bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the protein glycosylation defect of a temperature‐sensitive cell cycle mutant by the use of mutant cells overexpressing the human epidermal growth factor receptor after transfection of the gene
Authors: Feige Jean‐Jacques , Scheffler Immo E.
Published: February 2005

A temperature‐sensitive mutant with a defect in glycoprotein synthesis and a cell cycle (G1)‐specific arrest at the nonpermissive temperature (Tenner et al., J. Cell. Physiol., 90:145–160, 1977; Tenner and Scheffler, J. Cell. Physiol., 98:251–266, 1979) was investigated further after a human epidermal growth factor (EGF) receptor gene had been transfected and amplified in these cells. While a temperature shift‐up lead to an immediate arrest in the biosynthesis of mature EGF receptor and its appearance on the plasma membrane, the observed turnover of the preexisting receptor was too slow to account for the arrest of DNA synthesis in these mutant cells. Tunicamycin could in fact mimic the effect of a temperature shift on the biosynthesis of EGF receptor, but it did not have the same rapid effect on DNA synthesis and cell cycle progression. These mutants have also been shown to induce a set of stress proteins or glucose‐regulated proteins, GRPs (Lee et al., J. Cell. Physiol., 129:277–282, 1986). The question is addressed whether the defect in glycoprotein synthesis is the primary defect and a possible cause of the induction of the GRPs, or whether a more basic defect at the level of the endoplasmic reticulum (ER) is responsible for the complex phenotype of the mutant. Our results argue in favor of a primary defect which indirectly affects N‐linked glycosylation of proteins, as well as several other functions associated with the ER. We hypothesize that the defect affects the calcium distribution between ER and cytosol, since the calcium ionophore A23187 has an effect similar to that of a temperature shift.
Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar
Authors: Kato Yukio , Iwamoto Masahiro , Koike Tatsuya
Published: February 2005

The effect of fibroblast growth factor (FGF) on the growth of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony‐forming efficiency of FGF‐exposed chondrocytes was similar to that of Rous sarcoma virus‐transformed chondrocytes (15–20%). Other mitogenic agents tested, such as epidermal growth factor, insulin, insulin‐like growth factor‐I, and platelet‐derived growth factor, induced very low levels of colony formation. The induction of growth in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage‐characteristic proteoglycan. FGF did not induce growth in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5‐bromodeoxyuridine. In addition, FGF did not induce growth in soft agar of primary fibroblasts and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates growth of differentiated chondrocytes in soft agar.
Spontaneous reversal of polarity of the voltage across LLC‐PK1 renal epithelial cell sheets
Authors: Mullin James M. , O'Brien Thomas G.
Published: February 2005

While sterilely monitoring transepithelial voltage (potential difference) across LLC‐PK cell sheets over a 24‐hr period, we noted that the apical‐negative, transepithelial voltage, a key property of the LLC‐PK1 renal epithelial cell line, reverses polarity to become apical‐positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC‐PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)‐negative PD, the apical‐positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4‐acetamido‐4‐isothiocynato‐stilbene‐2,2‐disulfonic acid (SITS). A pH gradient existed across apical‐positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature‐sensitive apical‐negative PD, the apical positive PD was unaffected by brief exposure to 4°C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical‐positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+‐sugar cotransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose‐free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 × 106 cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical‐positive PD values, a sizable gradient of Cl– did exist, directed apical to basoiateral. This gradient, coupled with anion‐selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.
EGF‐dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI‐38 cells
Authors: Brooks Katherine M. , Phillips Paul D. , Carlin Cathleen R. , Knowles Barbara B. , Cristofalo Vincent J.
Published: February 2005

Tyrosine‐specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI‐38 cells is EGF‐dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI‐38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P‐40 (NP‐40). Differences in the level of receptor phosphorylation in young vs. senescent NP‐40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP‐40 or chloroform‐methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP‐40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.
Human bronchial epithelial cells synthesize cholesterol sulfate during squamous differentiation in vitro
Authors: Rearick James I. , Hesterberg Thomas W. , Jetten Anton M.
Published: February 2005

