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CHO Cells (cho + cell)
Kinds of CHO Cells Terms modified by CHO Cells Selected AbstractsEffect of Doxycycline-Regulated ERp57 Expression on Specific Thrombopoietin Productivity of Recombinant CHO CellsBIOTECHNOLOGY PROGRESS, Issue 1 2003Sun Ok Hwang In an attempt to increase the specific thrombopoietin (TPO) productivity ( qTPO) of recombinant Chinese hamster ovary (rCHO) cells (TPO-33), the effect of expression level of ERp57, an isoform of protein disulfide isomerase, on qTPO was investigated. To regulate ERp57 expression level, the Tet-Off system was first introduced in TPO-33 cells and stable Tet-Off cells (TPO-33-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated ERp57 expression system (TPO-33-ERp57) were obtained by cotransfection of pTRE-ERp57 and pTK-Hyg expression vectors into TPO-33-Tet-Off cells and subsequent screening by Western blot analysis of ERp57 and an enzyme-linked immunosorbent assay of secreted TPO. Western blot analysis showed that ERp57 expression level in TPO-33-ERp57 cells could be regulated tightly by the addition of different concentrations of doxycycline to a culture medium. A doxycycline concentration of 1 ,g/mL, which did not influence cell growth and TPO production of TPO-33-Tet-Off cells, was high enough to suppress the ERp57 expression to a basal level. Compared with the basal level, a 1.7-fold increase in ERp57 expression level was obtained in the absence of doxycycline. This increased expression level of ERp57 resulted in a 2.1-fold increase in qTPO without growth inhibition, probably as a result of the chaperone-like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that qTPO of rCHO cells can be increased by elevating the expression level of ERp57. [source] Effects of Cloned Gene Dosage on the Response of Recombinant CHO Cells to Hyperosmotic Pressure in Regard to Cell Growth and Antibody ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2001Joon Soo Ryu The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (,) and increased specific antibody productivity (qAb) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in qAb and less reduction in , at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and qAb, indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G1 -phase arrest, suggesting that the increase of cell population in G1 -phase may contribute in part to enhanced qAb at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in qAb at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage. [source] Functional Studies of Synthetic Gramicidin Hybrid Ion Channels in CHO CellsCHEMBIOCHEM, Issue 5 2007Ryszard Wesolowski Abstract The function of a gramicidin hybrid ion channel in living Chinese hamster ovary (CHO) cells was investigated by the patch clamp method. The synthetic ion channel 1 consists of two cyclohexyl ether amino acids that link two minigramicidin strands. With 1 at a concentration of 1.0 ,M, an increase in the whole-cell membrane conductance was observed after 1.37 min. The conductance showed larger currents when Cs+ was used as charge carrier than when Na+ and K+ were used. In single-channel recordings with Cs+ as charge carrier, the substance showed comparable single-channel amplitudes in the membrane of living cells and artificial black lipid bilayers. In addition to functioning as a cation channel, compound 1 appeared to be a water channel. Exposure of the CHO cells to an extracellular hypoosmotic solution did not substantially change the cell volume. Extracellular hypoosmotic conditions in the presence of 1 increased the cell size to 146.5,% that of the control. Thus, the synthetic hybrid channel 1 can function as a cation channel with some Cs+ specificity, and as a water channel in CHO cells. [source] Thyroid hormone receptor , can control action potential duration in mouse ventricular myocytes through the KCNE1 ion channel subunitACTA PHYSIOLOGICA, Issue 2 2010A. Mansén Abstract Aims:, The reduced heart rate and prolonged QTend duration in mice deficient in thyroid hormone receptor (TR) ,1 may involve aberrant expression of the K+ channel ,-subunit KCNQ1 and its regulatory ,-subunit KCNE1. Here we focus on KCNE1 and study whether increased KCNE1 expression can explain changes in cardiac function observed in TR,1-deficient mice. Methods:, TR-deficient, KCNE1-overexpressing and their respective wildtype (wt) mice were used. mRNA and protein expression were assessed with Northern and Western blot respectively. Telemetry was used to record electrocardiogram and temperature in freely moving mice. Patch-clamp was used to measure action potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster ovary (CHO) cells. Results:, KCNE1 was four to 10-fold overexpressed in mice deficient in TR,1. Overexpression of KCNE1 with a heart-specific promoter in transgenic mice resulted in a cardiac phenotype similar to that in TR,1-deficient mice, including a lower heart rate and prolonged QTend time. Cardiomyocytes from KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at higher KCNE1 levels. Conclusion:, The bradycardia and prolonged QTend time in hypothyroid states can be explained by altered K+ channel function due to decreased TR,1-dependent repression of KCNE1 expression. [source] Complementary expression and heterophilic interactions between igLON family members neurotrimin and LAMPDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2002Orlando D. Gil Abstract Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 190,204, 2002 [source] Centrosome amplification induced by the antiretroviral nucleoside reverse transcriptase inhibitors lamivudine, stavudine, and didanosineENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 8 2009Mia Yu Abstract In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division. Environ. Mol. Mutagen., 2009. © 2009 Wiley-Liss, Inc. [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] The Synthesis of the Dimethyl Ester of Quino[4,4a,5,6- efg]-Annulated 