bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uptake of biotin by human hepatoma cell line, Hep G2: A carrier‐mediated process similar to that of normal liver
Authors: Said Hamid M. , Ma Thomas Y. , Kamanna Vaijinath S. 
Published: February 2005

AbstractLittle is known about the cellular and molecular regulation of the uptake process of the water‐soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human‐derived liver cells, Hep G2. Uptake to biotin by Hep G2 cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na+ gradient‐dependent as indicated by studies of Na+ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of 3H‐biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G2 cells with sulfhydryl group inhibitors (e.g., p‐chloromer‐curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G2 cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na+ uptake in Hep G2 cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na+‐gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G2 cells for in‐depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley‐Liss, Inc.This article is a US Government work, and as such, is in the public domain in the United State of America
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In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: A juxtacrine model for migration and adhesion of osteoclast precursors
Authors: Formigli Lucia , Orlandini Sandra Zecchi , Benvenuti Susanna , Masi Laura , Pinto Antonio , Gattel Valter , Bernabei Pietro Antonio , Robey Pamela Gehron , Collin‐Osdoby Patricia , Brandi Maria Luisa 
Published: February 2005

AbstractThe role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone‐derived endothelial and preosteoclastic cells. Insulin‐like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell‐specific and mediated through the binding of vitronectin and fibronection receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny. © 1995 Wiley‐Liss, Inc.
Relationship between actions of transforming growth factor (TGF)‐β and cell surface expression of its receptors in clonal osteoblastic cells
Authors: Takeuchi Yasuhiro , Fukumoto Seiji , Matsumoto Toshio 
Published: February 2005

AbstractVarious osteoblastic cell lines were examined for the relationship between the presence of cell‐surface transforming growth factor (TGF)‐β receptors and the synthesis of matrix proteins with their responsiveness to TGF‐β. Treatment with TGF‐β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3‐E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF‐β1 markedly stimulated the synthesis of decorin in MC3T3‐E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF‐β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF‐β1 enhanced fibronectin synthesis. TGF‐β1 did not show any of these effects in UMR 106 cells. Receptor cross‐linking studies revealed that only MC3T3‐E1 and MG 63 cells had both types I and II signal‐transducing receptors for TGF‐β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3‐E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF‐β1 under these conditions. UMR 106 cells with no response to TGF‐β1 had neither of the signal‐transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF‐β1, that the responsiveness of osteoblastic cells to TGF‐β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF‐β1 on fibronectin synthesis can develop with little binding of TGF‐β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell‐surface receptors for TGF‐β determines the responsiveness of osteoblastic cells to TGF‐β and that changes in cell‐surface TGF‐β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley‐Liss, Inc.
Reciprocal induction of tumor necrosis factor‐α and interleukin‐β activity mediates fibronectin synthesis in coronary artery smooth muscle cells
Authors: Molossi Silvana , Clausell Nadine , Rabinovitch Marlene 
Published: February 2005

AbstractWe previously demonstrated an immune‐inflammatory response associated with increased expression of interleukin (IL)‐β and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL‐β was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co‐induction of IL‐β and tumor necrosis factor (TNF)‐α has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF‐α expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]‐methionine radiolabeling and gelatin‐sepharose extraction. As previously shown with IL‐β, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF‐α antibodies, and exogenous TNF‐α upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF‐α‐stimulated fibronectin production was downregulated to or below control levels in the presence of IL‐β antibodies. Likewise, IL‐β‐stimulated fibronectin synthesis was downregulated to control levels when TNF‐α neutralizing antibodies were added. Combining TNF‐α and IL‐β enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL‐β and TNF‐α activity and provide the first demonstration of a ‘cytokine loop’ modulating matrix production. © 1995 Wiley‐Liss, Inc.
Volume regulation in leukocytes: Requirement for an intact cytoskeleton
Authors: Downey Gregory P. , Grinstein Sergio , Sue‐A‐Quan Andrea , Czaban Barbara , Chan Chi Kin 
Published: February 2005

AbstractAnimal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL‐60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL‐60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F‐actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F‐actin content of the different cells was determined by NBD‐phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 ± 0.01 for CB and 0.48 ± 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL‐60 cells or human neutrophils. To study the effects of an increase in F‐actin on volume regulation, neutrophils were treated with the chemoattractant f‐Met‐Leu‐Phe or with an antibody (Ab) to β2 integrins followed by a cross‐linking secondary Ab. Despite an increase in F‐actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation. © 1995 Wiley‐Liss, Inc.
Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line
Authors: Delezay Olivier , Verrier Bernard , Mabrouk Kamel , van Rietschoten Jurphaas , Fantini Jacques , Mauchamp Jean , Gerard Corinne 
Published: February 2005

