Science.gov

Sample records for hamster ovary cho-k1

  1. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. PMID:26762773

  2. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line).

    PubMed

    Zwolak, Iwona

    2015-08-01

    This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity

  3. Analysis of possible genotoxicity of the herbicide flurochloridone and its commercial formulations: Endo III and Fpg alkaline comet assays in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Nikoloff, Noelia; Larramendy, Marcelo L

    2016-02-01

    Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold(®) and Rainbow(®) were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold(®) or Rainbow(®) induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold(®)-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold(®)-, or Rainbow(®)-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold(®) exposure are due to the excipients contained within the technical formulation rather than FLC itself. PMID:26921020

  4. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    SciTech Connect

    Kramer, J.M. . Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  5. Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis.

    PubMed Central

    Vallis, K. A.; Reglinski, J.; Garner, M.; Bridgeman, M. M.; Wolf, C. R.

    1997-01-01

    A cell line (MRc40) resistant to the model quinone compound, menadione, has been isolated from a parental Chinese hamster ovary cell line (CHO-K1). The known relationship between menadione toxicity and glutathione (GSH) depletion led us to investigate whether the mechanism of resistance of MRc40 was related to alteration in GSH homeostasis. Intracellular concentrations of GSH and cysteine (CySH) were twofold and 3.2-fold greater in MRc40 than in CHO-K1. Following exposure to menadione, GSH and CySH were depleted, but subsequent recovery of thiols was more rapid and of greater magnitude in MRc40 than in CHO-K1. Twelve hours after exposure to menadione, the concentrations of GSH and CySH were 9.7- and 4.2-fold greater in MRc40 than in CHO-K1. Using nuclear magnetic resonance (NMR) spectroscopy, we observed the in situ removal of menadione from cell suspensions of CHO-K1 and MRc40. However, only in CHO-K1 did we observe concomitant depletion of NMR-visible GSH. We conclude that the perturbation of GSH metabolism contributes to the resistant phenotype and is an important characteristic of menadione-resistant CHO cells. PMID:9328145

  6. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

    PubMed Central

    Hanot, Camille C.; Choi, Young Suk; Anani, Tareq B.; Soundarrajan, Dharsan; David, Allan E.

    2015-01-01

    Superparamagnetic iron-oxide nanoparticles (SPIONs) show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells). We evaluated the effect of particle diameter (50 and 100 nm) and polyethylene glycol (PEG) chain length (2k, 5k and 20k Da) on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS). Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications. PMID:26729108

  7. G-protein-dependency of orexin/hypocretin receptor signalling in recombinant Chinese hamster ovary cells.

    PubMed

    Kukkonen, Jyrki P

    2016-08-01

    Multiple signalling pathways for orexin receptors have been discovered, and most thoroughly mapped in Chinese hamster ovary K1 (CHO-K1) cells. It is also known that orexin receptors can couple to the G-protein families Gi, Gs and Gq. However, the connection between the G-proteins and the downstream signals is only vaguely established, and we now set out to resolve this for human orexin receptors expressed in CHO-K1 cells. Adenylyl cyclase (AC), phospholipase A2, C and D, and diacylglycerol lipase activities were assessed by precursor radiolabelling and chromatographic separation, and calcium by fluorescent methods. Pertussis toxin, cholera toxin and the cyclic depsipeptide, UBO-QIC a.k.a. FR900359, were used to assess the involvement of Gi-, Gs- and Gq-family G-proteins, respectively. Calcium elevations as well as activation of the phospholipases and diacylglycerol lipase were dependent on Gq, as they were fully blocked by UBO-QIC. The low-potency AC activation fully depended on Gs. Surprisingly, the assumed Gi-dependent inhibition of AC was (fully or partially) inhibited by UBO-QIC, in opposition to the previous findings of no sensitivity of Gi proteins to UBO-QIC. Orexin receptor signalling is indeed mostly Gq-driven in CHO-K1 cells, even with respect to the less clearly mapped cascades such as phospholipase A2 and C and calcium influx, underlining the importance of Gq even under physiological conditions. AC regulation warrants more studies. PMID:27237973

  8. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    SciTech Connect

    Hirano, Seishiro; Fujitani, Yuji; Furuyama, Akiko; Kanno, Sanae

    2012-02-15

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC{sub 50} value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  9. Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

    PubMed Central

    Knox, Aaron J.; Graham, Christine; Bleskan, John; Brodsky, Gary; Patterson, David

    2009-01-01

    Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells. PMID:19007868

  10. [Stable expression of human anti-IL-33 scFv-IgG1Fc fusion protein in CHO k1 cells].

