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Ovary Cells (ovary + cell)
Kinds of Ovary Cells Selected AbstractsLimitations to the Development of Humanized Antibody Producing Chinese Hamster Ovary Cells Using Glutamine Synthetase-Mediated Gene AmplificationBIOTECHNOLOGY PROGRESS, Issue 3 2006Seung Chul Jun Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS) into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 ,M of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (qAb) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long-term cultures in the presence of the corresponding concentrations of MSX, qAb of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased qAb. Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification. [source] Effect of Simultaneous Application of Stressful Culture Conditions on Specific Productivity and Heterogeneity of Erythropoietin in Chinese Hamster Ovary CellsBIOTECHNOLOGY PROGRESS, Issue 4 2004Sung Kwan Yoon A single stressful culture condition induced by hypoosmotic stress (210 mOsm kg,1), low culture temperature (32 °C), or NaBu addition (1 mM) resulted in a 1.8- to 2.2-fold enhancement of specific erythropoietin (EPO) productivity (qEPO) of recombinant Chinese hamster ovary (rCHO) cells compared to normal culture condition (37 °C and 310 mOsm kg,1). Simultaneous application of these stressful conditions further enhanced qEPO up to approximately 5-fold. However, the quality of EPO was affected by stressful culture conditions. The proportion of acidic isoforms of EPO under a single stressful condition was 2.8,13.8% lower than that under normal culture condition. Simultaneous application of the stressful conditions further decreased the portion of acidic isoforms but not significantly. Despite 5-fold enhancement of qEPO, the portion of acidic isoforms under the simultaneous application of stressful culture conditions was 12.9,21.6% lower than that under normal culture condition. Taken together, these results suggest the potential of simultaneous application of different stressful culture conditions to the production phase of two-stage culture, where cell growth and production phases are separated, for improved EPO production. [source] Acidosis Impairs the Protective Role of hERG K+ Channels Against Premature StimulationJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 10 2010B.Sc., CHUN YUN DU M.B. Acidosis and the hERG K+ Channel.,Introduction: Potassium channels encoded by human ether-à-go-go-related gene (hERG) underlie the cardiac rapid delayed rectifier K+ channel current (IKr). Acidosis occurs in a number of pathological situations and modulates a range of ionic currents including IKr. The aim of this study was to characterize effects of extracellular acidosis on hERG current (IhERG), with particular reference to quantifying effects on IhERG elicited by physiological waveforms and upon the protective role afforded by hERG against premature depolarizing stimuli. Methods and Results: IhERG recordings were made from hERG-expressing Chinese Hamster Ovary cells using whole-cell patch-clamp at 37°C. IhERG during action potential (AP) waveforms was rapidly suppressed by reducing external pH from 7.4 to 6.3. Peak repolarizing current and steady state IhERG activation were shifted by ,+6 mV; maximal IhERG conductance was reduced. The voltage-dependence of IhERG inactivation was little-altered. Fast and slow time-constants of IhERG deactivation were smaller across a range of voltages at pH 6.3 than at pH 7.4, and the contribution of fast deactivation increased. A modest acceleration of the time-course of recovery of IhERG from inactivation was observed, but time-course of activation was unaffected. The amplitude of outward IhERG transients elicited by premature stimuli following an AP command was significantly decreased at lower pH. Computer simulations showed that after AP repolarization a subthreshold stimulus at pH 7.4 could evoke an AP at pH 6.3. Conclusion: During acidosis the contribution of IhERG to action potential repolarization is reduced and hERG may be less effective in counteracting proarrhythmogenic depolarizing stimuli. (J Cardiovasc Electrophysiol, Vol. 21, pp. 1160-1169) [source] Teicoplanin-dependent antibodies: detection and characterizationBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2005Stephen F. Garner Summary There are only a few reports of thrombocytopenia associated with clinical doses of teicoplanin, a glycopeptide antibiotic used against Gram-positive bacteria. We investigated 39 patients receiving teicoplanin; 31 were thrombocytopenic with platelet counts between 1,105 × 109/l and 8 were not thrombocytopenic. We identified 14 thrombocytopenic cases (45%) and two (25%) non-thrombocytopenic cases with IgG teicoplanin-dependent platelet-reactive antibodies. Use of glycoprotein (GP) capture enzyme-linked immunosorbent assay with platelets and GPIIb/IIIa