bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of alterations in endothelial cell volume on transendothelial albumin permeability
Authors: Shepard J. M. , Goderie S. K. , Brzyski N. , Del Vecchio P. J. , Malik A. B. , Kimelberg H. K. 
Published: February 2005

AbstractWe examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C‐urea) decreased twofold and remained decreased over a 30‐minute time‐span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125l‐albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125l‐albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.
Transmembrane electrical characteristics of cultured human skeletal muscle cells
Authors: Iannaccone Susan T. , Li Kai‐Xun , Sperelakis Nicholas 
Published: February 2005

AbstractSkeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3–4 days, and fusion of myoblasts was observed in 5–10 days. Multinucleated myotubes were impaled under high optical magnification, at 37°C, with conventional glass microelectrodes. The mean resting potential was −44.4 mV ± 2.4 (n = 399); −33 ± 2.3 mV at 9 days (n = 10) vs −48 ± 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M ω (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 ± 2.1 mV with a mean maximum rate of rise (±Vmax) of 72.1 ± 7.5 V/s. The mean overshoot was 13.9 ± 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5–100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.
Changes in erythroid membrane proteins during erythropoietin‐mediated terminal differentiation
Authors: Koury M. J. , Bondurant M. C. , Rana S. S. 
Published: February 2005

AbstractMembrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia‐inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. α and β spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulsechase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two‐dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remains in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP‐treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as α and β spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.
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

AbstractA 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

AbstractThe 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

AbstractWhile 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

AbstractTyrosine‐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

AbstractEpithelial 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

AbstractThe 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

AbstractA 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

AbstractThe 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

AbstractSmooth 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

AbstractThe 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.