AbstractIn this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT‐29‐D4. Isotopic tracer flux measurements show that differentiated HT‐29‐D4 cells possess a sodium‐dependent α‐methyl‐D‐glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D‐glucose, D‐galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium‐dependent AMG transporter with a Km of 1.2 ± 0.12 mM and a Vmax of 3.24 ± 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 ± 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402‐Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT‐29‐D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT‐29‐D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference. © 1995 Wiley‐Liss, Inc.
Intraphagosomal oxygen in stimulated macrophages
Authors: James Philip E. , Grinberg Oleg Y. , Michaels George , Swartz Harold M. 
Published: February 2005

AbstractA new electron paramagnetic resonance (EPR)‐based method was developed to obtain selective information on pO2 in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen‐sensitive probe (4‐(Trimethylammonium) 2,2,6,6‐tetramethylpiperidine‐d17‐1‐oxyl iodide (d‐Cat1)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐d16‐1‐oxyl (15N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley‐Liss, Inc.
Mechanical stimulation of skeletal muscle increases prostaglandin F2α production, cyclooxygenase activity, and cell growth by a pertussis toxin sensitive mechanism
Authors: Vandenburgh Herman H. , Shansky Janet , Solerssi Rosa , Chromiak Joseph 
Published: February 2005

AbstractRepetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long‐term production of prostaglandin F2α, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P <.005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch‐activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's Km, with little change in its Vmax. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen‐inducible isoform of cyclooxygenase (PGHS‐2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch‐induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch‐induced cyclooxygenase activation. Pertussis toxin also inhibited stretch‐induced increases in PGF2α production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein‐dependent process involving the activation of cyclooxygenase, an immediate early gene product. © 1995 Wiley‐Liss, Inc.
Evidence that cell survival is controlled by interleukin‐3 independently of cell proliferation
Authors: Berridge Michael V. , Horsfield Julie A. , Tan An S. 
Published: February 2005

AbstractHemopoietic cell proliferation is controlled by a set of polypeptide growth factors and regulatory molecules that bind to cell surface receptors inducing cellular responses. Maintenance of a viable state, cell growth, DNA synthesis and mitosis are basic properties of proliferating cells, but links between growth factor receptors and each of these cellular outcomes are poorly understood. Most studies have monitored DNA synthesis as a measure of progression through the cell cycle or directly measured viable cell numbers, but cell survival per se as an output of receptor activation by ligand, has received little attention. In this study we have used a bone marrow‐derived murine cell line that is dependent on interleukin‐3 for growth, to investigate the relationship between DNA synthesis and a biochemical marker of cell survival, reduction of the tetrazolium salt, MTT. We show that at times up to 6 hr, continued DNA synthesis, RNA synthesis, protein synthesis, and mitochondrial respiration are not necessary for background or IL‐3‐stimulated MTT reduction. Furthermore, dibutyryl cyclic AMP promoted background and IL‐3‐dependent MTT reduction while simultaneously inhibiting DNA synthesis. These results provide evidence that IL‐3 controls events involved in MTT reduction and cell survival independently of DNA synthesis. © 1995 Wiley‐Liss, Inc.
De novo synthesis of glutathione is required for both entry into and progression through the cell cycle
Authors: Poot Martin , Teubert Heidi , Rabinovitch Peter S. , Kavanagh Terrance J. 
Published: February 2005

AbstractTo study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH‐3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose‐dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO‐induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit. © 1995 Wiley‐Liss, Inc.
Mitogenic activity of neu differentiation factor/heregulin mimics that of epidermal growth factor and insulin‐like growth factor‐I in human mammary epithelial cells
Authors: Ram Tracy G. , Kokeny Kristine E. , Dilts Cheryl A. , Ethier Stephen P. 
Published: February 2005

