bluuebunny/crossref_metadata_2025 huggingface dataset:
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

In 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

Cycloheximide 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

The 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

We 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

Bovine 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

The 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

Actin 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

We 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.
Novel marine alkaloids from the tunicate Eudistoma sp. are potent regulators of cellular growth and differentiation and affect cAMP‐mediated processes
Authors: Shochet Nava R. , Rudi Amira , Kashman Yoel , Hod Yaacov , El‐Maghrabi M. Raafat , Spector Ilan
Published: February 2005

Six novel alkaloids that contain a fused tetracyclic pyrido[2,3,4‐kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cels flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virustransformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well‐characterized cAMP‐mediated processes involved in hepatic glucose metabolism–inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and “reverse transformation”. Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that the Eudistoma alkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems. © 1993 Wiley‐Liss, Inc.
Expression of a squamous cell marker, the spr1 gene, is posttranscriptionally down‐regulated by retinol in airway epithelium
Authors: An Gang , Tesfaigzi Johannes , Carlson Don M. , Wu Reen
Published: February 2005

Vitamin A (retinol) is required for the normal mucociliary differentiation of respiratory epithelium. A depletion of vitamin A promotes squamous cell metaplasia. To understand how vitamin A suppresses squamous cell differentiation, the expression of a squamous cell differentiation marker, the small proline‐rich protein gene (spr1), was studied in cultured monkey tracheobronchial epithelial (TBE) cells. The expression of the spr1 gene was inhibited about 40 fold by retinol. The mRNA levels of the spr1 gene started to decline within 6 h of retinol treatment and reached a minimum level after 7 days. The inhibition by retinol was concentration dependent and did not require concurrent protein synthesis. The inhibition of the spr1 mRNA by retinol was not due to a decrease in the transcription rate of its gene but due to a decrease in its stability, as determined by nuclear run‐on assays and mRNA half‐life measurement, respectively. This result was further supported by a DNA transfection study using a chimeric construct containing the spr1 promoter region and the chloramphenicol acetyltransferase (CAT) reporter gene. The CAT activity in transfected cells was not inhibited by retinol. These results suggest that spr1 gene expression is posttranscriptionally down‐regulated by retinol. © 1993 Wiley‐Liss, Inc.
Oxygen tension regulates neutrophil adhesion to human endothelial cells via an LFA‐1‐dependent mechanism
Authors: Ginis Irene , Mentzer Steven J. , Faller Douglas V.
Published: February 2005

Extravasation of leukocytes at the sites of ischemia‐reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO2 of 50 torr for 3 hours, increased 2.5‐fold, 2‐, and 1.5‐fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell‐associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia‐induced adhesion was inhibited by LFA‐1‐specific (CD 11 a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM‐1 ligand for the LFA‐1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA‐1 molecule on edothelial cells which can be affected by hypoxic environments.
Lipophilic impurity of phenol red is a potent cation transport modulator
Authors: Hopp Laszlo , Bunker Clareann H.
Published: February 2005

Previously, we described substantial alterations in the Na+ and K+ homeostasis of human skin fibroblasts following removal of fetal bovine serum (FBS). Herein, we report that FBS removal per se does not cause any cellular ionic changes unless a lipophilic impurity of commercial phenol red preparations is present. This substance accelerates 86Rb+ efflux four to seven times, causes a four to eight time increase in cellular Na+, and a 40–70% reduction in cellular K+ contents. FBS (10%) or albumin (0.8%) appears to bind the impurity thus inhibiting its action. The increased cellular Na+ and decreased K+ contents do not return to baseline within 4 hours following the removal of the phenol red extract. However, albumin completely reverses the cellular cationic changes that develop during a 2 hour exposure of the cells to the free substance. The reversibility of its action by albumin suggests that the substance exerts its effect on or within the cell membrane and not intracellularly. Among seven different cell lines tested the 86Rb+ efflux from, and the Na+‐K+ contents of, COS‐7 and Hs68 cells also responded to unpurified phenol red in a way similar to human fibroblasts. The amount of the phenol red contaminant is manufacturer dependent. As little as 0.5 μM phenol red, from one vendor, was sufficient to elicit response in the 86Rb+ efflux. Given that the impurity is unlikely to be more than a small fraction of phenol red, it seems to be a potent ionic transport modulator. Based on these results, the presence of commercial phenol red in serum‐free growth or test media, including the increasing variety of chemically defined culture media, should be considered as a potential confounding factor in measurements that depend on intracellular Na+‐K+ homeostasis. The findings of such earlier studies may need to be reconsidered if the cells were exposed to unbound phenol red. We recommend that, until the manufacturers further refine their product, phenol red be purified by ether extraction before its use. The evaluation of the potential physiologic or pharmacologic relevance of this potent cation transport modulator awaits its isolation. © 1993 Wiley‐Liss, Inc.
Effect of stretch on growth and collagen synthesis in cultured rat and lamb pulmonary arterial smooth muscle cells
Authors: Kulik Thomas J. , Alvarado Stephen P.
Published: February 2005