Epithelial cells of the airways can, under pathological conditions, undergo squamous metaplasia. The accumulation of cholesterol sulfate has recently been described as a new marker for squamous cell differentiation in rabbit tracheal epithelial cells. We now report that normal human bronchial epithelial cells in culture metabolically incorporated [35S]‐sulfate and [3H]‐mevalonate into material indistinguishable from cholesterol sulfate by the criteria of solubility in organic solvents, behavior on ion‐exchange chromatography, susceptibility to solvolysis, and behavior on thin‐layer chromatography before and after solvolysis. The accumulation of cholesterol [35S]‐sulfate correlated well with squamous cell differentiation (as measured by cross‐linked envelope formation), which occurred when the cells reached confluency. The increase in the level of cholesterol sulfate could be inhibited by the inclusion of retinoic acid in the cell‐culture medium. The addition of phorbol‐12‐myristate‐13‐acetate or the presence of high Ca2+ concentration in the medium stimulated the accumulation of cholesterol sulfate. An increased activity of cholesterol sulfotransferase seems to account for the cholesterol sulfate accumulation. The original observation of cholesterol sulfate accumulation during squamous differentiation thus extends across species lines and strengthens the suggestion that the cholesterol sulfate may play an important role in this type of differentiation. Moreover, cholesterol sulfate provides a sensitive biochemical marker to study this pathway of differentiation of human bronchial epithelial cells.
Endothelial cell response to pulsed electromagnetic fields: Stimulation of growth rate and angiogenesis in vitro
Authors: Yen‐Patton G. P. Ameia , Patton Wayne F. , Beer Donna M. , Jacobson Bruce S
Published: February 2005

The effects of pulsed electromagnetic fields on the repopulation rate of denuded regions of endothelial cell monolayers and on endothelial cell reorganization into complex vessellike structures was monitored in vitro by using human umbilical vein and bovine aortic endothelial cells. A small (20–40%) but statistically significant enhancement in growth rate of partially denuded endothelial cell monolayers as determined by tritiated thymidine incorporation was observed in the presence of pulsed electromagnetic fields. Morphologically, endothelial cells entering the denuded regions were observed to be elongated, often connecting end to end to form a mycelial or “sprouting” pattern when exposed to pulsed electromagnetic fields. This was in contrast to cells outside of the field which had a more cuboidal morphology. Complete disruption of the endothelial cell monolayer by passaging the cells with EDTA trypsin resulted in reorganization of some of the cells into three‐dimensional vessellike structures after as little as 5–8 hours in the presence of the pulsed electromagnetic field. This reorganization occurred in the presence of heparin, endothelial cell growth factor, and a competent fibronectin matrix. Vas‐cularization for comparable cultures outside of the field did not occur during the time‐course of the experiments. Discrete stages of neovascularization were observed in the presence of the field that were qualitatively similar to stages of angiogenesis observed in vivo.
Preadipocyte differentiation in vitro: Identification of a highly active adipogenic agent
Authors: Hiragun Akiyoshi , Sato Mayumi , Mitsui Hiromi
Published: February 2005

A highly active adipogenic agent was identified in an in vitro adipose conversion system. This agent, ADD 4743 (or ADD), was synthesized by Takeda Chemical Industries (Osaka) as a 3‐hydroxy derivative of an oral antidiabetic agent, ciglitazone, and has been presumed to be an active metabolite of the latter substance. When ST 13 mouse preadipose cells were treated with micromolar concentrations of ADD they rapidly and uniformly converted into lipid‐accumulating adipocytelike cells within 8–11 days after cell seeding. The degree of adipose conversion and lipid accumulation induced by ADD far exceeded those of the previously known inducing agents such as indomethacin plus insulin. The highly potent adipogenic activity of ADD was confirmed with two other preadipose cell lines (3T3 L1 and RMT rat preadipose cells). In addition to adipogenic activity, ADD inhibited cell proliferation of preadipose cells specifically. Activity of ADD induced lipid accumulation and growth inhibition of ST 13 cells, exhibiting very similar dose‐response relationships. Cell proliferation or triacylglycerol content of nonadipocytic mesenchymal cells or epithelial cells were not affected by ADD. These observations strongly suggest that ADD‐induced growth inhibition is not due to the nonspecific toxicity of the drug but is tightly associated with the adipocytic character of the treated cells. The present observation provides evidence that ADD would be a powerful agent in studies that involve preadipocyte differentiation.
Temporal relationship of the prolactin‐dependent LH‐induced LH receptor to the LH stimulus
Authors: Hussein Mohamed O. , Zipf William B.
Published: February 2005

