bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complex pattern of insulin‐like growth factor binding protein expression in primary rat osteoblast enriched cultures: Regulation by prostaglandin E2, growth hormone, and the insulin‐like growth factors
Authors: McCarthy Thomas L. , Casinghino Sandra , Centrella Michael , Canalis Ernesto 
Published: February 2005

AbstractPrimary osteoblast‐enriched (Ob) cultures from fetal rat bone synthesize insulinlike growth factor (IGF) I and IGF‐II, which each enhance Ob function. While a number of agents modulate IGF‐I production, IGF‐II is constitutively expressed in this culture model. Independent of their expression, however, the activity of the IGFs can be modified by a small group of proteins termed IGF binding proteins (IGFBPs), but little is known about the regulation of individual IGFBPs that are synthesized by Ob cells. Northern blot analysis revealed that serum‐deprived primary rat Ob cells. Northern blot analysis revealed that serum‐deprived primary rat Ob cells express transcripts encoding IGFBP‐2, IGFBP‐3, IGFBP‐4, IGFBP‐5, and IGFBP‐6, but undetectable levels of IGFBP‐1 transcripts. Western ligand blots of Ob culture medium probed with 125I‐IGF‐I or 125I‐IGF‐II showed predominant IGFBPs migrating at 30/32 kDa, with minor bands at 24 and 38–47 kDa. Western antibody analysis identified IGFBP‐2 and IGFBP‐5 within the 30/32 kDa complex, while gel mobility shift on SDS‐PAGE following deglycosylation determined that IGFBP‐3 comprised the 38–47 kDa complex. By Northern analysis, 6 h treatment with prostaglandin E2 (PGE2), growth hormone (hGH), IGF‐I, or IGF‐II revealed a complex pattern of regulatory effects on steady‐state IGFBP transcript expression. PGE2 increased the transcript levels of IGFBP‐3, IGFBP‐4, and IGFBP‐5, (∼22‐, ∼2‐, and ∼4‐fold respectively), but had no effect on IGFBP‐2 or IGFBP‐6 transcripts. hGH enhanced IGFBP‐3 and IGFBP‐5 transcripts (each approximately twofold). IGF‐I and IGF‐II had no effect on IGFBP‐2 steady‐state transcript levels but enhanced the level of IGFBP‐5 transcripts (approximately fourfold). By Western ligand blot analysis, 24 h treatment with PGE2 elevated the 24 and 38–47 kDa IGFBPs and to a lesser extent the 30/32 kDa complex, hGH elevated the 38–47 kDa IGFBPs, and IGF‐I and IGF‐II each increased the 30/32 kDa IGFBP complex. Therefore, a comparison of results obtained from Northern, Western ligand, and Western antibody studies indicates that multiple IGFBPs are expressed by primary rat Ob cultures. While IGFBP‐2 and IGFBP‐6 synthesis in Ob cultures is relatively unaffected by short‐term treatment with PGE2, hGH, or the IGFs, these agents modify IGFBP‐3, IGFBP‐4, and IGFBP‐5 expression with individual patterns of effects. In addition, some changes in IGFBP polypeptide levels that are independent of alterations in transcript expression may result from the formation of complexes between IGFs and certain IGFBPs, which could serve to store IGFs for future utilization in the formation phase of bone remodeling. © 1994 Wiley‐Liss, Inc.
Hematopoietic progenitor cells of transgenic mice with increased copper/zinc‐superoxide dismutase activity are resistant to tumor necrosis factor
Authors: Sakashita Akiko , Koeffler H. Phillip , Epstein Charles J. , Carlson Elaine 
Published: February 2005