7-Demethyl-8-deethylmesoporphyrin and Three of Its Isomers with Unprecedented peri -Condensed Quinoline Porphyrin Structures.EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 19 2004Molecules with Outstanding Properties as Sensitizers for Photodynamic Therapy in the Far-Red Region of the Visible Spectrum Abstract The mesoporphyrin dimethyl ester nickel complex has been formylated via the Vilsmeier method. The four possible mono meso-formyl derivatives were isolated and characterized. Wadsworth,Emmons coupling with the anion of (diethylphosphono)acetonitrile converted these aldehydes into the four novel meso acrylonitriles. Brief treatment of these acrylonitrile systems in hot trichloroacetic acid resulted in the formation of four achiral porphyrin derivatives with unprecedented nickel complexes of quino-fused porphyrins. Subsequent removal of the nickel gave four quino-porphyrin free bases: quino[4,4a,5,6- efg]-annulated 7-demethyl-8-deethylmesoporphyrin dimethyl ester 6a, 2,-(methoxycarbonyl)quino[4,4a,5,6- jkl]-annulated 12-demethyl-13-de[2,-(methoxycarbonyl)ethyl]mesoporphyrin dimethyl ester 6b, 2,-(methoxycarbonyl)quino[4,4a,5,6- qrs]-annulated 18-demethyl-17-de(2,-methoxycarbonylethyl)mesoporphyrin dimethyl ester 6c and quino[4,5,6,7- abt]-annulated 2-demethyl-3-deethylmesoporphyrin dimethyl ester 6d. The structures of these systems were unambiguously determined via mass spectroscopy and a plethora of NMR techniques. In the same way, etioporphyrin and octaethylporphyrin were converted into the corresponding peri -condensed quinoporphyrins as products, which shows that the formation of novel pericondensed quino-porphyrins is a general reaction in the porphyrin series and will have a wide scope in this field. Also, a plausible reaction mechanism for the formation of the quinoporphyrin systems was derived. As a first test for the use of these systems as sensitizers in far-red phototherapy, the quantum yield of singlet oxygen generation by 6a in toluene was studied. This quantum yield is 0.77, which is even higher than the singlet oxygen generation by sensitized meso-tetraphenylporphyrin. Secondly, when Chinese Hamster ovary (CHO) cells were incubated in medium which contained up to 15 ,g/ml of 6a, the survival rate of the cells in the dark is complete within experimental error, showing that under these conditions, 6a is not toxic to CHO cells. When CHO cells incubated in medium containing 6a in concentrations of 1 ,g/ml and higher were treated with white light of intensity 30 mW/cm2 for 15 minutes, complete cell death was observed. Based on these facts, we expect that all four achiral systems will show very promising properties to form the basis of a photodynamic therapy in far-red light. The fact that these systems are achiral is an additional bonus for medical applications. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locusFEBS JOURNAL, Issue 3 2003Potencial role in the glucocorticoid response Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein , (C/EBP,), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBP, cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (,350/,311 and ,628/,608), increase reporter gene expression when cotransfected with a C/EBP, expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBP, and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBP, accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBP, binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBP,, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBP, participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids. [source] Engineering of a monomeric and low-glycosylated form of human butyrylcholinesteraseFEBS JOURNAL, Issue 2 2002Expression, characterization, crystallization, purification Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source] Coupling of endothelin receptors to the ERK/MAP kinase pathway,FEBS JOURNAL, Issue 20 2001Roles of palmitoylation Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ETA and ETB. To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G, protein binding capacity. Endothelin-1 stimulation of wild-type ETA or ETB induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ETA and ETB mutants failed to stimulate ERK. A truncated ETB lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G,q, but not of G,i or G,s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ETA - and ETB -mediated ERK activation whereas blockage of phospholipase C-, completely abrogated endothelin-promoted ERK activation through ETA and ETB in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G,q/phospholipase C-,/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway. [source] Effect of cadmium on the relationship between replicative and repair DNA synthesis in synchronized CHO cellsFEBS JOURNAL, Issue 22 2000Gaspar Banfalvi Repair and replicative DNA synthesis were measured at different stages of the cell cycle in control and cadmium-treated Chinese hamster ovary (CHO-K1) cells. Cells were synchronized by counterflow centrifugal elutriation. Elutriation resulted in five repair and four replication subphases. On Cd treatment, repair synthesis was elevated in certain subphases. Replicative subphases were suppressed by Cd treatment, with some of the peaks almost invisible. The number of spontaneous strand breaks measured by random oligonucleotide primed synthesis assay showed a cell-cycle-dependent fluctuation in control cells and was greatly increased after Cd treatment throughout the S phase. Elevated levels of the oxidative DNA damage product, 8-oxodeoxyguanosine, were observed after Cd treatment, with the highest level in early S phase, which gradually declined as damaged cells progressed through the cell cycle. [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] The structure of the TGF-, latency associated peptide region determines the ability of the proprotein convertase furin to cleave TGF-,sJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Makoto Kusakabe Abstract The TGF-, family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-,1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-,2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-,2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-,2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-,2 or a chimeric TGF-, consisting of the TGF-,1 LAP region, the TGF-,2 cleavage site and the TGF-,2 mature peptide. The constructs included a c- myc epitope tag in the N-terminal region of the mature peptide. The TGF-,s produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-,2, but efficiently processed the chimeric TGF-,. This indicates that the insensitivity of both forms of TGF-,2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-,1 and -,2 may be part of the mechanism that generates isoform-specific functions of the TGF-,s. J. Cell. Biochem. 