bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts
Authors: Jamieson Gordon A. , Etscheid Beth G. , Muldoon Leslie L. , Villereal Mitchel L. 
Published: February 2005

AbstractMitogen c stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]‐thymidine. We have determined previously that pretreatment with the tumor‐promoting phorbol ester 12–0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) inhibits mitogen‐stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen‐stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen‐stimulated inositol phosphate release occurs at 1–2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate‐stimulated inositol phosphate release and Na influx. In addition, Orthovanadate was determined to increase intra‐cellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura‐2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus‐mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA‐mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]‐thymidine. These results are contrasted with those obtained in WI‐38 cells. A cell‐type in which (1) orthovanadate does not stimulate inositol phosphate release, (2) TPA has minimal or no inhibitory effect on early growth factor induced cellular responses (i.e., inositol phosphate release, intracellular Ca mobilization, activation of Na/H exchange), (3) TPA stimulates Na/H exchanger activity, without activating inositol phosphate release, and (4) TPA promotes (unlike in HSWP cells) the incorporation of [3H]‐thymidine.
Transferrin and iron in cultured chick embryonic neurons: A comparison between human and chick transferrins
Authors: Hyndman Arnold G. , Zeevalk Gail D. 
Published: February 2005

AbstractTransferrin was not required for the short‐term survival of cultured chick retinal neurons. Both human and chick transferrin failed to enhance the in vitro survival of 8‐ or 11‐day embryonic chick retinal neurons when cultured in a defined medium. Furthermore, maintenance of neurons in the presence of chick transferrin antibody did not alter in vitro survival. Retinal neurons, however, could bind and internalize human or chick transferrin when assayed for by fluorescence immunohistochemical techniques. Binding and internalization of chick transferrin appeared to be greater than human transferrin. Iron uptake was measured in cultures maintained in the absence of transferrin. After incubation with 59FeCI3, iron uptake was 3.5 ± 1.1 fmoles/cell. The presence of chick transferrin antibody did not significantly alter the amount of iron uptake occurring in this assay. In a comparison of human and chick transferrin mediated iron uptake, chick transferrin was 50% more effective than human transferrin in transporting iron. This study demonstrates that cultured embryonic retinal neurons are not dependent on transferrin for survival or iron uptake, although they actively bind and internalize transferrin. Results also demonstrate that whereas cultured chick retinal neurons can bind and utilize human transferrin, they do so with less efficiency than chick transferrin.
Thrombin‐induced adherence of neutrophils to cultured endothelial monolayers: Increased endothelial adhesiveness
Authors: Bizios Rena , Lai Linda C. , Cooper Jeffrey A. , Del Vecchio Peter J. , Malik Asrar B. 
Published: February 2005

AbstractWe examined the effects of α‐thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10−8 M) resulted in a time‐dependent increase in neutrophil adherence to the endothelium. The response was concentration‐dependent with a maximal response at 10−8 M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell‐derived matrix. The adherence response was inhibited in the presence of α‐thrombin that had been inactivated with anti‐thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti‐thrombin III or hirudin simultaneously with α‐thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin‐induced adherence of neutrophils to the endothelial monolayer. Addition of 10−8 M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10−8 M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 ± 2.3% vs. 12.9 ± 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin‐induced neutrophil adherence.
Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration
Authors: Iwamoto Y. , Graf J. , Sasaki M. , Kleinman H. K. , Greatorex D. R. , Martin G. R. , Robey F. A. , Yamada Y. 
Published: February 2005

AbstractLaminin is a basement membrane‐specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine‐amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell‐binding site and migration site on laminin share a common sequence that is unique to laminin.
Changes in inositol polyphosphate‐sensitive calcium exchange in aortic smooth muscle cells in vitro
Authors: Fittingoff Marianne , Krall J. Frederick 
Published: February 2005

AbstractCells that expressed the muscle‐specific intermediate filament protein desmin were cultured from the aorta of Fischer 344 rats. When the cultured cells were extracted with digitonin, they accumulated 45Ca2+ from the incubation medium in a manner that was stimulated by ATP and released subsequently by exposure to the Ca2+ ionophore A23187. Ca2+ bound in the presence of ATP was also released by exposure to inositol 1,4,5‐trisphosphate (IP3). Like contraction in some kinds of smooth muscle, IP3 released Ca2+ in either the absence or the presence of the ATPase‐inhibitor ruthenium red. When the responsiveness of digitonin‐extracted cells cultured from 3‐, 12‐, and 24‐month‐old rats was compared, cells from the youngest group released only about one‐half as much Ca2+ as cells from the 12‐ or 24‐month‐old rats. The results suggest that in the rat there are changes during maturation in the responsiveness to inositol polyphosphates of intracellular compartments that sequester Ca2+ for stimulus‐contraction coupling in the aortic smooth muscle cell. These changes, characterized in smooth muscle cells in vitro, might contribute to the way vascular responsiveness is regulated in vivo.
Transforming growth factor‐beta modulates the expression of osteoblast and chondroblast phenotypes in vitro
Authors: Rosen David M. , Stempien Sue A. , Thompson Andrea Y. , Seyedin Saeid M. 
Published: February 2005

