bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells
Authors: Opas Michal , Dziak Ewa , Fliegel Larry , Michalak Marek 
Published: February 2005

AbstractIn the present study we have demonstrated the presence of calreticulin, a major Ca2+‐sequestering protein of nonmuscle cells, in a variety of cell types in tissue culture. The protein localizes to the endoplasmic reticulum in most cell types and also to the nuclear envelope or nucleoli‐like structures in some cell types. Calreticulin is enriched in the rough endoplasmic reticulum, suggesting a possible involvement in protein synthesis. Calreticulin terminates with the KDEL‐COOH sequence, which is likely responsible for its endoplasmic reticulum localization. Unlike some other KDEL proteins, calreticulin expression is neither heat‐shock nor Ca2+‐shock dependent. Using a variety of metabolic inhibitors, we have shown that the pool of calreticulin in L6 cells has a relatively slow turnover and a stable intracellular distribution. In proliferating muscle cells in culture (both L6 and human skeletal muscle) calreticulin is present in the endoplasmic reticulum, and additional intranuclear staining is observed. When fusion of the L6 cells is inhibited with either a high serum concentration or TGF‐β TPA, the nucleolar staining by anticalreticulin antibodies is diminished, although the presence of calreticulin in the endoplasmic reticulum remains unchanged. In contrast, in differentiated (i.e., fused) muscle cells neither intranuclear nor intracellular staining for calreticulin is present. We conclude, therefore, that calreticulin is abundant in the endoplasmic reticulum in proliferating myoblasts, while it is present in only small amounts in sarcoplasmic reticulum membranes in terminally differentiated myotubes. We propose a model for the domain structure of calreticulin that may explain the differential subcellular distribution of this protein. Because of its widespread distribution in nonmuscle tissues, we postulate that calreticulin is a multifunctional protein that plays an important role in Ca2+ sequestering and thus that it is the nonmuscle analog of calsequestrin.
Time course of IL1 and IL6 synthesis and release in human bronchial epithelial cell cultures exposed to toluene diisocyanate
Authors: Mattoli S. , Colotta F. , Fincato G. , Mezzetti M. , Mantovani A. , Patalano F. , Fasoli A. 
Published: February 2005

AbstractWe have previously demonstrated that human bronchial epithelial cells release appreciable amounts of interleukin 1 (IL1) and interleukin 6 (IL6) when exposed to toluene diisocyanate (TDI) in vitro. TDI is an inflammatory and asthmogenic stimulus presumed to act at least in part through immunological mechanisms. The epithelial cell‐derived IL1 and IL6 can promote T cell activation and proliferation in culture, and if this also happens in vivo they may contribute to the persistence of the inflammatory response of the bronchial mucosa observed in TDI‐sensitive asthmatics. In this study, we confirmed the release of biologically active IL1β and IL6‐like substances from bronchial epithelial cells exposed to isocyanates in vitro, and related the rate and the magnitude of the cytokine secretion with the pattern of IL1β and IL6 gene expression and the extent of epithelial cell injury. In the epithelial cell cultures exposed to TDI, there was a parallel, progressive increase in the expression of IL6 mRNA and in the secretion of IL6 protein between 48 hours and 6 days after exposure. By contrast, although increasing amounts of biologically active IL1β were detected in the supernatants of TDI‐exposed epithelial cells throughout the 6‐day period following exposure, augmented levels of IL1β mRNA were only evident 6 days after exposure, suggesting that TDI exposure might have initially affected the enzymatic cleavage of the intracellular IL1β precursor and the mechanisms which regulate the secretion of mature IL1β.
Localization of cellular transglutaminase on the extracellular matrix after wounding: Characteristics of the matrix bound enzyme
Authors: Upchurch Herbert F. , Conway Eugene , Patterson M. K. , Maxwell Merle D. 
Published: February 2005

AbstractExtending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI‐38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4°C or 37°C. Prior incubation of exogenous TGase with guanosine 5′‐triphosphate (GTP), guanosine 5′‐diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2‐to 3‐fold. The Ca++‐dependent change was a concentration‐dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM‐bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.
The signaling potential of the receptors for insulin and insulin‐like growth factor I (IGF‐I) in 3t3‐l1 adipocytes: Comparison of glucose transport activity, induction of oncogene c‐fos, glucose transporter mRNA, and DNA‐synthesis
Authors: Weiland M. , Bahr F. , Höuhne M. , Schürmann A. , Ziehm D. , Joost H. G. 
Published: February 2005

AbstractThe receptors for insulin and insulin‐like growth factor I (IGF‐I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF‐I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA‐synthesis, and expression of proto‐oncogene c‐fos in 3T3‐L1 adipoytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, K1 of IGF‐I > 500 nM; IGF‐I receptor, KD = 1.1 nM, K1 of insulin = 191 nM). Induction of proto‐oncogene c‐fos mRNA by insulin and IGF‐I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF‐I, 3.9 nM). Similarly, both insulin and IGF‐I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF‐I, 4.0 nM), and glucose transport activity (EC50 of insulin, 1.7 nM; IGF‐I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3‐L1 cells the α‐subunits of insulin and IGF‐I receptors have an equal potential to stimulate a metabolic and a mitogenic response.
Inhibition of human erythroid colony‐forming units by interleukin‐1 is mediated by gamma interferon
Authors: Means Robert T. , Dessypris Emmanuel N. , Krantz Sanford B. 
Published: February 2005