AbstractThe mechanism of growth inhibition mediated by tumor necrosis factor (TNF) is unclear. Since recent data strongly suggested that generation of superoxide is a key step in cytotoxicity of TNF, we reasoned that cells expressing high levels of enzymes that degrade superoxide radicals would be resistant to TNF. Therefore, we examined the TNF‐sensitivity of bone marrow progenitor cells of transgenic mice that expressed the gene for human copper zinc‐superoxide dismutase (CuZn‐SOD). The CuZn‐SOD is a key enzyme in the metabolism of superoxide radicals. Heterozygous and homozygous transgenic mice had 3‐ and 5‐fold increased levels of CuZn‐SOD activity, respectively. Bone marrow cells of transgenic and nontransgenic mice were plated in soft gel culture with TNF (0.01–100 ng/ml). TNF inhibited myeloid colony formation supported by either granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or G‐CSF from nontransgenic mice in a dose‐dependent manner. In contrast, the myeloid clonal growth of homozygote transgenic mice was not inhibited by TNF at concentrations up to 100 ng/ml. As expected, the effects of TNF on erythroid clonogenic cells, which do not produce superoxide, and the action of transforming growth factor‐β on myeloid progenitor cells, were similar in both transgenic and nontransgenic mice. These results suggest that the mechanism of TNF‐mediated growth inhibition of hematopoietic cells occurs through production of superoxide. © 1994 Wiley‐Liss, Inc.
Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential
Authors: Culty Martine , Shizari Mehran , Thompson Erik W. , Underhill Charles B. 
Published: February 2005

AbstractIn the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85–200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes‐1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan. © 1994 Wiley‐Liss, Inc.
Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture
Authors: Desruisseau S. , Alquier C. , Depetris D. , Gruffat D. , Chabaud O. 
Published: February 2005

AbstractPorcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum‐free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of 3H‐mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive. © 1994 Wiley‐Liss, Inc.
Changes in cytoskeletal proteins and their mRNAs during maturation of human erythroid progenitor cells
Authors: Wickrema Amittha , Dai Chun‐Hua , Krantz Sanford B. , Koury Stephen T. 
Published: February 2005

AbstractWe have used highly purified human early erythroid progenitors to study changes in cytoskeletal proteins during their maturation and terminal differentiation. When erythroid progenitors at the burst‐forming unit‐erythroid (BFU‐E) stage of development are grown in the presence of erythropoietin, the cells mature and terminally differentiate into reticulocytes during a 14–15‐day culture period. We have shown by immunofluorescence that spectrin is present in day 3 BFU‐E, at which time proteins band 3, ankyrin, and band 4.1 cannot be detected. Ankyrin and band 4.1 were detected in the majority of the cells by day 7 of culture, at the colony‐forming unit (CFU)‐E stage, whereas only 15% of the cells were positive for band 3 protein on day 7 of culture. The mRNA level for spectrin was already at its maximum on day 8 whereas the mRNAs for band 3, ankyrin, and band 4.1 were just beginning to accumulate. After enucleation, spectrin, band 3, ankyrin, and band 4.1 fluorescence were all associated with the reticulocytes. Actin was localized at the constriction between the extruding nucleus and the incipient reticulocyte in enucleating erythroblasts suggesting a key role for actin in the enucleation of human erythroblasts. Our investigations have also shown that purified human erythroid progenitors cultured in serum‐free suspension media are capable of enucleating without the requirement of an extracellular matrix. These results demonstrate that the synthesis and expression of major cytoskeletal proteins in the human erythrocyte membrane occur in an asynchronous manner and that the remodeling of the membrane skeleton begins at a very early stage during erythrocyte development. © 1994 Wiley‐Liss, Inc.
Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact
Authors: Nanno Masanobu , Hata Masahiro , Yagi Hideki , Itoh Tsunetoshi , Doi Hideyuki , Satomi Susumu , Sakata Tsuneaki , Suzuki Ryuji 
Published: February 2005