AbstractTransforming growth factor β (TGF‐β) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261:5693, 1986). Here we report the effects of bovine TGF‐β on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF‐β resulted in a dose and time‐dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF‐β. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF‐β resulted in decreased production of cartilage‐specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF‐β. Modulation of these differentiated markers by TGF‐β could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF‐β. These results indicate that TGF‐β inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF‐β may be mediated through interactions between the extracellular matrix and cytoskeletal elements.
Direct effects of wheat germ agglutinin on inositol phosphate formation and cytosolic‐free calcium level in intestine 407 cells
Authors: Sjölander Anita 
Published: February 2005

AbstractThe interaction between dietary lectins, especially wheat germ agglutinin (WGA), and intestinal cells has been implicated in the pathogenesis of celiac disease. The present study was undertaken to investigate the immediate effects following such an interaction. Direct WGA‐stimulation of Intestine 407 cells leads to an immediate rise in the cytosolic‐free calcium concentration. The major part of this lectin‐induced rise is due to an influx of calcium across the plasma membrane into the cytosol. However, WGA‐exposure also results in an immediate mobilization of calcium from intracellular stores, most likely mediated via the simultaneous increase of inositol trisphosphate formation in these cells. The transduction mechanism described for WGA in these intestinal cells is not very sensitive towards pertussin toxin, indicating that if a G‐protein is involved, it differs from those of most other systems. The suggested role for WGA in changing the functional and structural properties of intestinal cells might involve increases of inositol phosphate and cytosolic‐free calcium levels.
Transformation of single myeloid precursor cells by the malignant histiocytosis sarcoma virus (MHSV): Generation of growth‐factor‐independent myeloid colonies and permanent cell lines
Authors: Klingler K. , Johnson G. R. , Nicola N. A. , Arman G. , Kluge N. , Ostertag W. 
Published: February 2005

AbstractDirect single‐cell assays for oncogenic transformation are available for fibroblasts but not for other cell types. Using malignant histiocytosis sarcoma virus (MHSV), a member of the ras family of retroviruses, in vivo‐infected granulocyte/macrophage and macrophage precursor cells lost the requirement for externally added hematopoietic growth factors. Factor‐independent growth was demonstrated by colony‐transfer experiments. More than 25% of the independent colonies were established as permanent macrophage cell lines following a phase of adaptation to tissue culture conditions. Factor‐independent colony growth was also obtained by in vitro infection of single cells. As many as 50% of all myeloid precursor cells were target cells for MHSV as measured by this assay. About 2 × 10−3 of these colony‐forming cells acquired growth factor independence and immortality after in vitro infection. Cell lines derived from these colonies did not require adaptation to tissue culture conditions.
Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line
Authors: Garbi Corrado , Zurzolo Chiara , Bifulco Maurizio , Nitsch Lucio 
Published: February 2005

AbstractWe examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL‐5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electromicroscopy using the protein A‐colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL‐5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.
Demonstration that some of the nonhistone proteins, inducible to translocate into the nucleus, are glycosylated
Authors: Polet Herman , Molnar Janos 
Published: February 2005

AbstractCon A, NaF, and eserine (lysosomotropic agents) induced marked translocation of acidic [3H] nonhistone proteins (NHP) from the cytoplasm to the nucleus in lymphocytes prelabeled with [3H]‐2‐mannose. The nuclear [3H] NHP contents were 38–120% higher in cells treated with these agents than in control cells. Tunicamycin, a strong inhibitor of N‐glycosylation via the dolichol pathway, caused a concentration‐dependent inhibition of [3H]‐2‐mannose incorporation into the nuclear [3H] NHP. Considerable amounts of nuclear [3H] NHP from lymphocytes labeled with either [3H]‐2‐mannose or [3H] leucine, bound specifically to Con A‐Sepharose and could be eluted by α‐methyl mannoside. Con A and NaF caused also nuclear translocation of acidic [3H] NHP in cells labeled with [3H] glucosamine, [3H] galactose, or [3H] fucose. Fractionation of the nuclear proteins by isoelectric focusing in a pH gradient of 2.5–6.5 showed that multiple species of acidic NHP were labeled with each of the four 3H‐sugars. These results indicate that a fraction of the acidic nuclear NHP are N‐glycosylated proteins and that gene activation and mitogenesis are associated with the translocation of these glycoproteins to the nucleus. Considering the known intracellular traffic of nascent glycoproteins our results suggest that at least some of the acidic NHP are synthesized and glycosylated in the endoplasmic reticulum and the Golgi (secretory pathway). It is likely that these proteins, after completion of synthesis and glycosylation, emerge from the trans‐stack of the Golgi packaged in vesicles and accumulate in the cytoplasm. Induction of nuclear translocation of such NHP by various agents may be mediated by a vesicular transport mechanism.
Characterization of granulocytes and mast cells in cultures of mouse bone marrow stimulated with interleukin‐3
Authors: Bender James G. , Van Epps Dennis E. , Stewart Carleton C. 
Published: February 2005