transfected Chinese Hamster Ovary cells as well as flow cytometry with GP-deficient platelets indicated that the GPIIb/IIIa complex is a major target antigen of these antibodies. [source] Expression of the E. coli fpg protein in CHO cells lowers endogenous oxypurine clustered damage levels and decreases accumulation of endogenous Hprt mutations,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 5 2006Sunirmal Paul Abstract Endogenous DNA damage clusters,two or more oxidized bases, abasic sites, or strand breaks within about 20 base pairs on opposing strands,can accumulate in unirradiated mammalian cells, and may be significant origins of spontaneous detrimental biological effects. Factors determining the levels of such endogenous clusters are largely unknown. To determine if cellular repair genotype can affect endogenous cluster levels in mammalian cells, the authors examined cluster levels, growth rates, and mutant frequencies in Chinese hamster ovary cells expressing the Escherichia coli glycosylase fpg protein, whose principal substrates are oxidized purines. In cells expressing high levels of fpg protein, the levels of oxypurine clustered damages were decreased while those of oxypyrimidine clusters and abasic clusters were unchanged. Furthermore, in these cells, the growth rates were increased and the level of spontaneous background mutants in the hypoxanthine guanine phosphoribosyl transferase gene was decreased. These results suggest that endogenous clusters are potentially detrimental DNA damages, and that their levels,as well as the detrimental consequences of their presence,can be effectively reduced by increased cellular activity of specific DNA repair proteins. Environ. Mol. Mutagen., 2006. Published 2006 Wiley-Liss, Inc. [source] Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity testsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2002M. Vijayaraj Reddy Abstract 3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the 32P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts. Environ. Mol. Mutagen. 40:1,17, 2002. © 2002 Wiley-Liss, Inc. [source] Antioxidant and anti-inflammatory activities of melanocortin peptidesEXPERIMENTAL DERMATOLOGY, Issue 9 2004J. W. Haycock ,-Melanocyte-stimulating hormone (,-MSH) has previously been identified as a potent anti-inflammatory agent in various tissues including the skin. It operates by binding to the melanocortin-1 receptor (MC-1R) which results in the elevation of cyclic AMP. ,-MSH opposes the action of several proinflammatory cytokines including tumour necrosis factor-, (TNF-,). We have shown that ,-MSH can inhibit TNF-,-stimulated activation of nuclear factor-,B (NF-,B) in human cultured melanocytes, melanoma cells, keratinocytes, fibroblasts, Schwann cells and olfactory ensheathing cells. It also inhibits TNF-,-stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in many of these cells and can inhibit peroxide-stimulated activation of glutathione peroxidase, suggesting an antioxidant role. ,-MSH is also able to stimulate intracellular calcium release in keratinocytes and fibroblasts (which do not readily show detectible cyclic AMP elevation) but only in the presence of PIA (an adenosine agonist). The carboxyl terminal tripeptides KPV/KP-D-V are reported to be the minimal sequences necessary to convey anti-inflammatory potential, but evidence on how they act is not fully known. Stable transfection of Chinese hamster ovary cells with MC-1R suggests that the KPV peptides operate by this receptor, at least by elevating intracellular calcium. Elevation of cyclic AMP by these tripeptides has not been detected in any cell type studied; however, calcium elevation can inhibit TNF-,-stimulated NF-,B activity (as for cyclic AMP). In conclusion, the MSH peptides convey anti-inflammatory and antioxidant activity in many cell types in skin and nerve, by counteracting proinflammatory cytokine signalling. The KPV peptides appear to act functionally via the MC-1R and can also elevate intracellular calcium. [source] Regulated expression by PPAR, and unique localization of 17,-hydroxysteroid dehydrogenase type 11 protein in mouse intestine and liverFEBS JOURNAL, Issue 18 2007Yasuhide Yokoi 17,-Hydroxysteroid dehydrogenase type 11 (17,-HSD11) is a member of the short-chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17,-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-, PPAR, in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141,4146]. In this study, we characterized 17,-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17,-HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-, agonist induced 17,-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17,-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse-chase experiment suggested that 17,-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17,-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17,-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-, agonist. Taken