bluuebunny/crossref_metadata_2025_split huggingface dataset:
Accumulation of insulin‐like growth factor binding protein‐3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro
Authors: Goldstein Samuel , Moerman Elena J. , Baxter Robert C. 
Published: February 2005

AbstractWe have found that insulin‐like growth factor binding protein‐3 (IGFBP‐3) accumulates to higher levels in medium conditioned by a strain of normal fibroblasts at late passage (LP) and a strain derived from subjects with Werner syndrome (WS) of premature aging, compared to medium conditioned by the same normal cells at early passage (EP) (Goldstein et al., Proc. Natl. Acad. Sci. USA, 88:9680–9684, 1991). To explore the generality of this phenomenon with respect to chronological age of donor (in vivo aging) and LP (in vitro senescence) we assayed IGFBP‐3 in medium conditioned by 18 normal fibroblast strains at EP and LP and two WS strains at the midpoint of their curtailed replicative lifespans and assessed IGFBP‐3 mRNA levels in cells by Northern analysis. The lowest accumulations of IGFBP‐3 were found in medium conditioned by fetal cells with progressively increasing amounts postnatally; direct correlations between IGFBP‐3 levels and donor age were seen in EP cells 3 days after subculture (during logarithmic growth) r = 0.80, P < 0.001, and 7 days after subculture (at confluence) r = 0.77, P < 0.001. With two exceptions, conditioned medium of cell strains accumulated more IGFBP‐3 at LP; IGFBP‐3 levels correlated with chronological age after 3 days, r = 0.50, P = 0.05, and after 7 days, r = 0.75, P < 0.001. IGFBP‐3 content of WS culture medium fell within the range of LP normal cells. Cumulative IGFBP‐3 levels were inversely proportional to the thymidine labeling index, a measure of proliferative vigor. With some exceptions IGFBP‐3 mRNA levels were commensurate with the amount of IGFBP‐3 accumulated in the medium, suggesting that distal translational and posttranslational mechanisms also regulate IGFBP‐3 production in some strains. The trend toward augmented IGFBP‐3 output of fibroblasts as a direct function of chronological age and in vitro senescence and as an inverse function of proliferative vigor is consistent with the known inhibitory effect of excess IGFBP‐3 on IGF‐mediated DNA synthesis and the reduced regenerative potential that is evident during biological aging in vivo. © 1993 Wiley‐Liss, Inc.
Effect of pH and ATP on the equilibrium density of lysosomes
Authors: Mayorga Luis S. , De Veca Mercedes G. , Colombo Maria I. , Bertini Francisco 
Published: February 2005

AbstractLysosomes are membrane bound structures that accumulate and hydrolyze material internalized by the endocytic pathway. A very conspicuous property of this subcellular compartment is its relatively high equilibrium density. The actual mechanism that regulates lysosomal density is poorly understood. In an attempt to gain knowledge on the factors that regulate lysosomal density we have assessed the equilibrium density of lysosomal markers after in vitro incubation of a lysosome‐enriched subcellular fraction. Incubation at pH 6 for 10 min at 37°C causes a density shift of several lysosomal markers to light density regions of Percoll gradients. Addition of ATP was able to prevent the acid‐induced density shift. Pretreatment of the vesicles with N‐ethylmaleimide (NEM) or trypsin inhibited the effect of ATP. Working in intact cells, ATP depletion, a condition that causes cytoplasmic acidification, also decreases lysosomal density. The results indicate that at low pH lysosomal density is preserved by an active process that requires ATP and membrane associated proteins. © 1993 Wiley‐Liss, Inc.
Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells
Authors: Liu Shu Ming , Magnusson Karl‐Eric , Sundqvist Tommy 
Published: February 2005

AbstractThe macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid‐phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC‐dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid‐phase endocytosis and that microfilaments hinder this process. © 1993 Wiley‐Liss, Inc.
Modulation of the immunophenotype of ovarian cancer cells by N,N‐dimethylformamide and transforming growth factor‐β1
Authors: Grunt Thomas W. , Oeller Helga , Somay Canatay , Dittrich Evelyn , Fazeny Barbara , Mannhalter Christine , Dittrich Christian 
Published: February 2005