AbstractWe have previously shown that a fetal liver‐derived epithelial cell clone, FHC‐4D2, could support hematopoiesis in vitro through its colony‐stimulating factor (CSF) activities in a short‐term culture. In this study, since FHC‐4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC‐4D2 could exert hematopoietic supporting activity in a long‐term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC‐4D2 cells. This clone could maintain the colony‐forming unit of granulocytes and macrophages (CFU‐GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC‐4A3, could not support the survival of CFU‐GM, even for 1 week. In addition to BM CFU‐GM, the FHC‐4D2 clone also supported the survival of FL CFU‐GM, burst‐forming unit of erythroid cells (BFUe), and colony‐forming unit of mixed progenitors (CFU‐Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC‐4D2 cells in the coculture, the comparable number of CFU‐GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU‐GM were present in both nonadherent and adherent cells to the FHC‐4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU‐GM. CFU‐GM derived from the adherent cells formed larger colonies and contained more bipotential CFU‐GM than the nonadherent population. When BM cells from mice given 5‐fluorouracil were cocultured with FHC‐4D2 cells under the limiting dilution condition, interleukin‐3 (IL‐3)‐responsive CFU‐GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL‐3. From these data we conclude that the FHC‐4D2 clone could generate and maintain IL‐3‐responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL‐3‐unresponsive immature hematopoietic cells into CFU‐GM (progenitors responsive to IL‐3) and in triggering the self‐renewal of CFU‐GM. © 1994 Wiley‐Liss, Inc.
Effect of tyrosine kinase inhibition on basal and epidermal growth factor‐stimulated human Caco‐2 enterocyte sheet migration and proliferation
Authors: Basson Marc D. , Turowski Gregory , Zarif Abdul , Modlin Irvin M. , Beidler David R. , Jena Bhanu P. , Madri Joseph A. 
Published: February 2005

AbstractMucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation‐blocked cell migration using human Caco‐2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2, 5‐dihydroxymethylcinnamate, which block kinase ATP‐binding and substrate‐binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF‐stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2, 5‐dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation‐blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein‐linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5‐dihydroxymeth‐ylcinnamate blocked changes in the α2 integrin subunit organization which may modulate EGF‐stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase‐dependent modulation of cell‐matrix interaction inhibits EGF‐stimulated migration. © 1994 Wiley‐Liss, Inc.
Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in swiss 3T3 cells
Authors: Kiehne Karlheinz , Rozengurt Enrique 
Published: February 2005

AbstractVasopressin and bradykinin bind to receptors coupled to GTP‐binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca2+ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vaso‐pressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long‐term activation of protein kinase C, like vasopression or phorbol 12, 13‐dibutyrate. Down‐regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long‐term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of Mr = 110,000–130,000 and Mr = 70,000–80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca2+ ‐mobilizing peptides on mitogenesis may be more general than previously thought. © 1994 Wiley‐Liss, Inc.
Cytokine production and DNA synthesis by Human peripheral Lymphocytes in response to Palmitic, Stearic, Oleic, and Linoleic acid
Authors: Karsten S. , Schäfer G. , Schauder P. 
Published: February 2005

AbstractEffects of palmitic, stearic, oleic, and linoleic acid on mitogen‐induced DNA synthesis, on production of IL‐1β, IL‐2, IFN‐gamma, and TNF‐α, and on IL‐2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiologica concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN‐gamma at concentrations that diminished TNF‐α production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN‐gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL‐2R expression correlated with the production of IL‐2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA. © 1994 Wiley‐Liss, Inc.
Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix
Authors: Simpson D. G. , Terracio L. , Terracio M. , Price R. L. , Turner D. C. , Borg T. K. 
Published: February 2005

AbstractCellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac α1β1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo‐like phenotype. Individual NHCs displayed an elongated, rod‐like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue‐like organizational pattern was dependant upon specific interactions between the NHCs and the collagen‐based matrix that were mediated by the cardiac α1β1 integrin complex. This conclusion was supported by a variety of expermental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac α1, or β1 integrin chain inhibited the expression of the tissue‐like pattern of organization. Neither cell‐to‐cell contact or contractile function were necessary to induce the formation of the rod‐like cell shape. However, beating activity was necessary for the assembly of a well‐differentiated myofibrillar apparatus. These data suggest that the cardiac α1 β1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix. © 1994 Wiley‐Liss, Inc.
Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch
Authors: Arora Pamela D. , Bibby Kathryn J. , McCulloch Christopher A. G. 
Published: February 2005

AbstractCalcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (< 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca2+]i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura‐2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca2+]i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch‐activated (SA) channels and L‐type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), indicating that IP3 sensitive pools, IP3 insensitive pools, G5α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of Giα and Goα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca2+ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin‐sensitive subunits of G‐proteins, and actin filaments. © 1994 Wiley‐Liss, Inc.