together, these data indicate that 17,-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17,-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments. [source] Soluble LDL-R are formed by cell surface cleavage in response to phorbol estersFEBS JOURNAL, Issue 3 2004Michael J. Begg A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4,-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins. [source] Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin bindingFEBS JOURNAL, Issue 3 2002Christoph Böhme The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with ,2,6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal ,1,6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in >,90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively ,2,3-linked N -acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin,Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans. [source] Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cellsbut not in Chinese hamster ovary cellsFEBS JOURNAL, Issue 15 2000Ingrid M. Van den Nieuwenhof We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N,N,- diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a ,1,4- N- acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form. [source] Biochanin A induction of sulfotransferases in ratsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2010Yue Chen Abstract Biochanin A (BCA) is a dietary isoflavone present in red clover (Trifoliumn pretense) and many herbal products. BCA has been reported to have chemopreventive actions against various cancers including prostate, breast, colon cancer, and so on. Sulfotransferases are a family of phase II drug-metabolizing enzymes, which are important for xenobiotic detoxification and regulation of biological signaling molecule biological activities. Sulfotransferase gene expressions are regulated by different hormones and xenobiotics. Improper regulation of sulfotransferases leads to improper functions of biological signaling molecules, which in turn can cause cancer or other diseases. BCA inhibits the enzyme activities of the phase I drug-metabolizing enzymes CYP1A1 and CYP1B1 in Chinese hamster ovary cells and induces the phase II drug-metabolizing enzymes UDP-glucuronosyltransferases in human prostate cancer cells. BCA induction of sulfotransferases has not been studied. This investigation evaluates the in vivo regulation of sulfotransferases at protein and mRNA levels in the liver and intestine of Sprague-Dawley rats treated with BCA (0, 2, 10, and 50 mg/kg/day) for 7 days. Our experimental results demonstrate for the first time that chronic BCA treatment can significantly induce the expression of rat sulfotransferase 1A1 (rSULT1A1, AST-IV), sulfotransferase 2A1 (rSULT2A1, STa), and rat estrogen sulfotransferase (rSULT1E1, EST) in rat liver and intestine. Our Western blot results are in good agreement with real-time RT-PCR data, suggesting that BCA induction of sulfotransferases occurs at the transcriptional level. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:102,114, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20318 [source] Extracellular Acidosis Modulates Drug Block of Kv4.3 Currents by Flecainide and QuinidineJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2003Suresh Singarayar M.D. Introduction: As a molecular model of the effect of ischemia on drug block of the transient outward potassium current, the effect of acidosis on the blocking properties of flecainide and quinidine on Kv4.3 currents was studied. Methods and Results: Kv4.3 channels were stably expressed in Chinese hamster ovary cells. Whole-cell, voltage clamp techniques were used to measure the effect of flecainide and quinidine on Kv4.3 currents in solutions of pH 7.4 and 6.0. Extracellular acidosis attenuated flecainide block of Kv4.3 currents, with the IC50 for flecainide (based on current-time integrals) increasing from7.8 ± 1.1 ,Mat pH 7.4 to125.1 ± 1.1 ,Mat pH 6.0. Similar effects were observed for quinidine (IC50 5.2 ± 1.1 ,Mat pH 7.4 and22.1 ± 1.3 ,Mat pH 6.0). Following block by either drug, Kv4.3 channels showed a hyperpolarizing shift in the voltage sensitivity of inactivation and a slowing in the time to recover from inactivation/block that was unaffected by acidosis. In contrast, acidosis attenuated the effects on the time course of inactivation and the degree of tonic- and frequency-dependent block for both drugs. Conclusion: Extracellular acidosis significantly decreases the potency of blockade of Kv4.3 by both flecainide and quinidine. This change in potency may be due to allosteric changes in the channel, changes in the proportion of uncharged drug, and/or changes in the kinetics of drug binding or unbinding. These findings are in contrast to the effects of extracellular acidosis on block of the fast sodium channel by these agents and provide a molecular mechanism for divergent modulation of drug block potentially leading to ischemia-associated proarrhythmia.