AbstractExposure of HOC‐7 ovarian adenocarcinoma cells to regulators of cell differentiation caused inducer‐dependent alterations of the antigenic pattern of the cells. Immunocytochemistry revealed that N,N‐dimethylformamide (DMF) elevated the membrane staining for epidermal growth factor (EGF)‐receptor and for desmoplakins I and II. DMF also stimulated cytoplasmic and surface labeling for CA 125 and the deposition of fibronectin into the extracellular matrix. Stimulation of fibronectin was also seen after addition of transforming growth factor (TGF)‐β1. These responses were quantified using a fixed‐cell, enzyme‐linked immunosorbent assay (ELISA) and revealed that DMF dose‐dependently induced expression of EGF‐receptor, CA 125, fibronectin, and desmoplakins I and II. TGF‐β1 stimulated fibronectin and desmoplakins I and II only. Production of EGF and TGF‐α was not affected by these inducers. Immunocytochemistry, ELISA and Western blotting showed that both inducers caused down‐regulation of myc oncoproteins. DMF was more effective in changing the immunophenotype of HOC‐7 cells than TGF‐β1. Desmoplakins I and II demonstrated elevated epithelial differentiation, whereas fibronectin indicated stimulation of extracellular matrix formation. Elevated EGF‐receptor could not compensate for the growth inhibition induced by DMF. The expression of myc oncoproteins was inversely related to cell proliferation. CA 125, however, seems to be unrelated to cell growth. © 1993 Wiley‐Liss, Inc.
Intracellular calcium oscillations induced in a T‐cell line by a weak 50 Hz magnetic field
Authors: Lindströum Ewa , Lindströum Per , Berglund André , Mild Kiell Hansson , Lundgren Erik 
Published: February 2005

AbstractApplied weak magnetic fields have been shown to affect cellular activity on several levels, but the mechanisms involved remain elusive. We have decided to study an early signal transduction event in the human T cell line Jurkat; oscillations of free [Ca2+]i, of the type seen by crosslinking the CD3 complex. Cells were exposed to a 50 Hz, 0.1 mT, sinusoidal magnetic field while intracellular free calcium was measured in individual cells, using fura‐2 as a probe. An acute response was observed with oscillatory increases in [Ca2+]i, which subsided when the field was turned off. The effect of the magnetic field on [Ca2+]i was comparable to that achieved by an anti‐CD3 monoclonal antibody. © 1993 Wiley‐Liss, Inc.
Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil‐mediated antibody‐dependent cellular cytotoxicity
Authors: Cao Dongrong , Boxer Laurence A. , Petty Howard R. 
Published: February 2005

AbstractIn this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5′ (or 6′) carboxyl‐2′,7′‐dichlorodihy‐drofluorescein (H2‐CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H2‐CDCF‐conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H2‐CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil‐mediated antibody‐dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H2‐CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethyl‐rosamine within tumor cells. Using two‐color automated epifluorescence micros‐copy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (<12) sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant‐mediated damage. © 1993 Wiley‐Liss, Inc.
Interleukin‐3 inhibits cycloheximide of C‐Jun mRNA in human monocytes: Possible role for a serine/threonine phosphatase
Authors: Rao Padmini , Mufson R. Allan 
Published: February 2005

AbstractCycloheximide is a strong inducer of the c‐jun protooncogene mRNA at concentrations (≤50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30–60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin‐3 (IL‐3) (10 ng/ml) is also a modest inducer of the c‐jun gene in these cells; however, in combination with cycloheximide, IL‐3 dramatically reduces the c‐jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL‐3 is observed at both 30 and 60 min after simultaneous addition of both IL‐3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL‐3 restored the level of c‐jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL‐3 to inhibit cycloheximide induction of c‐jun in monocytes. © 1993 Wiley‐Liss, Inc.
Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells
Authors: Low Boon Chuan , Ross Ian K. , Grigor Murray R. 
Published: February 2005

AbstractThe uptake of L‐leucine and L‐lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ·Na+ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y+ transporter. © 1993 Wiley‐Liss, Inc.
In vitro DNA synthesis of mouse hepatocytes stimulated by tumor necrosis factor is inhibited by glucocorticoids and prostaglandin D2 but enhanced by retinoic acid
Authors: Satoh Motonobu , Yamazaki Masatoshi 
Published: February 2005