There are no studies of the effect of stretch in cultured pulmonary vascular smooth muscle, and some data suggest that a stretch‐mediated increase in connective tissue synthesis in pulmonary arteries is mediated by the endothelium. To investigate whether stretch can serve as a growth stimulus in this smooth muscle, we studied two types of cultured pulmonary arterial smooth muscle cells (a multiply passaged clonal line of rat cells [PAC1], and early passage lamb cells [EPTC]). Cells were grown on a collagen‐coated silicone surface and subjected to repetitive stretch (0.33–0.5 Hz; 10–20% strain). The relative rates of total RNA, DNA, protein, and soluble collagen synthesis were determined using 3H precursors, and c‐fos and collagen mRNAs by Northern blot analysis. Stretch caused no significant change in the rate of RNA synthesis in either PAC1 cells (+9%) or EPTC (—3%). The relative rate of total protein synthesis was decreased by stretch (6% in PAC1 cells and 36% in EPTC [both NS]) as was the rate of collagen synthesis (−24% in EPTC [NS]). In EPTC, the percentage of 3H‐thymidine labeled cells was modestly increased with 24 h stretch (17 ± 5.7%; P ≤.001), but trichloroacetic acid (TCA) precipitated 3H‐thymidine was unaltered by stretch, and the number of cells not significantly changed with stretch. c‐fos mRNA expression was only inconsistently induced by stretch x30 min in EPTC, and not at all in PAC1 cells. Expression of mRNA for α1 (I) and α1 (III) collagen was not changed significantly by 24 h or 48 h of stretch. We conclude that stretch does not serve as a significant growth stimulus in cultured pulmonary vascular smooth muscle cells in this system. These findings do not rule out the possibility that stretch is a growth stimulus for these cells under different conditions, but do suggest that other models will be needed to determine if and how mechanical stimuli affect growth of pulmonary vascular smooth muscle. © 1993 Wiley‐Liss, Inc.
Iron absorption by CaCo 2 cells cultivated in serum‐free medium as in vitro model of the human intestinal epithelial barrier
Authors: Halleux Christine , Schneider Yves‐Jacques
Published: February 2005

A cell culture system consisting of confluent monolayer of human enterocyte‐like CaCo 2 cells, cultivated in a serum‐free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of 55ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time‐ and concentration‐dependent process, largely specific from the apical pole. When 55ferric citrate is added at the apical pole, radioiron appears at the basal pole and the clearance rate is ∼four times higher than in the opposite direction; the amounts of 55Fe increase with the concentration in iron citrate and the duration of incubation. At least two concurrent mechanisms could be involved in iron absorption across monolayers of CaCo 2 cells. A first route would correspond to a paracellular passage of the metal from the apical to the basal pole. The second route would involve a selective intake of iron at the apical pole and could require a reduction of ferric iron, prior to the entry. Iron accumulated by the cells would, for a minor part, be stored within ferritin, whereas the major part would be excreted at the basolateral pole, either as low molecular weight material of undetermined chemical composition but from which iron is easily mobilized by apotransferrin or associated with neosynthesized apotransferrin. Vesicular transport and protein synthesis seem to be required. © 1994 Wiley‐Liss, Inc.
Abnormalities of phosphoprotein gene expression in three osteopetrotic rat mutations: Elevated mrna transcripts, protein synthesis, and accumulation in bone of mutant animals
Authors: Shalhoub V. , Bettencourt B. , Jackson M. E. , Mackay C. A. , Glimcher M. J. , Marks S. C. , Stein G. S. , Lian J. B.
Published: February 2005

