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Hamster Ovary (hamster + ovary)
Kinds of Hamster Ovary Terms modified by Hamster Ovary Selected AbstractsOpposite effects of overexpressed myosin Va or heavy meromyosin Va on vesicle distribution, cytoskeleton organization, and cell motility in nonmuscle cellsCYTOSKELETON, Issue 3 2008Robbin D. Eppinga Abstract Myosin Va, an actin-based motor protein that transports intracellular cargos, can bundle actin in vitro. Whether myosin Va regulates cellular actin dynamics or cell migration remains unclear. To address this, we compared Chinese Hamster Ovary (CHO) cells that stably express GFP fused to either full length mouse myosin Va (GFP-M5) or heavy meromyosin Va (GFP-M5,). GFP-M5 and GFP-M5, co-immunoprecipitate with CHO myosin Va and serve as overexpression of wild-type and dominant negative mutants of myosin Va. Compared to non-expressing control cells, GFP-M5-overexpressing cells have peripheral endocytic vesicles, spread slowly after plating, as well as produce robust interior actin stress fibers, myosin II bundles, and focal adhesions. However, these cells display normal cell migration and lamellipodial dynamics. In contrast, GFP-M5,-expressing cells have perinuclear endocytic vesicles, produce thin interior actin and myosin bundles and contain no interior focal adhesions. In addition, these cells spread rapidly, migrate slowly and display reduced lamellipodial dynamics. Similarly, neurite outgrowth is compromised in neurons cultured from transgenic Drosophila that express M5,-dsRed and in neurons cultured from Drosophila that produce a tailless version of endogenous myosin V. Together, these data suggest that myosin Va overexpression induces actin bundles in vivo whereas the tailless version fails to bundle actin and disrupts cell motility. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [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] TLS-CHOP target gene DOL54 expression in liposarcomas and malignant fibrous histiocytomasPATHOLOGY INTERNATIONAL, Issue 8 2002Hideharu Domoto Downstream of the gene for the liposarcoma-associated fusion oncoprotein 54 (DOL54) is a target gene of the myxoid liposarcoma and round cell liposarcoma (M-LPS/RC-LPS) oncogene, TLS/FUS-CHOP. The DOL54 gene product is closely associated with adipogenic differentiation. DOL54 overexpression resulted in tumorigenicity when Chinese Hamster Ovary (CHO) cells were injected subcutaneously into nude mice. The biological significance of DOL54 expression for human malignant soft tissue tumors, however, has not yet been investigated. We examined TLS-CHOP and DOL54 expression in M-LPS/RC-LPS, well-differentiated liposarcoma and malignant fibrous histiocytoma (MFH), a tumor whose cellular origin has not been determined. We observed DOL54 expression in 50% of M-LPS/RC-LPS cases (in which TLS-CHOP was also expressed) and 33% of MFH cases, suggesting that a portion of MFH lesions may either derive from adipocytic precursor cells or have the potential to undergo adipogenic differentiation. In this manner, M-LPS/RC-LPS and MFH lesions may share tumorigenic characteristics, resulting from the unscheduled expression of DOL54. [source] Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1ANIMAL GENETICS, Issue 3 2001E. Meijerink In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an ,(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl- ,(1,2)galactopyranoside; the enzyme did not use phenyl- , - D -galactopyranoside (phenyl- , -D-Gal) as an acceptor. Because the ,(1,3)GalNAc transferase gene product requires an ,(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the ,(1,2)fucosyltransferase gene product is necessary for the functioning of the ,(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. Abbreviations: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fuc,(1,2)Gal: fucosyl- ,(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Gal,(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl- , -D-Gal: phenyl- , - D -galactopyranoside; R: Gal,1-4Glc,1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine [source] Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometricsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Boyan Li Abstract The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous-based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins. The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for "in-house" sample handling, tracking, and quality control. Biotechnol. Bioeng. 2010;107: 290,301. © 2010 Wiley Periodicals, Inc. [source] Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Donglin Guo Abstract An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine,guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity. Biotechnol. Bioeng. 