    PubMed

    Ye, Yingchun; Nian, Siji; Wang, Xu; Wu, Tong; Xu, Wenfeng; Yuan, Qing

    2016-05-01

    Objective To construct two different eukaryotic expression vectors of human anti-interleukin 33 (IL-33) single-chain antibody fragment (scFv-Fc) to transfect Chinese hamster ovary (CHO) k1 cells and select the stably and high-level expressed cell lines to improve the expression level of the fusion protein. Methods The previously constructed recombinant plasmid pcDNA3.1/SP-scFv-Fc was digested to obtain SP-scFv-Fc fragments, and the fragments were inserted into the plasmid PMH3(EN) to construct recombinant plasmid PMH3(EN)/SP-scFv-Fc. The plasmids PMH3(EN)/SP-scFv-Fc and pcDNA3.1/SP-scFv-Fc were separately transfected into CHO k1 cells. The transcription and translation level of the SP-scFv-Fc were detected by reverse transcription PCR (RT-PCR) and Western blotting, respectively. The stably and high-level expressed cell lines were screened by Dot blotting. The expression level and binding activity of the expressed scFv-Fc were measured by ELISA. Results The recombinant plasmid PMH3(EN)/SP-scFv-Fc was successfully constructed and the size of the inserted SP-scFv-Fc was about 1560 bp. The RT-PCR results showed that the SP-scFv-Fc was successfully transfected into CHO k1 cells. The scFv-Fc proteins could be secreted into the cultural supernatant and specifically bind to human IL-33 and anti human IgG1 Fc antibody. The expression level of scFv-Fc in plasmid PMH3(EN) was higher than that in plasmid pcDNA3.1. After four rounds of screening, the stably and high-level expressed cell strains were obtained. The expression level of the scFv-Fc was about 10 mg/L. The competitive ELISA results showed that the expressed scFv-Fc fusion proteins could inhibit the binding of IL-33 to ST2. Conclusion The anti-IL-33 scFv-Fc proteins were highly expressed in CHO k1 cells. PMID:27126936

  11. Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency.

    PubMed

    Vliet, Lydia K; Wilkinson, Terry G; Duval, Nathan; Vacano, Guido; Graham, Christine; Zikánová, Marie; Skopova, Vaclava; Baresova, Veronika; Hnízda, Aleš; Kmoch, Stanislav; Patterson, David

    2011-01-01

    Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency. PMID:20884265

  12. Molecular characterization of the AdeI mutant of Chinese hamster ovary Cells: a cellular model of adenylosuccinate lyase deficiency

    PubMed Central

    Vliet, Lydia K.; Wilkinson, Terry G.; Duval, Nathan; Vacano, Guido; Graham, Christine; Zikánová, Marie; Skopova, Vaclava; Baresova, Veronika; Hnízda, Aleš; Kmoch, Stanislav; Patterson, David

    2010-01-01

    Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (Ade I) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and Ade I cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in Ade I ADSL. This substitution lies in the “signature sequence” of ADSL, inactivates the enzyme, and validates Ade I as a cellular model of ADSL deficiency. PMID:20884265

  13. Profiling highly conserved microrna expression in recombinant IgG-producing and parental Chinese hamster ovary cells.

    PubMed

    Lin, Nan; Davis, Angela; Bahr, Scott; Borgschulte, Trissa; Achtien, Katherine; Kayser, Kevin

    2011-07-01

    MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY™ miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT-PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44-derived CHO lines producing a recombinant human IgG. We observed that miR-221 and miR-222 were significantly downregulated in all IgG-producing cell lines when compared with parental DG44, whereas miR-125b was significantly downregulated in one IgG-producing line. In another IgG-producing line, miR-19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let-7b and miR-221 were significantly downregulated. In parental CHO K1, let-7b, miR-15b, and miR-17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. PMID:21692195

  14. Proteomic Analysis of Chinese Hamster Ovary Cells

    PubMed Central

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E.; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N.; Krag, Sharon S.; Cole, Robert N.; Palsson, Bernhard O.; Zhang, Hui; Betenbaugh, Michael

    2013-01-01

    In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most a 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using CHO genome exclusively which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. 504 of the detected proteins included N-acetylation modifications and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  15. Proteomic analysis of Chinese hamster ovary cells.