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of 125l‐labeled hCG was investigated in hypophysecto‐mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle‐stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased 125l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone‐induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection.

Seven days pretreatment with FSH or GH at 100 μg/day plus 10‐μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats.

These studies suggest that the induction or “up‐regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre‐exposure to prolactin.
Serum and growth factors regulate expression of a 43 kDa protein in smooth muscle cell cultures
Authors: Millis Albert J. T. , Hoyle Marian
Published: February 2005

Smooth muscle cells respond to injury and the presence of serum factors by modulating from a quiescent contractile cell to a motile synthetic phenotype. To evaluate the biochemical response to serum exposure, we examined the proteins synthesized and secreted in response to serum. The most prominent effect of serum was the rapid production of a protein with an apparent molecular weight of 43 kDa. Removal of serum from the culture environment led to a cessation of 43 kDa protein production. The effect of exogenous heparin on 43 kDa protein production was also evaluated. Neither the 43 kDa protein nor a previously described 38 kDa protein was induced by heparin. Further, heparin treatment did not counteract the effects of serum. These studies demonstrate that an early response of vascular smooth muscle cells to serum is the production of this previously undescribed protein and that other modifications of the culture conditions did not affect its synthesis.
The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes
Authors: Luscinskas Francis W. , Mark Deiren E. , Brunkhorst Beatrice , Lionetti Fabian J. , Cragoe Edward J. , Simons Elizabeth R.
Published: February 2005

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex‐stimulated transmembrane depolarization and superoxide production, events which accompany—and have been used as indicators of—PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus‐induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K‐PRK) depolarized the cells and reduced the stimulus‐induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5‐(N,N‐dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one‐half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4CI (NH4CI‐PMNL) instead of H2O (H2O‐PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: (1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; (2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and (3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4CI lysis technique.
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

Mitogen 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

Transferrin 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

We 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

Laminin 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

Cells 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

Transforming 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

The 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

Direct 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

We 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

Con 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

Bone 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

We 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

The 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

Transforming 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.
Proliferation and differentiation of abelson virus‐infected murine myeloid leukemia cell lines does not involve an autocrine mechanism
Authors: Hines David L.
Published: February 2005

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus‐infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony‐forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth‐factor independent replication nor the spontaneous differentiation of Abelson virusinfected myeloid cells involves autocrine secretion of growth regulators.
Delayed processing/export of messenger RNA under conditions of reduced protein synthesis
Authors: Muralidhar M. G. , Johnson L. F.
Published: February 2005

The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorportion of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acidstarved cells.
Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na+/K+)‐ATpase activity
Authors: Schenerman Mark A. , Leister Kirk J. , Trachtenberg David K. , Racker Efraim
Published: February 2005