AbstractWe have recently shown that TNF is produced in liver rapidly after partial hepatectomy and that TNF can stimulate DNA synthesis of hepatocyte primary culture with its inhibition by interleukin 6 and transforming growth factor‐β, indicating a pivotal role of TNF and TNF‐driven cytokine induction in liver regeneration. We here examined the effects of biological or inflammatory mediators of low molecular weight on the in vitro DNA synthesis of hepatocytes stimulated by TNF. Simultaneous addition of dexamethasone markedly suppressed the growth‐stimulating action of TNF, maximally at 10−7 M and effectively at about 10−8 M. However, the growth‐stimulating effect of EGF was not affected by dexamethasone at all. Physiological glucocorticoids, corticosterone, and hydrocortisone showed virtually the same effect, but other steroid hormones, β‐estradiol, or progesterone did not. Retinoic acid at 10−7 M, however, enhanced TNF‐stimulated hepatocyte DNA synthesis and even more effectively the growth response to FGF. PGD2 at 20 μM was markedly suppressive but PGE2 was not. The addition of indomethacin enhanced the hepatocyte in vitro growth by TNF and EGF at 2–20 μM. These results indicate that the growth of hepatocytes stimulated by TNF is up and down‐regulated by inflammatory mediators and hormones as well as cytokines and suggest the biological significance of TNF and TNF‐driven inflammatory reactions in liver regeneration. © 1993 Wiley‐Liss, Inc.
Senescence of aortic endothelial cells in culture: Effects of basic fibroblast growth factor expression on cell phenotype, migration, and proliferation
Authors: Augustin‐Voss Hellmut G. , Voss Anne K. , Pauli Bendicht U. 
Published: February 2005

AbstractBovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long‐term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early‐passage BAEC (passages 5–15) rapidly produce dense, cobblestone‐like monolayers. Their growth beyond the monolayer configuration is characterized by the formation of an irregular network of spindle‐shaped, crisscrossing BAEC growing either on top or beneath the monolayer, and by the assembly of elongated BAEC into well‐differentiated capillary‐like tubes. In contrast, senescent BAEC (passages 35–45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary‐like tubes but not the crisscrossing networks of their early‐passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic fibroblast growth factor (bFGF) which elevates the migratory and proliferative capacities of early‐passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine growth regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures. © 1993 Wiley‐Liss, Inc.
Insulin receptor characterization and function in bovine aorta endothelial cells: Insulin degradation by a plasma membrane, protease‐resistant insulin receptor
Authors: Milton Shirlette G. , Knutson Victoria P. 
Published: February 2005

AbstractThe functional significance of the insulin receptor on bovine aorta endothelial (BAE) cells is not well defined. The insulin receptor expressed on BAE cells does not mediate insulin hormonal effects and does not mediate the transcytosis of insulin from the apical to the basolateral domain of the cell monolayer. To assess the role of the insulin receptor on BAE cells, the physical characteristics of the BAE cell receptor were investigated, and the time‐dependent interaction of insulin and insulin degradation products with BAE cell monolayers was quantitated. The BAE cell insulin receptor was found to be highly resistant to the proteolytic action of trypsin, pronase, and proteinase K at either 4°C or 37°C. This resistance may permit the receptor to maintain insulin binding capabilities in spite of the high concentrations of proteases which are normally present in blood. Scatchard analysis of cell‐surface and total cellular insulin receptor demonstrated dissociation constants similar to values obtained with other cells and tissues. However, whereas other cells and tissues contain an intracellular pool of receptor that ranges from 20–40% of the total cellular receptor content, no intracellular population of insulin receptors was detected in BAE cells. Upon incubation of intact BAE cell monolayers with insulin, no endocytosis of cell‐surface insulin receptor could be demonstrated. However, insulin degradation by the BAE cells was readily quantitated, at a rate of 16.3 fmol/106 cells/h at an insulin concentration of 2 nM. This rate of degradation was not inhibited by chloroquine, which inhibits insulin degradation in fibroblasts, hepatocytes, and adipocytes, nor by phenylarsine oxide, which inhibits endocytosis. Bacitracin inhibited insulin binding to the cell monolayers and inhibited insulin degradation with identical IC50 values (80 μM). These data suggest that in BAE cells, insulin degradation occurs in the absence of receptor‐mediated endocytosis and is mediated by binding of insulin to its receptor. Therefore, it is concluded that the functional role of the insulin receptor expressed in BAE cells is to bind blood‐borne insulin at the plasma membrane of the cell and thereby facilitate the degradation of insulin at the BAE cell plasma membrane. © 1993 Wiley‐Liss, Inc.
Differential regulation of actin polymerization following activation of resting T lymphocytes from young and aged mice
Authors: Brock Mary Anne , Chrest Francis 
Published: February 2005

