bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin augments osteoclast resorption‐stimulating activity in serum
Authors: Fuller K. , Chambers T. J. , Gallagher A. C.
Published: February 2005

Increased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long‐term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet‐poor plasma‐derived serum, heparin, in the range 25–100 μg/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption‐stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor‐p, platelet‐derived growth factor, insulin‐like growth factors I or II, acidic or basic fibroblast growth factors, fibfonectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin‐binding factor. The ability of glycosaminoglycans (GACs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin‐2‐sulfate, chon‐droitin‐4‐sulfate, and chondroitin‐6‐sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor invoked in the locd and systemic regulation of osteoclastic bone resorption.
Extracellular calcium‐dependent regulation of transmembrane calcium fluxes in murine keratinocytes
Authors: Reiss Michael , Lipsey Lewis R. , Zhou Zhao‐Ling
Published: February 2005

Because the level of extracellular Ca2+ is an important stimulus for differentiation of epidermal cells in vitro, we characterized the extracellular Ca2+ ‐dependent transmembrane Ca2+ fluxes in BALB/MK mouse keratinocytes. Increasing levels of extracellular Ca2+ ranging from 0.07 to 1.87 mM, stimulated the rate of 45Ca2+ uptake into these cells 10‐ to 70‐fold and doubled the rate of 45Ca2+ efflux. The divalent cations, Ni2+ and Co2+, were able to block the influx of Ca2+, but dihydropyridines and verapamil were not. Furthermore, 10 to 100 μM of the trivalent cation La3+ induced a dose‐dependent 2‐ to 100‐fold increase of Ca2+ uptake, independently of the level of extracellular Ca2+. These observations suggest that keratinocytes possess a cell‐surface “Ca2+ ‐receptor,” activation of which stimulates the influx of 45Ca2+ through a type of voltage‐independent, receptor‐operated Ca2+ channels. Epidermal growth factor induced an accumulation of 45Ca2+ of a much smaller magnitude than elevations of the level of extracellular Ca2+, without a detectable increase of Ca2+ efflux. Thus, the divergent cellular responses of keratinocytes to EGF and extracellular Ca2+ may be due, in part, to the distinct changes in transmembrane Ca2+ fluxes that these two stimuli generate. Treatment of cells with type β transforming growth factor led to a gradual 6‐fold increase of the Ca2+ ‐activated rate of Ca2+ uptake over a period of 4 hours, but reduced the Ca2+ efflux by approximately 50% within 10 minutes. Thus, type β transforming growth factor apparently stimulates Ca2+ influx indirectly, but may control the differentiation of keratinocytes by direct inhibition of Ca2+ efflux pumps.
Effects of a sialoglycopeptide on early events associated with signal transduction
Authors: Toole‐Simms W. E. , Loder D. K. , Fattaey H. K. , Johnson T. C.
Published: February 2005

A sialoglycopeptide (SGP), isolated and purified from bovine cerebral cortex cells, was studied in regard to early signal transduction events associated with the cell cycle. Previously shown to be a potent antagonist to a variety of mitogens, the SGP abrogated the ability of 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) to elicit an alkalinization of 3T3 cell cytosol, but only when added minutes prior to, or simultaneously with, the tumor promoter. 3T3 cell TPA‐mediated Ca2+ mobilization was also inhibited by the SGP although the inhibitor itself did not bind Ca2+ in a cell‐free assay. The results are discussed in light of the already known kinetics of interaction between the SGP, various mitogens, and the calcium ionophore A23187 with regard to the pivotal events leading to the decision of a cell to divide or not to divide.
Colony stimulating factor‐1 stimulates diacylglycerol generation in murine bone marrow‐derived macrophages, but not in resident peritoneal macrophages
Authors: Veis Nurin , Hamilton John A.
Published: February 2005

Colony stimulating factor‐1 (CSF‐1) stimulates DNA synthesis in murine bone marrow‐derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol‐4,5‐bisphosphate hydrolysis is not associated with CSF‐1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF‐1 transiently elevated both [3H]myristoyl‐ and [3H]arachidonyl‐diacylglycerol (DAG) in BMM in a dose‐dependent fashion. CSF‐1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF‐1 in macrophages. Other mitogenic agents, 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl‐ and [3H]arachidonyl‐DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor‐α (TNF‐α) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF‐α increased only [3H]arachidonyl‐DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.
The NH2‐terminal extension of high molecular weight bFGF is a nuclear targeting signal
Authors: Quarto Natalina , Finger Fern P. , Rifkin Daniel B.
Published: February 2005