    PubMed

    Baycin-Hizal, Deniz; Tabb, David L; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N; Krag, Sharon S; Cole, Robert N; Palsson, Bernhard O; Zhang, Hui; Betenbaugh, Michael

    2012-11-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  16. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis. PMID:26803706

  17. Cell growth stimulating effect of Ganoderma lucidum spores and their potential application for Chinese hamster ovary K1 cell cultivation.

    PubMed

    Li, Ding; Zhong, Qi; Liu, Tingting; Wang, Jufang

    2016-06-01

    In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production. PMID:26921102

  18. Cytoprotective effect of resveratrol diastereomers in CHO-K1 cells exposed to beauvericin.

    PubMed

    Mallebrera, B; Brandolini, V; Font, G; Ruiz, M J

    2015-06-01

    Beauvericin (BEA) causes cytotoxicity, lipid peroxidation and reactive oxygen species in CHO-K1 cells. Resveratrol (RSV) is a polyphenol with multiple biological properties, including antioxidant effects. RSV has two forms: trans and cis. The aims of this study were to determine the cytoprotective effect of trans-RSV and diastereomers mixtures (50:50 trans/cis-RSV and 70:30 trans/cis-RSV) incubated alone and in combination with BEA in ovarian (CHO-K1) cells. The results demonstrated that cell viability increases (from 9% to 77%) when they were exposed to low concentration of RSV. Moreover, when the cells were pre-treated with RSV and then exposed to BEA, a cytoprotective effect (from 25% to 76%) and a ROS production diminution (from 27% to 92%) were observed, with respect to cells exposed to BEA without previous RSV exposure. RSV pre-treatment decreased the MDA levels (from 15% to 37%) when it is compared with cells exposed only to BEA. Therefore, it can be concluded that RSV could reduce the toxicological risk produced by BEA when they are in combination. PMID:25843362

  19. Multi-omic profiling -of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

    PubMed

    Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2015-11-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+) , adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. PMID:25995028

  20. Quantitative mutagenesis and mutagen screening with Chinese hamster ovary cells

    SciTech Connect

    Hsie, A.W.; San Sebastian, J.R.; Tan, E.L.

    1980-01-01

    A summary is presented on the development of a specific gene mutation assay, the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system, and the utilization of this system to study structure-activity relationship affecting cytotoxicity and gene mutation by various carcinogens. Then, preliminary development and validation of a Multiplex CHO System for the simultaneous determination of chromosome aberration, sister chromatid exchange in addition to cytotoxicity and gene mutation is presented. The potential use of a CHO/human cell hybrid system for measuring chromosomal deletion and loss is discussed.

  1. Homologous desensitization of human histamine H₃ receptors expressed in CHO-K1 cells.

    PubMed

    Osorio-Espinoza, Angélica; Escamilla-Sánchez, Juan; Aquino-Jarquin, Guillermo; Arias-Montaño, José-Antonio

    2014-02-01

    Histamine H₃ receptors (H₃Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H₃Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H₃R (hH3R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [(35)S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [(3)H]-N-methyl-histamine ([(3)H]-NMHA) to intact cells and modified the sub-cellular distribution of H₃Rs, as detected by sucrose density gradients and [(3)H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl₂ and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH₃Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles. PMID:24161268

  2. Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

    PubMed

    Fujimoto, Yasuyuki; Kamakura, Aya; Motohashi, Yu; Ohashi-Kobayashi, Ayako; Maeda, Masatomo

    2011-01-01

    The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-β-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol. PMID:21212514

  3. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins. PMID:8552660

  4. Cytotoxic effects induced by patulin, sterigmatocystin and beauvericin on CHO-K1 cells.

    PubMed

    Zouaoui, Nidhal; Mallebrera, Beatriz; Berrada, Houda; Abid-Essefi, Salwa; Bacha, Hassen; Ruiz, Maria-Jose