AbstractActin polymerization accompan es receptor‐mediated responses and is correlated with motility‐related events. In T lymphocytes, there is a lateral redistribution of surface receptors into caps and aggregation of actin‐myosin in cytoplasmic subcaps, and these are impaired in T cells from aged individuals. This study documents marked changes in age‐related cytoskeletal actin filament function which may account for the reduced motility. Basal levels of filamentous actin (F‐actin) are significantly higher in purified G0 T cells from aged C57BL/6 mice, due to a preferential increase in the CD8+ subpopulation. Following activation of the resting T cells with Concanavalin A (Con A), F‐actin depolymerized in cells from young mice for 2 min, followed by rapid polymerization, reaching a plateau 200% above resting levels. In cells from 15–17‐month‐old mice, an attenuated depolymerization phase was seen for 45 sec, followed by little polymerization. No depolymerization or polymerization phases occurred in cells from aged mice. Phorbol 12 myristate 13‐acetate (PMA), which activates protein kinase C (PKC), bypassing receptor mediated signals, induced actin polymerization to 57% of the levels of that after Con A stimulation in cells from both young and old animals and partially eliminated the differences in actin filament assembly due to age. Perturbation of the cytoskeleton with cytochalasin E (CE) potentiated proliferation of Con A–stimulated T cells from aged mice but did not completely restore the deficit attributed to immunosenescence. The results show an age‐related impairment of cytoskeletal functions and suggest that differences in early signal transduction events contribute to the decrements in surface receptor motility and subsequent proliferation of T lymphocytes from older individuals. © 1993 Wiley‐Liss, Inc.
Induction of collagenase and stromelysin gene expression by mechanical injury in a vascular smooth muscle‐derived cell line
Authors: James Timothy W. , Wagner Robert , White Lori A. , Zwolak Robert M. , Brinckerhoff Constance E. 
Published: February 2005

AbstractWe describe a novel system for studying the production of matrix metalloproteinases types I and Ill (collagenase and stromelysin) by a vascular smooth muscle cell line (Rb‐1 cells) in response to mechanical injury. Highly confluent Rb‐1 cells are disrupted by passing a plastic comb around the plate to clear a series of circumferential paths, which are bordered by two ridges of displaced cells. Over the next 24 hours, cells migrate into the cleared areas. Northern blot analysis demonstrates the accumulation of mRNAs for collagenase and stromelysin during this process, although they are undetectable prior to injury. Cotreatment with all‐trans‐retinoic acid (10−6 M) markedly decreases the level of mRNAs induced by injury, whereas dexamethasone (10−7 M) causes only a slight reduction. In situ hybridization studies for stromelysin mRNA and immunohistochemical staining for collagenase protein on plates of injured cells showed the highest levels of stromelysin mRNA in cells in the ridges left by the injury; lower levels were observed in some cells migrating into the clear region. The same pattern of expression was observed when cells were stained with antiserum to collagenase protein. Nuclear run‐on assays demonstrated increases in transcription of stromelysin and collagenase genes following injury. Transient transfection of cells with a vector containing the luciferase gene driven by a wild‐type promoter comprising 1.8 kb of the 5′ flanking region of the rabbit collagenase gene showed increased activity associated with injury. We conclude that: (1) mechanical injury is associated with induction of mRNAs for the metalloproteinases collagenase and stromelysin, (2) retinoic acid effectively antagonizes this responses, and (3) the increase in steady state mRNA levels is, at least in part, transcriptionally mediated. Thus our data suggest a role for mechanical forces in initiating the changes in gene expression in vascular smooth muscle cells following arterial injury in vivo. © 1993 Wiley‐Liss, Inc.