(J Cardiovasc Electrophysiol, Vol. 14, pp. 641-650, June 2003) [source] A transient expression vector for recombinant protein production in Chinese hamster ovary cellsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2006Mimi ML Liao Abstract An expression vector was specifically designed for use in Chinese hamster ovary (CHO) cells to enhance the level of protein production in a transient expression system. Two key components that can increase protein production transiently are the promoter used to drive recombinant gene expression and the template copy number. In this study the modified and metal-inducible metallothionein (M2.6) promoter was shown to be superior to the human cytomegalovirus (CMV) and to the simian virus SV40 promoters. Plasmid replication was achieved using the Polyoma (Py) virus origin of replication (PyOri) and the Py Large T antigen (PyLT). An expression vector containing Py elements was shown to replicate extensively in CHO cells. The combination of the metal-inducible M2.6 promoter and episomal replication of the expression vector, named pPyOriLT resulted in elevated levels of transgene expression following transient transfection of CHO cells Copyright © 2005 Society of Chemical Industry [source] Agonist-Induced Internalization and Recycling of the Human A3 Adenosine ReceptorsJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Resensitization, Role in Receptor Desensitization Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6 -(4-amino-3-[125I]iodobenzyl)adenosine-5,- N -methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min -1. Agonist-induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min -1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization. [source] Nerve Growth Factor-Induced Differentiation Does Not Alter the Biochemical Properties of a Mutant Prion Protein Expressed in PC12 CellsJOURNAL OF NEUROCHEMISTRY, Issue 1 2000Roberto Chiesa Abstract : Insertional and point mutations in the gene encoding the prion protein (PrP) are responsible for familial prion diseases. We have previously generated lines of Chinese hamster ovary cells that express PrP molecules carrying pathogenic mutations, and found that the mutant proteins display several biochemical properties reminiscent of PrPSc, the infectious isoform of PrP. To analyze the properties and effects of mutant PrP molecules expressed in cells with a neuronal phenotype, we have constructed stably transfected lines of PC12 cells that synthesize a PrP molecule carrying a nine-octapeptide insertion. We report here that this mutant PrP acquires scrapie-like properties, including detergent insolubility, protease resistance, and resistance to phospholipase cleavage of its glycolipid anchor. A detergent-insoluble and phospholipase-resistant form of the mutant protein is also released spontaneously into conditioned medium. These scrapie-like biochemical properties are quantitatively similar to those seen in Chinese hamster ovary cells and are not affected by differentiation of the PC12 cells into sympathetic neurons by nerve growth factor. Moreover, there is no detectable effect of mutant PrP expression on the morphology or viability of the cells in either the differentiated or undifferentiated state. These results indicate that conversion of mutant PrP into a PrPSc -like form does not depend critically on the cellular context, and they suggest that mutant PrP expressed in cultured cells, even those having the phenotype of differentiated neurons, is not neurotoxic. [source] Investigation of ,2 -adrenoceptor subtype selectivity and organ specificity for bedoradrine (KUR-1246), a novel tocolytic beta-adrenergic receptor stimulantJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 3 2009Yoshihito Inoue Abstract Objectives:, The aim of this study was to evaluate the beta-adrenergic receptor (,-AR) selectivity, organ specificity and efficacy of delaying the onset of spontaneous delivery of bedoradrine (KUR-1246), a novel uterine relaxant. Methods:, ,-AR selectivity was evaluated in terms of the amount of cyclic adenosine monophosphate produced by bedoradrine, ritodrine and isoprenaline in Chinese hamster ovary cells expressing human ,1 -, ,2 -AR or ,3 -AR. Inhibition of contractions of the atrium, trachea and proximal colon by bedoradrine were compared with those of the uterus in pregnant rats using an organ bath method. Finally, the delaying effect of bedoradrine on spontaneous labor was evaluated by an in vivo study using term pregnant rats. Results:, EC50 values of bedoradrine for cyclic adenosine monophosphate production in Chinese hamster ovary cells via ,1 -, ,2 - and ,3 -AR were 2400 ± 30, 2.9 ± 0.10 and 363 ± 3 nmol/L, respectively, indicating that bedoradrine had 832- and 126-fold higher selectivity for ,2 -AR than