Osteoclast abnormalities that characterize osteopetrosis, a disorder of bone resorption, may derive from aberrant signals from the osteoblast or the bone matrix. In the present studies, both synthesis and the bone matrix content of the major bone phosphoprotein component, osteopontin, were found to be elevated in three osteopetrotic rat mutations (ia, op, and tl). In whole bone, a twofold increase in the content of the characteristic amino acid O‐phosphoserine for osteopontin occurred in op and tl mutant long bone, but a smaller (15%) and more variable increase was observed in ia mutant rat long bone. Extraction of the bone matrix components and partial purification by reverse phase chromatography showed a twofold increase in a phosphoprotein fraction relative to other noncollagenous components. Amino acid analysis and staining characteristics of SDS‐PAGE fractionated proteins indicated this to be osteopontin. Organ cultures of calvarial bone from 4 day ia osteopetrotic mutant and normal rats in the presence of 3H‐proline showed increased synthesis of this 60 kD protein, which was stimulated by vitamin D. Preparation of total cellular RNA from bone of 2‐ and 6‐weekold mutants and normal rats supported increased synthesis of osteopontin as reflected by hybridization with osteopontin cDNA probe, showing significantly higher levels of mRNA transcripts in ia (3–5 fold), tl (1.4–2 fold), and op (6–25 fold) mutant bone compared to normal littermates. The changes in osteopontin mRNA levels in mutant bone were also examined in relation to other growth and phenotype‐expressed genes. The findings of increased accumulation of osteopontin in osteopetrotic bone and increased synthesis by osteoblasts are interesting in light of the previously reported decrease in bone osteocalcin content (Endocrinology, 126:966, 1990), confirmed here by decreased osteocalcin mRNA transcripts. Such aberrations in the composition of skeletal extracellular matrix could be a reflection of or a contributing factor to the osteoclast abnormalities of some of these osteopetrotic disorders. © 1994 Wiley‐Liss, Inc.
Transient mitochondrial transcript level decay in oxidative stressed chondrocytes
Authors: Vincent Françoise , Corral‐Debrinski Marisol , Adolphe Monique
Published: February 2005

Steady‐state levels of 12S rRNA and NADH dehydrogenase subunit 4 mRNA (ND4) mitochondrial transcripts were measured on rabbit articular chondrocyte in culture. In pseudosynchronized chondrocytes, changes of mitochondrial RNA levels were observed during the progression of the cells in the cell cycle. Oxidative stress generated by the hypoxanthine‐xanthine oxidase system (HX‐XO) induced a transient decrease in the levels of both ND4 and 12S rRNA. Mitochondrial RNA levels recovered 24 h after the oxidative stress. These RNA level changes were not associated with modifications in the structure or the copy number of the mitochondrial genome. Furthermore, the decrease in the amount of the mitochondrial transcripts observed may be related to a transient inhibition of mitochondrial transcription since the treatment of cells with ethidium bromide (a mitochondrial transcription inhibitor) resulted in the same decrease in 12S rRNA level as HX‐XO treatment alone. © 1994 Wiley‐Liss, Inc.
Epitope mapping and functional studies with three monoclonal antibodies to the C‐KIT receptor tyrosine kinase, YB5.B8, 17F11, and SR‐1
Authors: Ashman Leonie K. , Bühring Hans‐Jörg , Aylett Gabriella W. , Broudy Virginia C. , Müller Claudia
Published: February 2005

Three monoclonal antibodies (MAbs) to the human c‐kit receptor tyrosine kinase (P145c‐kit), derived in independent laboratories, have been extensively used in studies of c‐kit expression and the role of its ligand, steel factor (SLF), in hemopoiesis and mast cell differentiation and function. In this study, the relationship between the epitopes they identify, and their effects on SLF binding, receptor internalization, and signal transduction are compared.

Epitope mapping studies carried out on the high P145c‐kit‐expressing cell line HEL‐DR showed that SR‐1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacting epitopes. SR‐1 potently blocked the binding of SLF to P145c‐kit on these cells and also on cells of the factor‐dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conversely, SLF partially blocked the binding of SR‐1 and YB5.B8 to cells, while binding of 17F11 was actually enhanced by SLF on some target cells. Preincubation of HEL‐DR and MO7e cells with MAbs prior to exposure to SLF revealed that 17F11 itself brought about partial down‐regulation of P145c‐kit and did not inhibit SLF‐mediated down‐regulation. SR‐1 caused minimal down‐regulation and inhibited SLF‐mediated receptor internalization. YB5.B8 had minimal effects on either cell line in this assay. To determine whether the antibodies had any agonist activity, they were compared with SLF for their ability to bring about receptor phosphorylation in intact MO7e cells. All three antibodies induced detectable tyrosine phosphorylation with 17F11 being the most effective, while YB5.B8 was the least effective. Finally, the ability of the antibodies to influence the proliferation of the MO7e cells was examined. As expected, SR‐1 potently inhibited the proliferative response to SLF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but reproducible agonist activity. © 1994 Wiley‐Liss, Inc.
Transforming growth factor β1 augments mitogen‐induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene both in swiss 3T3 cells and in murine embryo fibroblasts
Authors: Gilbert Rebecca S. , Reddy Srinivasa T. , Kujubu Dean A. , Xie Weilin , Luner Steven , Herschman Harvey R.
Published: February 2005