AbstractRecently, a family of growth factors has been described that activates erbB‐2 receptors. These factors, known as the neu differentiation factors (NDF) or heregulins (HRG), induce tyrosine phosphorylation of erbB‐2 receptors as a result of their direct interaction with either erbB‐3 or erbB‐4 receptors. Although it is known that expression of erbB‐2 receptors has relevance in human breast cancer progression, how erbB‐2, ‐3 and ‐4 receptors regulate mammary epithelial cell proliferation is not known. Therefore, experiments were carried out to study the mitogenic activity of NDF/HRG on the human mammary epithelial cell line MCF‐10A which can be cultured continuously under serum‐free conditions. MCF‐10A cells, like primary cultures of normal human mammary epithelial cells, express an absolute requirement for exogenous epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) for growth. The results of these experiments indicate that NDF/HRG can induce tyrosine phosphorylation of p185erbB‐2 in MCF‐10A cells and is mitogenic for these cells. This is consistent with the coexpression of erbB‐2 and erbB‐3 mRNA that we have observed in MCF‐10A cells. In addition, we found that NDF/HRG can substitute for either EGF or IGF‐I to stimulate proliferation of these cells. The ability to substitute for both EGF and IGF‐I is a unique property of NDF/HRG and is not shared by other members of the EGF or IGF family of growth factors, nor by other factors that we have studied. A striking isoform specificity was also observed which indicated that the β‐isoforms of NDF/HRG were greater than ten times more mitogenic than the α‐isoforms. We also examined the mitogenic activity of NDF/HRG on MCF‐10A cells that overexpress the erbB‐2 receptor as a result of infection with a retroviral vector containing the human c‐erbB‐2 gene (MCF‐10AerbB‐2 cells). These studies indicated that MCF‐10AerbB‐2 cells have increased sensitivity to the mitogenic effects of NDF/HRG and that these cells are responsive to the α‐isoforms of NDF/HRG at physiological concentrations. Thus, NDF/HRG is a dual specificity growth factor for human mammary epithelial cells, and the responsiveness of the cells to NDF/HRG is influenced by the level of expression of erbB‐2 receptors. © 1995 Wiley‐Liss, Inc.
Repression of tissue inhibitor of matrix metalloproteinase expression by all‐trans‐retinoic acid in rat bone cell populations: Comparison with transforming growth factor‐βT1
Authors: Overall Christopher M. 
Published: February 2005

AbstractRetinoids and transforming growth factor‐b̃1 (TGF‐b̃1) reduce the transcriptional activation of matrix metalloproteinases (MMPs) and increase the expression of the specific tissue inhibitor of MMPs (TIMP‐1) in fibroblasts. In contrast, all‐trans‐retinoic acid (retinoic acid) increases MMP expression in osteoblasts. Therefore, the mechanistic aspects of TIMP‐1 regulation by retinoic acid in primary cultures of rat calvarial bone cell populations were studied and compared with those of TGF‐b̃1 to determine if modulation of TIMP‐1 would augment MMP expression. Retionic acid was found to reduce TIMP‐1 mRNA levels after 24 and 72 hr of culture by up to 60% in a dose‐dependent manner. Maximal inhibition occurred at 10−6 M retinoic acid with half maximal repression at ∼5 × 10−8 M. To determine the half life of TIMP‐1 mRNA, the specific RNA polymerase II inhibitor DRB was added to cultures and the chase RNA analyzed by slot blots. TIMP‐1 mRNA had a half life of ∼14 hr and this was unaltered by retinoic acid treatment, suggesting that retinoic acid exerts its effects on TIMP‐1 transcriptionally. When retinoic acid was added to cycloheximide‐treated cultures TIMP‐1 mRNA levels were reduced at 5 hr compared with controls. This showed that ongoing protein synthesis was not required to mediate the retinoic acid repression of TIMP‐1 mRNA levels and supports the evidence that retinoic acid acts at the transcriptional level to reduce TIMP‐1 expression. In contrast, TGF‐b̃1 increased TIMP‐1 mRNA levels by 3.5‐fold at 24 hr to>10‐fold at 72 hr without alterations in mRNA stability indicating that transforming growth factor (TGF)‐b̃1 also acts at the transcriptional level to upregulate TIMP‐1 expression in bone cells. Thus, these studies have revealed that TIMP‐1 regulation by retinoic acid is different in osteoblasts from other cells and that retinoic acid has the property of generating resorptive and formative cell phenotypes in a tissue‐specific manner. In bone, reduced TIMP‐1 expression would favor bone matrix degradation and bone resorption that is a characteristic action of retinoids. © 1995 Wiley‐Liss, Inc.