for ,1 - and ,3 -AR. EC50 values of bedoradrine for the uterus, atrium, trachea and proximal colon were 1.01 ± 0.27, 2300 ± 356, 1610 ± 299 and 219 ± 23.5 nmol/L, respectively. Thus, bedoradrine was 2280-, 1590- and 217-fold more specific for the uterus than for the atrium, trachea and proximal colon, respectively. Bedoradrine delayed the spontaneous delivery of 21-day-pregnant rats in a dose-dependent manner. Conclusions:, Bedoradrine is a promising drug for the treatment of preterm labor in obstetrical practice because it has better selectivity for ,2 -AR and specificity for the uterus than currently used agents and may effectively delay spontaneous delivery. [source] Triton-X-100-modified polymer and microspheres for reversal of multidrug resistanceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2001Zhi Liu Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes. However, it interacts with cells in a non-specific way, causing cytotoxicity. This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects. When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent. Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized. Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CHRC5) was compared with that of free Triton X-100 solutions. The in-vitro effect of the products on 3H-vinblastine accumulation by CHRC5 cells was determined. Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations. Drug accumulation by CHRC5 cells was increased over two fold in the presence of T-MS or T-IN. These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers. [source] First cloning and functional characterization of a melatonin receptor in fish brain: a novel one?JOURNAL OF PINEAL RESEARCH, Issue 2 2002Pascaline Gaildrat Melatonin, a neuroendocrine transducer of photoperiod, influences a number of physiological functions and behaviors through specific seven transmembrane domains receptors. We report here the first full-length cloning and functional characterization of a melatonin receptor (P2.6) in a fish, the pike (Teleost). P2.6 encodes a protein that is ,80% identical to melatonin receptors previously isolated partially in non-mammals and classified as members of the Mel1b subtype; but, it shares only 61% identity with the full-length human Mel1b melatonin receptor (hMT2). Expression of P2.6 results in ligand binding characteristics similar to that described for endogenous melatonin receptors. Selective antagonists of the hMT2 (4-phenyl-2-propionamidotetraline and luzindole) were poor competitors of 2-[125I]iodomelatonin binding to the recombinant receptor. In Chinese hamster ovary cells expressing both the cystic fibrosis transmembrane conductance regulator chloride channel and P2.6 receptor, melatonin counteracted the forskolin induced activation of the channel. The results are best explained by a selective inhibition of the adenylyl cyclase. By reverse transcription-polymerase chain reaction, P2.6 mRNA appeared expressed in the optic tectum and, to lesser extent, in the retina and pituitary. In conclusion, these results, together with those of a phylogenetic analysis, suggest that P2.6 might belong to a distinct subtype group within the vertebrate melatonin receptor family. [source] Acute Alcohol Inhibits the Induction of Nuclear Regulatory Factor ,B Activation Through CD14/Toll-Like Receptor 4, Interleukin-1, and Tumor Necrosis Factor Receptors: A Common Mechanism Independent of Inhibitory ,B, Degradation?ALCOHOLISM, Issue 11 2002Pranoti Mandrekar Background Nuclear translocation and DNA binding of the nuclear factor ,B (NF-,B) is an early event in inflammatory cell activation in response to stimulation with bacterial components or cytokines. Cell activation via different receptors culminates in a common pathway leading to NF-,B activation and proinflammatory cytokine induction. We have previously shown that acute alcohol inhibits NF-,B activation by lipopolysaccharide (LPS) in human monocytes. Here we investigated whether acute alcohol treatment of human monocytes also inhibits NF-,B when induced through activation of the interleukin (IL)-1 or tumor necrosis factor (TNF) receptors. Methods Human peripheral blood monocytes were treated with LPS, TNF,, and IL-1, in the presence or absence of 25mM alcohol for 1 hr. NF-,B activation was determined by electrophoretic mobility shift assays using nuclear extracts. Inhibitory ,B, (I,B,) was estimated by Western blotting in cytoplasmic extracts. Chinese hamster ovary cells expressing human CD14 were treated with LPS in the presence or absence of alcohol to study NF-,B and I,B, regulation. Results Our results indicate that acute alcohol inhibits IL-1,- and TNF,-induced NF-,B activation. We further show in CD14/toll-like receptor 4,expressing Chinese hamster ovary cells the specificity of alcohol-mediated inhibition of NF-,B via the toll-like receptor 4/CD14 receptors. Inhibition of NF-,B by acute alcohol