Mouse embryo fibroblast cells (C3H‐10T½) and the methylcholanthrenetransformed derivative (MCA‐10T½) were treated with basal modified Eagle's medium (BME) containing 10% fetal bovine serum and varying concentrations of ouabain ranging from 0.05 mM to 0.7 mM for 16 h in culture. After replacing the ouabain‐containing medium with Earl's balanced salts solution, System A amino acid transport activity increased from approximately 40 to 500 pmol AIB accumulated · mg protein−1 · min−1 in the C3H‐10T½ cells and from approximately 300 to 700 pmol AIB accumulated · mg protein−1 · min−1 in the MCA‐10T½ cells. The (Na+/K+)‐ATPase pump activity also increased form approximately 12 to 46 nmol Rb+ accumulated · mg protein−1 · min−1 in the normal cells and from approximately 20 to 42 nmol Rb+ accumulated · mg protein−1 · min−1 in the transformed cells. System A and the (Na+/K+) ATPase activity were maximally increased at approximately0.4–0.6 mM ouabain in the normal cells in contrast to the transformed cells which were maximally stimulated at a concentration of approximately 0.2 mM ouabain. This treatment with ouabain increased the [Na+]i/[K+]i as measured by atomic absorption spectroscopy, and thereby decreased the Na+ and K+ electrochemical gradients. Our data show that the internal ion gradients inverted at a lower concentration of ouabain in the transformed cells compared to the normal cells. The ouabain‐induced increased in pump and System A activity shown here was used as a tool to further investigate the coordinated ion transport regulation in the control of cell growth.
Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells
Authors: Gonzalez Fernando A. , Gross David J. , Heppel Leon A. , Webb Watt W.
Published: February 2005

The response of cytosolic calcium [Ca2+]i to epidermal growth factor (EGF), fetal calf serum, and nucleotides was determined in individual A431 cells, using the fluorescent probe fura‐2 and quantitative digital video fluorescence microscopy. In the presence of 1 mM external Ca2+, EGF caused a rapid rise in [Ca2+]i, followed by a slower and variable decrease. The cells responded after a lag that varied from 10 to 30 seconds, and there was considerable cell‐to‐cell variation in extent of the rise in [Ca2+]i. A second challenge with EGF gave negative results. No response was obtained in nominally Ca2+‐free medium supplemented with 100 μM EGTA. Somewhat similar results were obtained with fetal calf serum except that a rise in [Ca2+]i was observed both in the presence and absence of external Ca2+. The A431 cells responded to external ATP with a rise in [Ca2+]i in less than 10 seconds, both in Ca2+‐containing and Ca2+‐free media. A coverslip with attached cells was mounted on a small chamber, allowing complete change of medium in 2 seconds. A nearly full response was obtained with only 10 seconds of contact of cells with ATP‐containing medium. After washing out ATP, there was little or no response to a second addition given 100 seconds after the first. However, a second response was obtained when the concentration of agonist was increased 10–20‐fold. These data favor the idea of receptor desensitization. Both homologous and heterologous receptor desensitization was observed. A transient rise in [Ca2+]i was also noted with UTP, while ITP and CTP were inactive.
Methotrexate‐poly(lysine) as a selective agent for mutants of chinese hamster ovary cells defective in endocytosis
Authors: Ryser Hugues J.‐P. , Mandel Richard , Hacobian Asteghik , Shen Wei‐Chiang
Published: February 2005

Methotrexate (MTX) covalently linked to poly(L‐lysine) [poly(Lys)] enters cells by endocytosis, is degraded in lysosomes and, upon liberation of small molecular methotrexate, is cytocidal to Chinese hamster cells in culture. This drug conjugate was used to select mutants resistant to MTX‐poly(Lys), which were examined for defects in endocytosis. Two mutants resistant to MYX‐poly(Lys) and sensitive to free MTX, MPL 3–4 and MPL 2–5, internalized the conjugate in normal fashion, but had a decreased ability to degrade it to small molecular drug. The magnitude of this defect in the two mutants correlated with their level of resistance. In addition, both mutants were cross resistant to diphtheria toxin and modeccin and hypersensitive to ricin. While MPL 3–4 internalized MTX‐poly(Lys) and inulin normally, it showed decreased endocytosis via the mannose‐6‐phosphate receptor and decreased uptake of 125I‐alpha‐2 macroglobulin. Acidification of subcellular fractions was measured using the partitioning of acridine orange. In MPL 3–4, the ATP‐driven acidification of the endosome‐containing cell fractions was slightly decreased (80% of controls), while acidification of the heavy lysosome‐containing fraction was normal. Complementation analysis using hybrids of MPL 3–4 × MPL 2–5 indicated that the mutations occurred at the same gene, but were expressed with different severity. This genotype is identical to that of the End 2 mutants described by Roff et al. (1986). Thus, surprisingly, mutants with identical genotypes were isolated independently by totally different selection procedures.