103: 311,320, 2008. © 2007 Wiley-Liss, Inc. [source] Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Wojciech Miltyk Abstract Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell,matrix and cell,cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation. Published 2005 Wiley-Liss, Inc. [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] Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentumJOURNAL OF NEUROCHEMISTRY, Issue 1 2007S. D. Clark Abstract Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 , 30 nmol/L; calcium mobility assay) and to be 10 000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 , 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures. [source] Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutationsJOURNAL OF NEUROCHEMISTRY, Issue 6 2007Norimasa Takahashi Abstract Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome. [source] Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowthJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Christine J. McNamee Abstract The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell,cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell,cell adhesion and recognition. [source] Characterization of an Antibody to the Human Melatonin mt1 ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2001L. M. Williams Abstract Melatonin acts via high affinity, G-protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C -terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt1338,352). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N -terminus (Flag-mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme-linked immunosorbent assay, was used to localize Flag-mt1 in stably transfected cells by immunofluoresence. Flag-mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2-(125I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag-mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag-mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells. [source] Role of Protein Kinases in the Prolactin-Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin ReceptorJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2000B. Sorin Abstract There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx. [source] Flocculation of biological cells: Experiment vs. theoryAICHE JOURNAL, Issue 7 2003Binbing Han Flocculation of biological cells is important in the biotechnology industry, as it could lead to improved efficiencies for bioreactor harvesting operations such as microfiltration. Experimental studies for flocculation of yeast and CHO cells using cationic polyelectrolytes suggest the existence of a steady-state, self-similar floc size distribution. The experimentally determined floc size distributions were modeled using a population balance approach. For flocculated yeast suspensions, the variation of the floc volume fraction with dimensionless particle diameter is predicted by the population balance model assuming a binary breakage distribution function. However, the variation of floc number fraction with dimensionless particle diameter is better predicted assuming a log normal fragment distribution function probably due to the presence of submicron-sized yeast cell debris. For CHO cell flocs, the floc volume and number fractions are predicted using a log normal fragment distribution function. CHO cells are far more fragile than yeast cells. Thus, individual CHO cells in a CHO cell floc can lyse leading to the formation of a number of small particles. [source] A novel functional role for the highly conserved , -subunit KVGFFKR motif distinct from integrin ,IIb,3 activation processesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2006K. AYLWARD Summary.,Background: The highly conserved integrin , -subunit membrane-proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin ,IIb,3. Previously, we have shown that a platelet permeable palmityl (pal)-peptide with this seven amino acid sequence can directly activate ,IIb,3 leading to platelet aggregation. Objectives: To investigate further the role of the KVGFFKR motif in integrin ,IIb,3 function. Methods: We used two sequence-specific complementary model systems, palmityl pal-peptides in platelets, and mutant ,IIb,3 -expressing Chinese Hamster Ovary (CHO) cell lines. Results: In platelets we show that the two phenylalanine amino acids in pal-KVGFFKR (pal-FF) peptide are critical for stimulating platelet aggregation. Pal-FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, ,IIb(F992A, F993A),3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that ,IIb,3 -mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these ,IIb,3 -mediated processes. Conclusion: In addition to regulating integrin ,IIb,3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence. [source] Involvement of the ,3 E749ATSTFTN756 region in stabilizing integrin ,IIb,3 -ligand interactionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003P. E. M. H. Litjens Summary., Platelet integrin ,IIb,3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the ,-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E,N peptide) and the T755NITYRGT762 domain (T,T peptide) of ,3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E,N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E,N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E,N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E,N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on ,IIb,3. T,T peptide did not affect these processes. In a model for outside-in integrin activation, E,N peptide disrupted the binding of CHO cells expressing ,IIb,3 to surface-bound ligand. Again, T,T peptide had no effect. We conclude that the E749ATSTFTN756 region of the ,3 -tail stabilizes the binding of soluble and surface-bound ligand to integrin ,IIb,3 via a mechanism that involves the phosphorylation of FAK. [source] ORIGINAL ARTICLE: Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co-culture with eukaryotic cellsMICROBIOLOGY AND IMMUNOLOGY, Issue 9 2010Mitsutoshi Senoh ABSTRACT VBNC Vibrio cholerae O139 VC-280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co-cultured with CHO cells. Other eukaryotic cell lines, including HT-29, Caco-2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC-280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co-culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed. [source] Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurementsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2009Aymeric Leray Abstract Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc. [source] Co-regulation of B-Myb expression by E2F1 and EGF receptor,MOLECULAR CARCINOGENESIS, Issue 1 2006Norihisa Hanada Abstract Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently over-expressed in human cancers and is associated with tumorigenesis, and increased tumor proliferation and progression. Also found in breast tumors with high levels is B-Myb, a transcription factor whose expression is activated by E2F1/3 at the late G1 phase and the level is sustained through the S phase. Recent reports suggest a casual correlation between EGFR and B-Myb expression in primary breast carcinomas. However, the mechanism for such co-expression remains un-investigated. Here, we report that EGFR is important for B-Myb expression and the underlying mechanism involves cooperated effects from EGFR and E2F1. EGF stimulation and forced expression of EGFR significantly increase B-Myb gene activity and such increase occurs in the G1 phase. EGF-induced B-Myb expression was not significantly suppressed following inhibition of PI-3K and ERK, two major EGFR downstream pathways. In contrast, we observed EGF-induced in vivo association of nuclear EGFR to the B-Myb promoter and the association is only detected at the G1/S phase and is abolished by EGFR kinase inhibitor. As EGFR lacks DNA-binding domain but contains transactivational activity and E2F1 activates B-Myb expression in the G1/S phase, we further reasoned that nuclear EGFR might cooperate with E2F1 leading to activation of B-Myb. Indeed, we found that EGFR co-immunoprecipitated with E2F1 in an EGF-dependent manner and that EGF activated in vivo binding of E2F1 to the B-Myb promoter. Consistently, forced expression of both EGFR and E2F1 in EGFR-null CHO cells greatly enhanced B-Myb promoter activity, compared to the vector control and expression of EGFR or E2F1 alone. Promoter mutagenesis studies showed that EGF-induced activation of B-Myb promoter required both E2F and EGFR target sites. In summary, our data suggest that deregulated EGFR signaling pathway facilitate tumor cell proliferation partly via EGFR interaction with E2F1 and subsequent activation of B-Myb gene expression. © 2005 Wiley-Liss, Inc. [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] Gene for porcine pregnancy-associated glycoprotein 2 (poPAG2): Its structural organization and analysis of its promoter,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Bozena Szafranska Abstract The pregnancy-associated glycoproteins (PAG) are abundant secretory products of the placental trophectoderm of ungulate species. They are structurally related to pepsin, having the capability to bind peptides. However, many cannot function as enzymes due to amino acid substitutions in and around the catalytic site. Here, we demonstrate that pigs, like cattle and sheep, but unlike equids, have multiple PAG genes. One of the transcribed porcine PAG (poPAG) genes, the one for poPAG2, was cloned. It had a nine-exon organization similar to that of other mammalian aspartic proteinase genes with an atypical TATA sequence. A total of 1.2 kbp upstream from exon 1 was sequenced. This region shared identity (>,65%) with the promoter regions of the bovine (bo) PAG1, boPAG2 and equine (eq) PAG genes, but not with other aspartyl proteinase genes, including that of pepsinogen A. Nor were there clear similarities to the promoters of other genes with trophoblast-specific expression. Of the different poPAG2 promoter constructs tested in transfection experiments in two human (JAr and JEG3) and one rat (Rcho) choriocarcinoma cell lines, only the shortest (,149 bp) was required to provide full expression of a luciferase reporter. Although this short promoter was not active in Cos-1 and L-929 cells, it was active in CHO cells, a transformed non-trophoblast hamster ovarian cell line. Co-transfection of Ets2 elevated the activity of this short promoter approximately six-fold in JAr cells, but, disruption of the two putative Ets sites did not alter the ability of Ets2 to transactivate the promoter. In the non-trophoblast cell lines, Ets2 failed to elicit any response. Ets2 responsiveness may be a common feature of most or all trophoblast-expressed genes, although in the case of poPAG2, the effect may be indirect. Mol. Reprod. Dev. 60: 137,146, 2001. © 2001 Wiley-Liss, Inc. [source] Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharidesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Johannes Stadlmann Abstract Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods. [source] | ||||||||||||||||||||||||||||||||||