was concomitant with decreased levels of the I,B, molecule in the cytoplasm of LPS, IL-1, and TNF,-activated monocytes. Conclusions These data suggest a unique, I,B,-independent pathway for the inhibition of NF-,B activation by acute alcohol in monocytes. Universal inhibition of NF-,B by acute alcohol via these various receptor systems suggests a target for the effects of alcohol in the NF-,B activation cascade that is downstream from I,B, degradation. Further, these results demonstrate that acute alcohol is a potent inhibitor of NF-,B activation by mediators of early (LPS) or late (IL-1, TNF,) stages of inflammation in monocytes. [source] Binding of platelet glycoprotein Ib, through the convex surface of leucine-rich repeats domain of glycoprotein IXJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2009X. MO Summary.,Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIb,, GPIb, and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIb, and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Objectives and methods: Guided by analysis of the LRR structure, we report a well-folded Ib,/IX chimera and its usage in dissecting GPIX function. Results: In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIb, ectodomain are replaced by their GPIX counterparts. Like GPIb, but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ib,/IX chimera, but not the GPIb, ectodomain, preserved its interaction with GPIb, as demonstrated by its native-like GPIb,-induced increase in surface expression and coimmunoprecipitation. Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIb,. [source] Overexpression of the partially activated ,IIb,3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet,like extensions in Chinese hamster ovary cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009E. SCHAFFNER-RECKINGER Summary.,Background: We have recently reported a novel mutation in the ,3 subunit of the platelet fibrinogen receptor (,IIb,3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated ,IIb,3D723H or ,IIb,3D723A salt bridge mutants, but not fully activated ,IIb,3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of ,IIb,3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+ -derived megakaryocyte proplatelets. Mutant ,IIb,3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type ,IIb,3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO ,IIb,3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO ,IIb,3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions. [source] Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib,IX complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007S.-Z. LUO Summary.,Background:,The glycoprotein (GP) Ib,IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIb, and GPIb, subunits interact and participate in complex assembly. Objectives and methods:,Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb,IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results:,Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb,IX complex. In contrast, replacing the GPIX TM domain (residues 132,153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIb,, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions:,The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb,IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex. [source] A novel Phe171Cys mutation in integrin ,IIb causes Glanzmann thrombasthenia by abrogating ,IIb,3 complex formationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2004N. Rosenberg Summary.,Background: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either ,IIb[glycoprotein (GP) IIb] or ,3 (GPIIIa) genes that lead to a lack or dysfunction of the integrin ,IIb,3 which serves as a fibrinogen receptor. Patients Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle. Objective: To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects. Results: Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of ,3, no ,IIb and no ,IIb,3 on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the ,IIb gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated ,IIb and wild-type ,3 failed to express ,IIb,3 as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the ,IIb,3 model, which was based on the crystal structure of ,v,3, indicated that Phe171 plays an essential role in the interface between the ,-propeller domain of ,IIb and the ,A domain of ,3. Conclusions: A novel Phe171Cys mutation in the ,IIb gene of patients with GT is associated with abrogation of ,IIb,3 complex formation. [source] Glycoprotein Ib,IX-mediated activation of integrin ,IIb,3: effects of receptor clustering and von Willebrand factor adhesionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2003M. Arya Summary., The interaction between the platelet glycoprotein (GP) Ib,IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to ,IIb,3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib,IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF,GP