Basic fibroblast growth factor (bFGF) is a member of the heparin‐binding growth factor (HBGF) family that includes at least seven species. These proteins are potent regulators of a number of cellular processes, including cell division and angio‐genesis. Multiple forms of bFGF exist differing only in the length of their NH2‐terminal extensions. These species of bFGF also have unique subcellular distributions. The smallest form (18 kD) occurs predominantly in the cytosol, while the higher molecular weight forms (22, 22.5, 24 kD) are associated with the nucleus and ribosomes. Here we report that the nuclear localization of the higher molecular weight forms of bFGF derives specifically from the amino acid sequences within the NH2‐terminal extension. This has been demonstrated by constructing a chimeric protein containing the NH2‐terminal extension of the highest molecular weight form of bFGF fused to β‐galactosidase (β‐gal). After transfection in a transient expression system, the chimeric protein accumulated in the nuclei of transfected cells, while the wild‐type β‐gal was found predominantly in the cytoplasm.
Stimulation of protein synthesis by internalized insulin
Authors: Miller David S. , Sykes Destiny B.
Published: February 2005

Previous studies showed that microinjected insulin stimulates transcription and translation in Stage IV Xenopus oocytes by acting at nuclear and cytoplasmic sites (Miller, D.S., 1988, 1989). The present report is concerned with the question of whether hormone, internalized from an external medium, can act on those sites to alter cell function. Both intracellular accumulation of undegraded 125I‐insulin and insulin‐stimulated 35S‐methionine incorporation into oocyte protein were measured. Anti‐insulin antiserum and purified anti‐insulin antibody were microinjected into the cytoplasm of insulin‐exposed cells to determine if insulin derived from the medium acted through internal sites. In cells exposed for 2 h to 7 or 70 nM external insulin, methionine incorporation was stimulated, but intracellular hormone accumulation was minimal and microinjected antibody was without effect. In cells exposed for 24 h, methionine incorporation again increased, but now accumulation of undegraded, intracellular hormone was substantial (2.6 and 25.3 fmol with 7 and 70 nM, respectively), and microinjected anti‐insulin antibody significantly reduced the insulin‐stimulated component of incorporation; basal incorporation was not affected. For cells exposed to 70 nM insulin for 24 h, inhibition of the insulin‐stimulated component was maximal at 39%. Thus under those conditions, about 40% of insulin's effects were mediated by the internal sites. Together, the data show that inhibition of insulin‐stimulated protein synthesis by microinjected antibody was associated with the intracellular accumulation of insulin. They indicate that when oocytes are exposed to external insulin, hormone eventually gains access to intracellular sites of action and through these stimulates translation. Control of translation appears to be shared between the internal sites and the surface receptor.
Amino acid‐rich medium (Leibovitz L‐15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum
Authors: Mitaka Toshihiro , Sattler Gerald L. , Pitot Henry C.
Published: February 2005

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum‐free, modified L‐15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L‐15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H‐thymidine incorporation in the L‐15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48–72 h and 72–96 h after plating at densities between 1.5 × 105 and 6.0 × 105 cells per 35‐mm dish. Even at a density of 9.0 × 105 cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48–72 h and 72–96 h. In addition, about 20% of the hepatocytes in Culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L‐15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.
Granular presence of terminin is the marker to distinguish between the senescent and quiescent states
Authors: Wang Eugenia , Tomaszewski George
Published: February 2005

We have previously identified statin, a nonproliferating‐cell‐specific nuclear protein of 57,000 dalton whose presence can be used to distinguish between growing and nongrowing cells. In this report we identify another protein, terminin, whose presence (by immunofluorescence microscopy) can be used to distinguish between temporarily and permanently growth‐arrested cells. Thus terminin is a marker to separate the senescent from the quiescent state. By means of an unique monoclonal antibody (mAb1.2), the presence of terminin is recognized as granules in the cytoplasm of in vitro aged fibroblasts; these granules are not found in serum‐starved, contact‐inhibited, growing, or transformed fibroblasts, except for those cells experiencing the initiation of apoptosis due to long‐term deprivation of nutrients. Preliminary histochemical studies show that terminin is also found in the superficial epithelial layer of the esophagus, where terminal differentiation is followed by apoptosis and sloughing off into the lumen. Biochemical characterization by Western blot shows the terminin antibody recognizing a protein of 84 kilodalton (kDa) in growing and quiescent cells, whereas in senescent cells a protein of 57 kDa is recognized; this result suggests that a senescence‐dependent protease may cleave the 84 kDa protein to 57 kDa. This proteolytic action seems to render the specific antigenic epitope exposed in its native state and accessible to the terminin antibody by immunofluorescence microscopy. It is this product of posttranslational modification in the form of a cytoplasmic 57 kDa protein that is the marker distinguishing between senescence and quiescence.
Cell polarity regulates the release of secretory component, the epithelial receptor for polymeric immunoglobulins, from the surface of HT‐29 colon carcinoma cells
Authors: Chintalacharuvu Koteswara R. , Piskurich Janet F. , Lamm Michael E. , Kaetzel Charlotte S.
Published: February 2005