Transforming growth factor‐beta (TGF‐beta), a potent cytokine, modulates a wide variety of biological responses. Among its actions, TGF‐beta can augment prostaglandin synthesis in several cell types. Although TGF‐beta alone has no effect on prostaglandin production in Swiss 3T3 cells, we find that TGF‐beta augments the ability of tetradecanoyl phorbol acetate (TPA) or serum to stimulate PGE2 production. The TIS10 gene is a primary response gene encoding a second form of prostaglandin synthase (PGS), the rate‐limiting enzyme in the biosynthesis of prostaglandins, thrornboxanes, and prostacyclins from arachidonic acid. TIS10/PGS‐2 expression is induced by mitogens in Swiss 3T3 cells. TGF‐beta also augments mitogen‐induced synthesis and accumulation of TIS10/PGS‐2 protein and induction of TIS10/PGS‐2 message in Swiss 3T3 cells. In contrast, TGF‐beta has little or no effect on the level of PGS‐1 (EC1.14.99.1) message, either alone or in concert with TPA or serum. TGF‐beta concentrations in the range of 0.01–0.10 ng/ml (0.4–4.0 pM) maximally enhance mitogen induction of TIS10/PGS‐2 message. TPA‐induced accumulation of unspliced TIS10/PGS‐2 transcript is augmented by TGF‐beta, suggesting that this cytokine exerts its effect on expression of the TIS10/PGS‐2 gene by transcriptional regulation. TGF‐beta also augments TPA‐induced prostaglandin production, TIS10/PGS‐2 antigen accumulation, and TIS10/PGS‐2 message induction in primary cultures of mouse embryo fibroblasts. Dexamethasone attenuates TGF‐beta enhancement of all these mitogen‐induced responses: PGE2 accumulation, appearance of TIS10/PGS‐2 protein and message, and accumulation of TIS10/PGS‐2 unprocessed transcript. © 1994 wiley‐Liss, Inc.
Amphiregulin‐dependent proliferation of cultured human keratinocytes: Autocrine growth, the effects of exogenous recombinant cytokine, and apparent requirement for heparin‐like glycosaminoglycans
Authors: Piepkorn Michael , Lo Christine , Plowman Greg
Published: February 2005

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum‐free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin‐like glycosaminoglycans in amphiregulin‐dependent growth. Keratinocytes plated at >103 cells/cm2 grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin‐blocking antibody indicated that ∼70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above ∼1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor‐dependent proliferation by 42%, exogenous amphiregulin‐dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1‐100 μg/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure‐function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin‐like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin‐like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding. © 1994 wiley‐Liss, Inc.
Endothelin‐1 acts as an autocrine growth factor for normal human keratinocytes
Authors: Tsuboi Ryoji , Sato Chiyo , Shi Chong‐Ming , Nakamura Tsukasa , Sakurai Takeshi , Ogawa Hideoki
Published: February 2005

