the media‐explant; the number of cells migrating through the 5 μm pores at the luminal side was diminished, suggesting that the adventitial tissue has an antiproliferative influence on SMCs. Moreover, it was found that in media‐explants near the filter with adventitia, the medial SMCs were in a better preserved condition than at the de‐endothelialised luminal side.As a control, cultures consisting of media‐explants were incubated without filters (i.e., explant organ cultures). The proliferates in the concavity (luminal side) exhibited a pattern of proliferating SMCs different from that of the cells at the abluminal convexity. This finding indicates that the different texture of vessel walls near the adventitia and near the lumen may also influence cell migration and proliferation.
Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet‐derived growth factor in osteoblast‐enriched cultures from fetal rat bone
Authors: Centrella Michael , McCarthy Thomas L. , Kusmik William F. , Canalis Ernesto 
Published: February 2005

AbstractPlatelet‐derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF‐A or PDGF‐B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF‐BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systemically in vivo, has not been reported. Therefore recombinant human PDGF‐BB, PDGF‐AB, and PDGF‐AA were evaluated in osteoblast‐enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half‐maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF‐BB, PDGF‐AB, and PDGF‐AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF‐BB being the most inhibitory. Binding studies with 125I‐PDGF‐BB or 125I‐PDGF‐AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I‐PDGF‐BB binding was preferentially displaced by PDGF‐BB (Ki ≈0.7 nM), less by PDGF‐AB (Ki ≈2.3 nM) and poorly by PDGF‐AA. In contrast, 125I‐PDGF‐AA binding was measurably reduced by PDGF‐AA (Ki ≈4.0 nM), but was more effectively displaced by PDGF‐BB or PDGF‐AB (each with Ki ≈0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF‐B subunit binding preferentially mediate these results in osteoblast‐enriched bone cell cultures.