2010;107: 163,171. © 2010 Wiley Periodicals, Inc. [source] Triple light chain antibodies: Factors that influence its formation in cell cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Natalia Gomez Abstract THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species,Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression,evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC),correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production,evaluated as the ratio of the cell-specific production rate of GSH (qGSH) to the cell-specific production rate of THIOMAB (qp),corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748,760. © 2009 Wiley Periodicals, Inc. [source] Using microarray technology to select housekeeping genes in Chinese hamster ovary cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009Scott M. Bahr Abstract In the present study, we have identified species-specific housekeeping genes (HKGs) for Chinese Hamster Ovary (CHO) cells using data from microarray gene expression profiling. HKGs suitable for quantitative RT-PCR normalization should display relatively stable expression levels across experimental conditions. We analyzed transcription profiles of several IgG-producing recombinant CHO cell lines under numerous culture conditions using a custom CHO DNA microarray platform and calculated relative expression variability from 124 microarrays. We selected a novel panel of candidate HKGs based on their low variability in expression from the microarray data. Compared to three traditional HKGs (Gapdh, Actb, and B2m), the majority of genes on this panel demonstrated lower or equal variability. Each candidate HKG was then validated using qRT-PCR. Final selection of CHO-specific HKGs include Actr5, Eif3i, Hirip3, Pabpn1, Vezt, Cog1, and Yaf2. The results reported here provide a useful tool for gene expression analyses in CHO cells, a critical expression platform used in biotherapeutics. Biotechnol. Bioeng. 2009; 104: 1041,1046. © 2009 Wiley Periodicals, Inc. [source] RNA interference of sialidase improves glycoprotein sialic acid content consistencyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Frederyk A. Ngantung Abstract An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-, produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-,, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. © 2006 Wiley Periodicals, Inc. [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] 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] Incorporation of vinylogous scaffolds in the C-terminal tripeptide of substance PCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2004S. Claudel Abstract:, Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Pro,(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Pro,((E) -CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Pro,((Z) -CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9,((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9, ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z) - or (E) -CH=C(CH3)- or (Z) -CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group. [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] Genetic variants of insulin receptor substrate-1 (IRS-1) in syndromes of severe insulin resistance.DIABETIC MEDICINE, Issue 10 2002Functional analysis of Ala513Pro, Gly1158Glu IRS- Abstract Aims To define further the role of IRS-1 mutations in human syndromes of severe insulin resistance. Methods The IRS-1 gene was scanned for mutations in 83 unrelated affected subjects and 47 unaffected individuals using fluorescent single-strand conformation polymorphism (fSSCP) analysis. A novel heterozygous mutation, Gly1158Glu, was found in one affected subject. Four and two subjects were heterozygous for the previously reported variants Gly972Arg and Ala513Pro, respectively. The previously identified variant Gly819Arg was found in one affected and one unaffected subject. While Gly972Arg has been described to alter the signalling properties of IRS-1, no functional studies of Ala513Pro or Gly1158Glu have been reported. Results Chinese hamster ovary (CHO) cells stably over-expressing the insulin receptor were transiently transfected with vectors expressing either wild-type, Glu1158 or Pro513 IRS-1. A modest increase in insulin-stimulated tyrosine phosphorylation of Glu1158 IRS-1 was observed. However, this did not result in any significant change in the association of Grb2 or the p85, subunit of PI3-kinase or of PI3-kinase activity. In parallel studies, the Pro513 IRS-1 variant was indistinguishable from wild-type IRS-1. Conclusions While subtle effects of these variants cannot be excluded in this system, it is unlikely that these variants are responsible for the extreme insulin resistance seen in the subjects harbouring them. Although IRS proteins play a central role in insulin signalling, functionally significant mutations in the IRS-1 gene are a rare cause of human syndromes of severe insulin resistance. [source] Bacterial and mammalian-cell genotoxicity of mixtures of chlorohydroxyfuranones, by-products of water chlorinationENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2004Jorma Mäki-Paakkanen Abstract The genotoxic responses of mixtures of four chlorohydroxyfuranones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro- 4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF), were compared with the genotoxicity of the individual compounds. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test), the in vitro Chinese hamster ovary (CHO) cell Hprt mutation assay, and in the CHO chromosome aberration test. When tested individually, the concentrations of the chemicals that were chosen for the mixtures induced no or only a modest increase in the genotoxic effects, and caused little or no cytotoxicity. In the Ames test, the genotoxic responses caused by the mixtures of CHFs did not follow simple additivity. Synergism was observed with strains TA97 and TA98, and antagonism with strain TA100. In the CHO/Hprt mutation assay, the mutagenic response of the mixtures was inconsistent, with near additivity seen with a mixture of CHFs that resulted in 12% cell survival. In contrast, the four CHFs together consistently caused more structural chromosome damage (mainly chromatid-type breaks and exchanges) compared to the sum of net effects of the four CHFs tested alone. Also, a potentiating effect was consistently seen for the cytotoxicity of the CHF mixtures both in the CHO/Hprt mutation assay and the chromosome aberration test. The present results indicate that the genotoxic effects of CHF mixtures can be greater than additive. Such effects may be worth considering in the cancer risk assessment of chlorinated drinking water. Environ. Mol. Mutagen. 43:217,225, 2004. © 2004 Wiley-Liss, Inc. [source] Cloning, distribution and functional analysis of the type III sodium channel from human brainEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2000Yu Hua Chen Abstract The type III voltage-gated sodium channel was cloned from human brain. The full-length cDNA has 89% identity with rat type III, and the predicted protein (1951 amino acids) has 55 differences. The expression pattern of human type III mRNA was determined in adult brain tissue and, in contrast to rat, was detected in many regions, including caudate nucleus, cerebellum, hippocampus and frontal lobe. The human type III channel was stably expressed in Chinese hamster ovary (CHO) cells and its biophysical properties compared to the human type II channel using identical conditions. The voltage dependence and kinetics of activation were found to be similar to that of type II. The kinetics of inactivation of the two human subtypes were also similar. However, type III channels inactivated at more hyperpolarized potentials and were slower to recover from inactivation than type II. When expressed in human embryonic kidney (HEK293T) cells, type III channels produced currents with a prominent persistent component, which were similar to those reported for rat type II [Ma et al. (1997) Neuron, 19, 443,452]. However, unlike type II, this was prominent even in the absence of coexpressed G-proteins, suggesting type III may adopt this gating mode more readily. The distinct properties of the channel, together with its wide distribution in adult brain, suggest that in humans, type III may have important physiological roles under normal, and perhaps also pathological conditions. [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] 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] Rough and smooth forms of fluorescein-labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentrationFEBS JOURNAL, Issue 8 2000Martha Triantafilou Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram-negative bacteria. Bacteria express either smooth LPS, which is composed of O-antigen (O-Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS-binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC-LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC-LPS to cells was both CD14- and LBP-dependent for LPS concentrations up to 100 ng·mL,1. At LPS concentrations higher than 100 ng·mL,1 we observed CD14/LBP-independent binding. CD14/LBP-dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti-CD14 antibody (MY4) or unlabelled LPS could outcompete it. [source] Cloning and molecular dissection of the 8.8 kb pig uroplakin II promoter using transgenic mice and RT4 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Deug-Nam Kwon Abstract Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.6 kb mUPII promoter was also detected in brain, kidney, and testis in some transgenic mouse lines. Here, we have cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to human erythropoietin (hEPO) or a luciferase gene. pUPII-luciferase expression vectors with various deletions of the promoter region were introduced into mouse fibroblast (NIH3T3), Chinese hamster ovary (CHO), and human bladder transitional carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high levels of luciferase activity in transiently transfected RT4 cells, whereas the 8.8 kb pUPII promoter region displayed only low levels of activity. The pUPII-hEPO expression vector was injected into the pronucleus of zygotes to make transgenic mice. To elucidate the in vivo molecular mechanisms controlling the tissue- and cell-specific expression of the pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII promoter fragments linked to the genomic hEPO gene were generated. An erythropoietin (EPO) assay showed that all nine transgenic lines carrying the 8.8 kb construct expressed recombinant human erythropoietin (rhEPO) only in their urethra and bladder, whereas two transgenic lines carrying the 2.1 kb pUPII promoter displayed hEPO expression in several organs including bladder, kidney, spleen, heart, and brain. These studies demonstrate that the 2.1 kb promoter contains the DNA elements necessary for high levels of expression, but lacks critical sequences necessary for tissue-specific expression. We compared binding sites in the 2.1 and 8.8 kb promoter sequences and found five peroxisome proliferator responsive elements (PPREs) in the 8.8 kb promoter. Our data demonstrated that proliferator-activated receptor (PPAR)-, activator treatment in RT4 cells induced the elevated expression of hEPO mRNA under the control of the 8.8 kb pUPII promoter, but not the 2.1 kb promoter. Collectively, our data suggested that all the major trans-regulatory elements required for bladder- and urethra-specific transcription are located in the 8.8 kb upstream region and that it may enhance tissue-specific protein production and be of interest to clinicians who are searching for therapeutic modalities with high efficacy and low toxicity. J. Cell. Biochem. 99: 462,477, 2006. © 2006 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] Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Natalia A. Dorofeeva Abstract The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC50 were 622 ± 34 ,M and 3.42 ± 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 ,M were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification. [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] Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-,1, an epidermal growth factor-like domainJOURNAL OF PEPTIDE SCIENCE, Issue 3 2008Taeko Kakizawa Abstract The human neuregulin 1-,1 (NRG1-,1, amino acid residues 176,246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-,1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Binding and functional affinity of some newly synthesized phenethylamine and phenoxypropanolamine derivatives for their agonistic activity at recombinant human ,3 -adrenoceptorJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2003Maruf Ahmed ABSTRACT ,3 -Adrenoceptor is the predominant ,-adrenoceptor in adipocytes and has drawn much attention during the investigation for anti-obesity and antidiabetes therapeutics. Thirteen new compounds have been evaluated for their potencies and efficacies as ,3 -adrenoceptor agonists on human ,3 - adrenoceptor expressed in COS-7 and Chinese hamster ovary (CHO) cells using radio ligand binding assay and cyclic AMP (cAMP) accumulation assay. Phenoxypropanolamine derivatives, SWR-0334NA (([E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy]acetic acid sodium salt), SWR-0335SA ((E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0342SA (S-(Z)-[4-[[1-[2-[(2-hydroxy,3-phenoxypropyl)]amino]ethyl]-1-pro-penyl]phenoxy] acetic acid ethanedioic acid), SWR-0348SA-SITA ((E)-[4-[5-[(3-phenoxy-2-hydroxy-propyl)amino]-2-hexene,3-yl] phenoxy]acetic acid ethanedioic acid) and SWR-0361SA ((E)-N-methyl-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl]phenoxy]acetoamide ethanedioic acid) showed higher agonistic activity for the ,3 -adrenoceptor. Among the compounds tested, SWR-0334NA exhibited full agonist activity (%Emax = 100.26) despite its lower binding affinity (pK1 = 6.11). Compounds SWR-0338SA((E)-[4-[5-[(2-phenyl-2-hydroxyethyl)amino]-2-pentene,3-yl]phenoxy]acetic acid ethanedioic acid), SWR-0339SA (S-(E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene,3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0345HA ((E)-2-methyl,3-[4-[2-(2-phenyl-2-hydroxyethyl-amino)ethoxy] phenyl]-2-propenoic acid ethyl ester hydrochloride), SWR-0358SA ((E)-(2-methoxy-ethyl)-[4-[5-[(3-phenoxy-2-hydroxypropyl) amino]-2-pentene,3-yl]phenoxy]acetoamide ethanedioic acid) and SWR-0362SA ((E)-1-[[[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene ,3-yl]phenoxy]-acetyl]carbonyl]piperidine ethanedioic acid) had moderate agonistic activity and were phenethylamine and phenoxypropanolamine derivatives. Compounds SWR-0065HA ([4-[2-[3-[[(3,4-dihydro-4-oxo-[1,2,4]-triazino(4,5-a)indol)-lyl]oxy]-2-hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR-0098NA ((E)-[4-[3-[(2-phenyl-2-hydroxyethyl)amino]-1-butenyl] phenoxy]-acetic acid sodium salt) and SWR-0302HA ([4-[[4-[2-(3-chlorophenoxy-2-hydroxypropyl)amino]-E-2-butenyl]oxy]phenoxy]acetic acid hydrochloride) had very low binding affinity towards ,3 -adreno-ceptors and they did not induce cAMP accumulation. We concluded that compounds SWR-0334NA, SWR-0335SA, SWR-0342SA, SWR-0348SA-SITA and SWR-0361SA were potential agonists of human ,3 - adrenoceptor. Further investigation on their selectivity towards ,3 -adrenoceptor could be useful for the exploration of the physiological properties of the ,3 -adrenoceptor. [source] Evaluation of the Effect of Ethanol's Toxic Metabolite Acetaldehyde on the Gastrointestinal Oligopeptide Transporter, PEPT1: In Vitro and in Vivo StudiesALCOHOLISM, Issue 1 2008Scott J. Fisher Background:, The effects of alcohol consumption and its subsequent metabolism on drug transport, absorption and pharmacokinetics are poorly understood. This study examines the effects of the ethanol metabolite, acetaldehyde, on the clinically relevant drug transporter, PEPT1. The metabolism of ethanol and the following acetaldehyde formation is thought to modulate the uptake capacity of PEPT1 within the gastrointestinal tract for a variety of clinically important peptidomimetic drug compounds. Methods:, Glycylsarcosine ([3H]-GlySar), a nonhydrolysable PEPT1 specific substrate was used in our studies. In vitro uptake studies were performed in the Caco-2 and Chinese hamster ovary (CHO)-hPEPT1 cell models, measuring cellular uptake of labeled compound against increasing levels of unlabeled compound in the presence of acetaldehyde. In vivo absorption of [3H]-GlySar was measured in male Sprague,Dawley rats that were treated with oral dose of ethanol/disulfiram (5 g/kg / 100 mg/kg) for 6 days. These results were compared to control rats treated with saline, ethanol alone or disulfiram alone. Results:, In vitro uptake of [3H]-GlySar in CHO-hPEPT1 cells treated with 1 mM acetaldehyde was significantly decreased (p < 0.05) as compared to untreated controls. The uptake of [3H]-GlySar in Caco-2 cell monolayers treated with 1 mM acetaldehyde was also significantly decreased as compared to the untreated control cells. In vivo absorption of [3H]-GlySar in ethanol treated rats, as measured by AUC0,12 hours were decreased by approximately 50% versus the control rat group. Conclusion:, The effects of acetaldehyde due to consumption of ethanol on the uptake and bioavailability of therapeutic drug compounds transported by the PEPT1 oligopeptide transporter have not been documented. In the present studies, we demonstrate that acetaldehyde significantly modulates PEPT1 function and, thereby, affects drug bioavailability. To our best knowledge, this is the first report on the effects of an ethanol metabolite on substrate absorption in the gastrointestinal tract, rather than interactions in the liver, which is an under-represented area of research in alcohol pathophysiology. [source] The glycoprotein Ib,,von Willebrand factor interaction induces platelet apoptosisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2010S. LI Summary.,Background: The interaction of glycoprotein (GP) Ib, with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIb,,VWF interaction. Objectives: To investigate whether the GPIb,,VWF interaction induces platelet apoptosis and the role of 14-3-3, in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb,IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin-induced GPIb,,VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIb, antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb,IX with GPIb, truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3,-binding site in GPIb,. Conclusions: This study demonstrates that the GPIb,,VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3, with the cytoplasmic domain of GPIb, is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. [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] Thrombin induces GPIb-IX-mediated fibrin binding to ,IIb,3 in a reconstituted Chinese hamster ovary cell modelJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2006D. PABÓN Summary.,Background: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. Objectives: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. Methods: We used Chinese hamster ovary (CHO) cells stably expressing human ,IIb,3 and/or GPIb-IX complexes (CHO- ,IIb,3 -IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. Results: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO- ,IIb,3 -IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against ,IIb,3 and GPIb,. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3, adaptor and calcium,calmodulin-dependent events are involved. Conclusions: The present data support a significant role of GPIb-IX and ,IIb,3 receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin. [source] | |||||||||||||||||||||||||||||||