The HT‐29 human colon carcinoma cell line differentiates in glucose‐free medium to an enterocytic phenotype. We previously isolated a series of HT‐29 subclones selected for high levels of expression of secretory component (SC), the epithelial receptor for polvmeric immunoglobulins. To develop a model system for studying effects of cell polarity on SC expression and release from the cell surface, the HT‐29.74 subclone was induced to differentiate in glucose‐free medium. Expression of SC was induced by glucose deprivation in both the parental HT‐29 cell line and, to an even greater extent, in the HT‐29.74 subclone. Prolonged glucose deprivation of HT‐29.74 cells resulted in morphological changes consistent with enterocytic differentiation. Metabolic radiolabeling of SC in differentiated HT‐29.74 cells indicated that proteolytic cleavage of membrane‐bound to free SC occurred both on the cell surface and intracellularly, possibly in a vacuolar apical compartment or intrapeithelial lumen. To study effects of cell polarity on SC release, differentiated HT‐29.74 cells were depolarized by culturing in low calcium medium. Within 2 hours after transfer of the cells into low calcium medium, a burst of SC release was observed concomitant with cell depolarization. Subsequently, release of SC declined significantly and remained low as long as cells were maintained in a depolarized state. The extent of cell depolarization could be controlled by varying the extracellular calcium concentration or by substituting the divalent cation Sr++, which partially prevents depolarization, for Ca++. In either case, the magnitude of the initial burst and subsequent decline in release of SC was proportional to the extent of cell depolarization. We conclude that cell polarity plays an important role in controlling the release of SC in intestinal epithelial cells, most likely by regulating the distribution of membranebound SC and SC protease, which are on the basolateral and apical cell surfaces, respectively, in differentiated cells.
Bovine aortic endothelial cells release hydrogen peroxide
Authors: Sundqvist T.
Published: February 2005

Endothelial cells grown on microcarriers are able to release H2O2 to the extracellular environment without any added stimulus. The extracellularly released H2O2 can be detected by luminol‐amplified chemiluminescence (CL) if horseradish peroxidase is added. The CL response can be reduced by catalase and blocked by superoxide dismutase, indicating that O2‐could be a precursor for H2O2. The CL kinetics, i.e., a long lag time followed by a rapid shift to a new level, indicate activation of an O2‐producing enzyme. The cells are also able to protect themselves from H2O2 stimulation by both catalase and the glutatione system. Bradykinin stimulates the H2O2 release, but if the effect is directly stimulatory or if it acts by reduction of the protective system is at present unclear. The extracellularly released H2O2 could be a cause of injury to the endothelial cells or to the subendothelial matrix.
Coordination of protein‐DNA interactions in the promoters of human H4, H3, and H1 histone genes during the cell cycle, tumorigenesis, and development
Authors: Van Wijnen André J. , Owen Thomas A. , Holthuis Joost , Lian Jane B. , Stein Janet L. , Stein Gary S.
Published: February 2005

Coordinate transcriptional control of replication‐dependent human H4, H3, and H1 histone genes was studied by comparing levels of H3 and H1 histone promoter binding activities with those of H4 histone promoter factor HiNF‐D during the cell cycle of both normal diploid and tumor‐derived cells, as well as in fetal and adult mammalian tissues. Both H3 and H1 histone promoters interact with binding activities that, as with HiNF‐D, are maximal during S‐phase but at low levels in the G1‐phase of normal diploid cells. However, these analogous DNA binding activities are constitutively maintained at high levels throughout the cell cycle in four different transformed and tumor‐derived cells. Downregulation of the H3 and H1 histone promoter factors in conjunction with HiNF‐D is observed in vivo at the onset of quiescence and differentiation during hepatic development. Hence, our results indicate a tight temporal coupling of three separate protein‐DNA interactions in different histone promoters during the cell cycle, development, and tumorigenesis. This suggests that a key oscillatory, cell‐growth‐control mechanism modulates three analogous histone gene promoter protein‐DNA interactions in concert. The derangement of this mechanism in four distinct tumor cells implies that concerted deregulation of these histone promoter factors is a common event resulting from heterogeneous aberrations in normal cell growth mechanisms during tumorigenesis. We postulate that this mechanism may be involved in the coordinate regulation of the human H4, H3, and H1 histone multigene families.
Transforming activity of mutant human p53 alleles
Authors: Slingerland Joyce M. , Benchimol Sam
Published: February 2005