AbstractIL‐1 inhibits erythropoiesis in vivo and in vitro. This inhibition was studied by comparing the effect of recombinant human IL‐1 (rhIL‐1) on highly purified CFU‐erythroid (E) generated from peripheral blood burst‐forming units‐erythroid (BFU‐E) (mean purity 44.4%) with its effect on unpurified marrow CFU‐E (mean purity 0.36%). Colony formation by marrow CFU‐E was significantly inhibited by rhIL‐1, while colony formation by highly purified CFU‐E was not inhibited. However, purified CFU‐E colonies were inhibited by rhIL‐1 in the presence of autologous T‐lymphocytes, and also by cell‐free conditioned medium prepared from T‐lymphocytes stimulated by rhIL‐1. This inhibitory effect was ablated by neutralizing antibodies to γinterferon (IFN), but not by antibodies to human IL‐1, tumor necrosis factor, or βIFN. Colony formation by highly purified CFU‐E was also inhibited by recombinant human γIFN (rhγIFN). IL‐1 and γIFN play significant roles in the pathogenesis of the anemia of chronic disease. These studies indicate that rhIL‐1 inhibits CFU‐E colony formation by an indirect mechanism involving T‐lymphocytes and requiring γIFN and that γIFN itself is most probably the direct mediator of this effect.
Evidence for receptor‐mediated calcium entry and refilling of intracellular calcium stores in FRTL‐5 rat thyroid cells
Authors: Törnquist Kid 
Published: February 2005

AbstractThe aim of the present study was to investigate the relationship between agonist‐induced changes in intracellular free Ca2+ ([Ca2+]j) and the refilling of intracellular Ca2+ stores in Fura 2–loaded thyroid FRTL‐5 cells. Stimulating the cells with ATP induced a dose‐dependent increase in ([Ca2+]j). The ATP‐induced increase in [Ca2+]j was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP‐induced increase in [Ca2+]j dependent on influx of Ca2+. In cells stimulated with ATP in a Ca2+‐free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]j; this increase was inhibited by Ni2+. In addition, the ATP‐induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]j. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]j was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL‐5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.
Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells
Authors: Meskini Nadia , Hosni Mohammed , Nemoz Georges , Lagarde Michel , Prigent Annie‐France 
Published: February 2005

AbstractCyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A‐activated mononuclear cells into lymphocyte‐enriched and monocyte‐enriched populations showed that the Con A‐induced increase of phosphodiesterase activity exclusively affected the lymphocyte‐enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A ( + 275%) than cyclic AMP phosphodiesterase activity (+ 75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 μg per 106 cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP‐inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP‐specific, Rolipramsensitive one. The non‐mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti‐CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.
SV40‐immortalization of rabbit articular chondrocytes: Alteration of differentiated functions
Authors: Thenet S. , Benya P. D. , Demignot S. , Feunteun J. , Adolphe M. 
Published: February 2005

AbstractCell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue–stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that proα1 (II) mRNA was undetectable, whereas proα1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re‐express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.
Short‐term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29‐D4 cells
Authors: Baghdiguian Stephen , Verrier Bernard , Marvaldi Jacques , Fantini Jacques 
Published: February 2005

AbstractSuramin is an anti‐cancer drug which induces the differentiation of the human colon cancer clone HT29‐D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29‐D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin‐free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29‐D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post‐differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29‐D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation.
Quantitative dissociation between EGF effects on c‐myc and c‐fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity
Authors: Hauguel‐Demouzon Sylvie , Csermely Peter , Zoppini Giacomo , Kahn C. Ronald 
Published: February 2005

AbstractThe exact relationship between EGF‐stimulated tyrosine phosphorylation, induction of the cellular proto‐oncogenes c‐myc and c‐fos, and DNA synthesis remains uncertain. Madin‐Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was −0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time‐dependent activation of c‐fos and c‐myc gene expression. However, the ED50 for these processes was ã10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto‐oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto‐oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose‐response studies, indicate that proto‐oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c‐fos and c‐myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF‐stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate‐limiting intermediate steps.
Epidermal growth factor and transforming growth factor‐alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM‐1) on cultured keratinocytes
Authors: Mitra Raj S. , Nickoloff Brian J. 
Published: February 2005

AbstractThe link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN‐γ). We studied the influence of keratinocyte mitogens such as transforming growth factor‐alpha (TGF‐α), epidermal growth factor (EGF), and somatomedin‐C (SM‐C) on the ligand binding of 32P‐labelled IFN‐γ to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN‐γ receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF‐α (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN‐γ receptor sites by TGF‐α/EGF was mitogen specific, as adding SM‐C (500 ng/ml) did not have any effect on ligand binding, although it similarily stimulated keratinocyte growth. The reduction in IFN‐γ receptors was time dependent, occurring primarily after 24–48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN‐γ receptors by TGF‐α/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA‐DR and intercellular adhesion molecule‐1 (ICAM‐1) induced by IFN‐γ, compared to actively growing TGF‐α/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF‐α/EGF but not SM‐C are capable of altering their response to IFN‐γ by decreasing their number of cell surface high affinity receptors for IFN‐γ.
Polarity in the transcytotic processing of apical and basal membrane‐bound peroxidase‐polylysine conjugates in MDCK cells
Authors: Taub Mitchell E. , Shen Wei‐Chiang 
Published: February 2005

AbstractA conjugate of horseradish peroxidase (HRP) to poly(L‐lysine) (PLL) was used to characterize a non‐lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal‐to‐apical, but not apical‐to‐basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag‐time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 μM) failed to inhibit this process, indicating that participation of the trans‐Golgi network (TGN) in the trafficking of internalized conjugate is not the rate‐determining step. Inhibition of HRP transport was found to be elicited by 50 μg/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface‐bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal‐to‐apical, but not apical‐to‐basal, transport pathway; and this compartment can be exploited for the transcytosis of membrane‐bound molecules.