Endothelin‐1 (ET‐1) is an endothelium‐derived 21 amino acid vasoconstrictor peptide possessing two intrachain disulfide bridges. Recently it has become evident that isoforms of ET (ET‐1, ‐2, and ‐3) have a wide range of pharmacological effects in various tissues and act as autocrine/paracrine factors. We demonstrate here that ET‐1 is secreted from normal human keratinocytes and may work as an autocrine growth factor through a specific receptor. In this study, human foreskin keratinocytes were cultured in serum‐free MCDB 153 medium. Cell growth and [3H] thymidine incorporation in low and high Ca++ concentration media was stimulated by ET‐1, ‐2, and ‐3 with similar potencies. The strongest response was observed at 10 nM ETs, whereas stimulatory activity was reduced at 100 nM. ETs suppressed keratinocyte differentiation as measured by reactivity with involucrin antibody. Plasminogen activator activity (mainly urokinase) in the medium was also stimulated by the addition of 10 nM ETs. ET‐1–like immunoreactivity measured by radioimmunoassay was 1.4 fmol/day/106 cells in non‐treated condition medium. Among the various cytokines, tumor necrosis factor–α (TNF‐α), interleukin‐1α, and transforming growth factor–β stimulated ET‐1 secretion in a dose‐dependent manner. The strongest response (ten‐fold) was observed upon the addition of 10 ng/ml TNF‐α. Scatchard plot analysis of [125I] ET‐1 binding to keratinocytes revealed the presence of a single class of high affinity receptors (KD 50 pM, 9 x 103 sites/cell). Binding was competitively inhibited by the addition of unlabeled ET‐1 and ‐2 with similar affinities and by ET‐3 with weaker affinity. ET‐1 mRNA expression in keratinocytes was detected by reverse transcription‐polymerase chain reaction and was increased by treatment with 10 ng/ml TNF‐α. These results suggest that ET‐1 acts as an autocrine growth factor for keratinocytes through a specific receptor. © 1994 wiley‐Liss, Inc.
Retinoic acid enhances adhesiveness, laminin and integrin β1 synthesis, and retinoic acid receptor expression in F9 teratocarcinoma cells
Authors: Ross Sharon A. , Ahrens Richard A. , De Luca Luigi M.
Published: February 2005

The teratocarcinoma‐derived F9 cells respond to retinoic acid (RA) and RA plus dibutyrylcyclic adenosine monophosphate (dcAMP) by differentiating into endoderm cells, which elaborate a laminin and type IV collagen‐rich matrix. We found that the induction of differentiation is accompanied by a small but consistent increase in cell adhesiveness to a variety of substrates, including laminin. Therefore we investigated biochemical mechanisms involved in this phenomenon. Endoglycosidase treatment showed that laminin contains complex and hybrid oligosaccharide structures. RA enhanced general biosynthesis of laminin without a specific increase in galactose incorporation: this sugar was mainly in polylactosamine structures in the A chain of laminin and as terminal galactose α 1,3 galactose in the B chain. Laminin receptor analysis showed that RA decreased laminin binding protein‐37 (LBP‐37) but increased the amount of β1 integrin, suggesting the involvement of β1 integrin in the attachment process. Northern blot analysis showed increased expression of retinoid receptors within hours of RA exposure. These studies demonstrate that RA increases cell to substrate interactions by increasing the biosynthesis of laminin and β1 integrin. These effects are most likely subsequent to the RA‐induced biosynthesis of the retinoid receptors. © 1994 wiley‐Liss, Inc.
Increased expression of heme oxygenase‐1 in human retinal pigment epithelial cells by transforming growth factor‐β
Authors: Krishnan Kutty R. , Nagineni Chandrasekharam N. , Kutty Geetha , Hooks John J. , Chader Gerald J. , Wiggert Barbara
Published: February 2005

Antibodies specific for heme oxygenase‐1 (HO‐1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor‐β1 (TGF‐β1) to induce the HO‐1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO‐1 in these cells in a time‐ and dose‐dependent manner. TGF‐β1 also increased the mRNA for HO‐1 in treated cells prior to the increase in HO‐1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF‐β1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor‐I, plateletderived growth factor, insulin‐like growth factor, transforming growth factor‐α, and epidermal growth factor did not show appreciable induction of HO‐1. Lipopolysaccharide, tumor necrosis factor‐α, and interferon‐γ were also not inducers, although TGF‐β2 effectively induced HO‐1. Heavy metal ions and thiol reagents were also highly potent inducers of HO‐1 in human RPE cells. The induction of HO‐1 by TGF‐β1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO‐1 can be induced by an important cytokine, TGF‐β1, causing an increase in the expression of both HO‐1 message and protein in specific neuroepithelial and fibroblast cells. © 1994 wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Peripheral and central vascular smooth muscle cells from rat lung exhibit different cytoskeletal protein profiles but similar growth factor requirements
Authors: Davies Paul , Patton Wayne
Published: February 2005