Mutant forms of the p53 gene have been shown to cooperate with an activated ras gene in transforming primary cells in culture. The aberrant proteins encoded by p53 mutants are thought to act in a dominant negative manner in these assays. In vivo data, however, reveal that where p53 has undergone genetic change in tumors, both alleles have been affected. We previously identified a case of human acute myelogenous leukemia (AML) in which both alleles of the p53 gene had undergone independent missense mutations (at codons 135 cys to ser and 246 met to val). In these blasts, p53 mutations appear to be acting recessively. We have assayed the transforming potential of these p53 mutations, as well as that of another mutation at codon 273, also identified in a human neoplasm. Both mutations from the AML blasts (codon 135 and codon 246) confer transforming ability on the mutant protein. While transformation assays may define functionally different subsets of p53 mutations, the overexpression phenotype of mutants in this assay may not accurately reflect the pathological effects of p53 mutations in vivo.
CD10 and CD44 genes of leukemic cells and malignant cell lines show no evidence of transformation‐related alterations
Authors: Kee Barbara L. , Dadi Harjit K. , Tran‐Paterson Rosette , Quackenbush Elizabeth J. , Andrulis Irene L. , Letarte Michelle
Published: February 2005

The expression of CD10/CALLA is associated primarily with childhood leukemia of pre‐B lymphocyte phenotype. We have compared the hybridization pattern of the CALLA gene from leukemic and normal cells digested with several restriction enzymes. No alterations were noticed with Eco Rl, Sac I, Pvu II, Eco RV, Hind III, and Msp I. Since CALLA is also found on other malignancies, we analyzed DNA samples prepared from cell lines derived from leukemia, lymphoma, glioblas‐toma, retinoblastoma, and neuroblastoma. Normal restriction patterns were observed for all the lines regardless of their CALLA phenotype. Having demonstrated previously that CALLA was structurally identical to neutral endopeptidase 3.4.24.11 (NEP), we have now established a correlation between surface expression of CALLA and NEP activity on leukemia samples and on several cell lines. Malignant cells tested expressed a functionally active enzyme and no gross alteration was present in the CALLA gene. The CD44 gene is expressed on most cells of hemopoietic origin and on > 95% of cases of acute lymphoblastic leukemia and acute myeloblastic leukemia studied. It is also expressed on normal astrocytes and on malignant cells of glioma/astrocytoma types. We now report that a similar pattern of hybridization was observed with Sac I, Pvu II, and Eco Rl for leukemic samples, normal cells, and malignant cell lines. A polymorphism was recently detected for CD44 using Hind III; leukemic cells and malignant lines also showed this normal polymorphism. Thus no deletion or insertion could be detected in the CD44 gene of leukemic cells and malignant lines, suggesting that no gross DNA alterations were involved. The correlation between surface expression and enzymatic activity of CD10/CALLA and the expression of CD44 on a variety of malignant cells would suggest that the structure and function of these two gene products are probably not altered by the process of transformation.
Clearance and fate of leukemia‐inhibitory factor (LIF) after injection into mice
Authors: Hilton Douglas J. , Nicola Nicos A. , Waring Paul M. , Metcalf Donald
Published: February 2005

Leukemia‐inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half‐life of 6‐8 min and a more prolonged second clearance phase. Clearance of 125I‐LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable 125I in the urine suggested that the kidneys were the major route of LIF clearance from the body.
Responsiveness of the increase in c‐fos mrna levels depends on the inducer and the cell's past
Authors: Tuijl Mark J. M. , Boon Johan A. Den , van Grunsven Wout M. J. , van Wijk Roeland
Published: February 2005

In this paper we show that in C3H10T1/2 mouse fibroblasts, the inducibility of c‐fos mRNA by heat shock or serum addition is strongly dependent on the cell's past. Four hours after a heat shock, a time point where the induced c‐fos mRNA has disappeared, c‐fos mRNA could not be induced again by a second heat shock. Four hours after serum addition, by which c‐fos was induced, a second serum addition also failed to induce c‐fos mRNA again. When, however, serum was added 4 hours after heat shock or heat shock was given 4 hours after serum addition, levels of c‐fos mRNA could be enhanced again. The induction by serum of c‐fos mRNA levels in thermotolerant cells might be related to their increased stimulation of DNA synthesis as compared to control cells.
Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in chinese hamster ovary cells
Authors: Lee Yong J. , Curetty Lindali , Corry Peter M.
Published: February 2005

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5°C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 μM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6–9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0–2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3–6 hr after heat shock, whereas that of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleasu.
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

In 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

We 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

Extending 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

The 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

IL‐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

The 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

Cyclic 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

Cell 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

Suramin 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

The 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

The 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

A 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.