AbstractBone marrow cells in liquid culture with interleukin 3 produce a population of non‐adherent granulocytes and mast cells. Flow cytometry was used to identify granulocytes and mast cells on the basis of the physical properties of perpendicular light scatter (PLS) and coulter volume (CV) as well as the expression lgE and CR3 receptors. Multicolor analysis indicated there were subpopulations of Thy 1.2 positive cells which transiently appeared in these cultures and also expressed lgE receptors, CR3 receptors or neither of these receptors. The data suggested a differentiation scheme in which Thy 1.2 positive precursor cells give rise to granulocytes and mast cells. Further evidence for this differentiation scheme was provided from CV vs. PLS distributions which showed increases in CV and PLS as Thy 1.2 positive cells differentiated into mast cells and decreases in CV and PLS as Thy 1.2 positive cells differentiated into granulocytes.
Inhibition of EGF processing in responsive and nonresponsive human fibroblastsx
Authors: Schaudies R. Paul , Wray H. Linton 
Published: February 2005

AbstractWe have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth‐responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies (Schaudies et al., 1985) have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des‐(53)‐EGF] or six [des (48–53)‐EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubatin system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30‐minutes incubation. However after 4‐hours incubation in the constant presence of 125I‐EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolable within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I‐EGF, and 125I‐EGF lacking the carboxyl terminal arginine were released from chloroquine‐treated cells in a ratio equal to that present in the intact cells. Incubations in leupeptin, proteolysis of EGF beyond the des‐(48–53)‐EGF was blocked; however, no large‐molecular‐weight species were released from the cells under these conditions.
Membrane potential of primitive red cells from chick embryo is a proton potential
Authors: Engelke Maria , Zingel Werner , Baumann Rosemarie 
Published: February 2005

AbstractThe membrane potential of primitive red cells from 4‐ and 6‐day old chick embryos has been determined using the fluorescent dye Dis‐C3‐(5). At day 4 the membrane potential Em was −44 mV for pH 7.4 and 20°C and −36 mV at day 6. Both values are far removed from the equilibrium potential for chloride, which is about −14 mV at day 6. Changes in the external potassium, sodium or chloride concentration were without effect on the membrane potential, except at very high potassium concentrations, where a small but significant depolarization was observed at day 6. The measurements gave the same results in the absence or presence of the anion exchange blocking agent DIDS. Three pieces of evidence indicate that the membrane potential of primitive red cells is primarily caused by an electrogenic H+ conductance: (1) The measured membrane potential of −36 mV at day 6 is close to the previously determined proton equilibrium potential (Baumann and Haller, 1983) EH+ of −36 mV. (2) Addition of the electrosilent Cl−/OH− exchanger tributyltin causes a significant depolarization of about 20 mV at day 4 and about 14 mV at day 6. (3) Measurement of hydrogen ion fluxes demonstrate a potential dependent proton conductance, which increases with depolarization. These results indicate that large qualitative differences exist with regard to the mechanisms involved in the generation of membrane potential and hydrogen distribution between red cell and plasma of embryonic and adult chicken.
TGF‐β inhibits growth factor‐induced DNA synthesis in hamster fibroblasts without affecting the early mitogenic events
Authors: Chambard Jean‐Claude , Pouysségur Jacques 
Published: February 2005

AbstractTransforming growth factor beta (TGF‐β) was found to inhibit (IC50 =0.1 ng/ml) α‐thrombin or FGF‐induced mitogenicity in G0‐arrested Chinese hamster lung fibroblasts. Growth factor‐stimulated cells became rapidly insensitive to TGF‐β addition during their progression through G0/G1 suggesting that an early step of the mitogenic response was the target of TGF‐β action. Surprisingly, none of the well characterized early mitogenic events commonly triggered by growth factors was found to be affected by TGF‐β addition. These responses included: phosphoinositide breakdown, activation of protein kinase C as determined by EGF receptor down‐modulation, subsequent rises in pHi, c‐fos, and c‐myc mRNA levels, ribosomal protein S6 phosphorylation, the increase in RNA and protein synthesis, induction of ornithine decarboxylase. Only the induction of thymidine kinase, a marker of entry in the S phase, was found to be repressed by TGF‐β, with maximal inhibition when TGF‐β was added early in G1. These results indicate that the inhibitory action of TGF‐β does not affect the growth factors signalling pathways but touches an early event different from those so far analyzed.