Ib,IX and fibrinogen,,IIb,3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib,IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell-permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib,IX(FKBP)2, and those between fibrinogen-coated beads and ,IIb,3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib,IX(FKBP)2 and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and ,IIb,3 was not changed by the clustering agent; however, the strength of single fibrinogen,,IIb,3 bonds increased significantly after ligation of GP Ib,IX(FKBP)2 by A1. These results demonstrate that GP Ib,IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib,IX can activate ,IIb,3, thereby increasing the strength of its interaction with fibrinogen. [source] Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitansMOLECULAR MICROBIOLOGY, Issue 5 2005Linsen Cao Summary The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5,- and 3,-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function. [source] Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzaeMOLECULAR MICROBIOLOGY, Issue 4 2000Mumtaz Virji Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs. [source] Chemistry and genotoxicity of caramelized sucroseMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 12 2006David D. Kitts Abstract Caramelization of a 1% sucrose solution at 180°C accompanied characteristic changes in pH, Mr, UV-absorbance, and fluorescence values as well as increased reducing power activity after 40,60 min. Similar changes occurred to sucrose heated at 150°C, after 150,240 min. Bioactivity of caramelized sucrose samples was tested for mutagenic activity, using Salmonella typhimurium strains TA-98 and TA-100, respectively, as well as the Saccharomyces D7 yeast strain for mitotic recombination and Chinese hamster ovary cells (CHO) to assess clastogenicity. Caramelized sucrose expressed no mutagenicity in the TA-98 strain, but gave positive (p < 0.05) results with the TA-100, base-pair substitution strain. Similarly, mitotic recombination in the Saccharomyces D7 yeast strain and clastogenic activity in CHO cells were induced when exposed to caramelized sucrose. In the all cases, preincubation with S-9 reduced (p < 0.05) the mutagenic activities of caramelized sucrose. Fractionation of the caramelized sucrose into volatile and nonvolatile compounds was performed and tested for clastogenicity using CHO cells. Volatile components contributed approximately 10% to total clastogenicity, which was enhanced by the presence of S-9. Nonvolatile components recovered, consisting of relatively lower Mr, gave highest (p < 0.05) clastogenic activity, denoting that higher Mr caramel colors are relatively free of this property. [source] Identification of shed proteins from chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databasesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2005Mamoun Ahram Abstract The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response. Chinese hamster ovary cells were chosen because they have been widely used for radiation studies and are reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and Fourier transform-ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analyses. Since the hamster genome has not been sequenced, MS data was searched against the mouse and human protein databases. Nearly 150 proteins identified by tandem mass spectrometry were confirmed by FT-ICR. When both types of MS data were evaluated, using a new confidence scoring tool based on discriminant analyses, about 500 proteins were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface and, hence were likely shed. However, estimates of quantitative changes, based on two independent MS approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using MS in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool. [source] Crystallization and preliminary X-ray analysis of the human vascular adhesion protein-1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Yvonne Nymalm Human vascular adhesion protein-1 (VAP-1) is a membrane-bound multifunctional glycoprotein with both adhesive and enzymatic properties. The protein belongs to the copper-containing amine oxidase (CAO) family, which use 2,4,5-trihydroxyphenylalanine quinone as a cofactor. Here, the crystallization and preliminary X-ray analysis of a mammalian CAO, human VAP-1, is reported. The protein was expressed in Chinese hamster ovary cells as a full-length form with an N-terminal transmembrane region and multiple glycosylation sites. Hexagonal crystals with unit-cell parameters a = b = 225.9, c = 218.7,Å, , = , = 90, , = 120° were obtained using the vapour-diffusion method. Data from three different crystals were collected at 100,K using synchrotron radiation and were processed to 3.2,Å resolution with 95.9% completeness and an Rmerge of 19.6%. [source] | ||||||||||||||||||||||||||||