In pulmonary vascular remodelling, the lining smooth muscle cells undergo various forms of growth involving cellular hypertrophy and hyperplasia. Differences in the growth pattern between central and peripheral regions suggested that cells from both should be obtained when investigating the cellular basis for the remodelling. Accordingly, we have obtained two smooth muscle cell types in culture: a cell from the central pulmonary artery (CC) and a cell morphologically similar to a pericyte (PC), from the periphery of the lung. Both cell types gave positive immunostaining for α‐smooth muscle isoactin. In vivo, the α‐isoactin was immunolocalized in the extracapillary vasculature. Quantitative two‐dimensional gel electrophoresis of cell extracts showed that PC express more vimentin and gelsolin than CC. Despite the differences between PC and CC in the expression of cytoskeletal proteins, their response to growth factors was similar. Both cell types increased DNA synthesis when stimulated by exogenous PDGF‐AB. This occurred in the absence of exogenous progression factors, but depended on a post‐competence, suramin‐sensitive mechanism that probably represents an autocrine progression factor. The cells were also stimulated by IGF‐1 alone, in the absence of exogenous competence factors. At an IGF‐1 concentration of 1 ng/ml, this response appeared specific for the IGF‐1 receptor and was sensitive to pretreatment with pertussis toxin, thus implicating a role for a G protein. © 1994 wiley‐Liss, Inc.
Critical role for intracellular calcium in tight junction biogenesis
Authors: Stuart Robert O. , Sun Adam , Panichas Michael , Hebert Steven C. , Brenner Barry M. , Nigam Sanjay K.
Published: February 2005

Using the Madin Darby canine kidney (MDCK) cell “calcium switch,” we have previously demonstrated that, as MDCK cells establish contact and ultimately form tight junctions, there are marked global and localized changes in intracellular calcium at the sites of cell‐cell contact (Nigam et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6162–6166). We have now examined whether intracellular Ca++ is critical to the biogenesis of tight junctions by chelating this ion and monitoring the formation of junctions by electrical, immunocytochemical, and biochemical criteria. Intracellular Ca++ was chelated with the cell‐permeant chelators, dimethyl‐BAPTA‐AM and BAPTA‐AM. By digital imaging of fura‐2 loaded cells, it was demonstrated that both agents efficiently chelated Ca++ during the “switch” in a dose‐dependent manner which paralleled their respective in vitro affinities for Ca++. Chelation of Ca++ during the switch markedly attenuated the development of transepithelial electrical resistance (TER), a measure of tight junction assembly. Immunofluorescent staining of the tight junctional protein, zonula occludens‐1 (ZO‐1), revealed that chelation of intracellular Ca++ retarded the movement of ZO‐1 from intracellular sites to the plasma membrane during the switch. During the development of tight junctions, a fraction of ZO‐1 redistributed from the Triton X‐100 soluble to the Triton X‐100 insoluble pool; chelation of Ca++ during the induction of cell–cell contact prevented this stabilization into the Triton X‐100 insoluble fraction. Taken together, these data indicate an important role for intracellular Ca++ in tight junction biogenesis and suggest a specific role for calcium in the early sorting and possible cytoskeletal association of tight junction components. © 1994 wiley‐Liss, Inc.
Responses of salivary acinar cells to intracellular alkalinization
Authors: Seagrave Jc. , Curry M. , Martinez J. R.
Published: February 2005

Responses of rat submandibular acini to intracellular alkalinization were investigated. Intracellular alkalinization was induced by addition of NH4Cl or methylamines, or by prepulse with Na butyrate. Only partial recovery occurred following Na butyrate prepulse or methylated amine addition, but full recovery was observed following addition of NH4Cl. The latter recovery was DIDS and dimethylamiloride‐insensitive but was inhibited by bumetanide or high [K+] and stimulated in Na+ free buffer and by ouabain. Acetylcholine stimulated recovery from NH4Cl‐ or Na butyrate pre‐pulse‐induced alkalinization and reduced the extent of alkalinization induced by methylated amines. Acetylcholine‐stimulated recovery from NH4Cl‐induced alkalinization was mimicked by substance P or ionomycin and was partially Ca2+‐dependent. This stimulated recovery was bumetanide‐insensitive but was partially sensitive to charybdotoxin. Taken together, these data indicate that in unstimulated cells, recovery from alkalinization induced by NH4Cl occurs by bumetanide‐sensitive transport of the NH4+ ion, that DIDS‐inhibitable anion transport contributes little to this recovery, and that acetylcholine and other Ca2+‐elevating agents accelerate recovery from NH4Cl‐induced alkaline challenge by a mechanism insensitive to bumetanide, DIDS, ouabain, and dimethylamiloride but sensitive to extracellular Ca2+ and to charybdotoxin. Partial recovery from alkaline challenge can also occur in the absence of NH4+ ions, and acetylcholine also stimulates this mode of recovery. Together, these data suggest that these cells have little intrinsic ability to recover from intracellular alkalinization and that the NH4+ ion may be a surrogate for K+ in at least two ion transport pathways. © 1994 wiley‐Liss, Inc.