    2016-03-01

    Mycotoxins are produced by different genera of fungi; mainly Aspergillus, Penicillium and Fusarium. The natural co-occurrence of beauvericin (BEA), patulin (PAT) and sterigmatocystin (STE) has been proved in feed and food commodities. This study investigates the cytotoxicity of individual and combined mycotoxins BEA, PAT and STE. The cytotoxicity on immortalized ovarian cells (CHO-K1) was evaluated using the MTT assay. After 24, 48 and 72 h, the IC50 values were 2.9 μM for PAT and ranged from 10.7 to 2.2 μM and from 25.0 to 12.5 μM for BEA and STE, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the three mycotoxin interaction's degree. Binary and tertiary combinations showed a dose dependent effect. At low fraction affected, mycotoxin combinations were synergetic; whereas, at higher fraction affected, the combinations showed additive effect. Our results indicate that the co-occurrence of low concentrations of mycotoxin in food may increase their toxic effects. PMID:26802678

  5. Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line.

    PubMed

    Zwolak, Iwona

    2016-06-01

    Previously, evaluation of sodium metavanadate (NaVO3) cytotoxicity after 24 h exposure of Chinese hamster ovary K1 (CHO-K1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (NR, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the Golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) and resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) tests (mitochondrial disruption). The trypan blue (TB) staining (plasma membrane permeability) showed cytotoxicity of NaVO3 at a much higher NaVO3 concentration than the above-mentioned assays. In the current study, using the same experimental approach, we have assessed the toxicity of vanadyl sulphate (VOSO4) and compared the obtained results with NaVO3 action. Unlike metavanadate, VOSO4 treatment at 24 h resulted in similar sensitivity of the NR and resazurin tests. Nevertheless, following the 48-h incubation with VOSO4, the NR test showed markedly higher sensitivity than the resazurin test when comparing the half maximal inhibitory concentration values (61 and 110 µM for the NR and resazurin test, respectively, p < 0.05). The TB staining method was the least susceptible for detecting vanadyl cytotoxicity at each exposure time point. In summary, both the NR and resazurin tests can be advocated as similarly sensitive in detection of VOSO4-induced cytotoxicity in the CHO-K1 cell line at 24 h. However, the longer incubation time with VOSO4 showed that the NR test is more sensitive than the resazurin assay. The differences in the results between the cytotoxicity tests employed probably arise from dissimilar susceptibility of the endpoints (targets) measured with these tests to the damage by vanadium. Considering this, the current and the previous studies highlight the role of lysosomes (and possibly the Golgi apparatus) apart from mitochondria

  6. Genotoxic effects of environmental estrogen-like compounds in CHO-K1 cells.

    PubMed

    Tayama, Sumiko; Nakagawa, Yoshio; Tayama, Kuniaki

    2008-01-01

    Some environmental estrogen-like compounds, such as bisphenol A (BPA), 4-nonylphenol (NP), 4-octylphenol (OP), propyl p-hydroxybenzoate (P-PHBA), and butyl p-hydroxybenzoate (B-PHBA), synthetic estrogen, diethylstilbestrol (DES), and natural estrogen, 17beta-estradiol (E2), were studied for their genotoxicity in CHO-K1 cells using sister-chromatid exchange (SCE), chromosome aberration (CA), and DNA strand break (comet) assays. Six of the chemicals, excluding E2, caused DNA migration in the comet assay and induced SCEs at one or more of the highest doses. Among the chemicals, OP produced an especially high incidence of SCEs. Structural CA was induced by five of the chemicals, excluding OP and NP, and BPA, E2, and DES also induced aneuploid cells. E2 and DES particularly increased the rate of polyploidy at high doses. The incidence of colchicine-mitosis-like (c-mitotic) figures suggesting spindle disrupting effects was also detected with five of the chemicals, excluding OP and NP, and six of the chemicals, excluding E2, caused endoreduplication (ERD), a form of nuclear polyploidization induced by block of cell cycle at G2 phase, at one or more high doses. Our present results suggest that OP and NP cause repairable DNA damage, including SCEs, and do not result in CA, while the damage caused by DES, BPA, P-PHBA, and B-PHBA results in the induction of CAs together with SCEs probably because of imperfect repair. We are unable to explain the observation that the DNA damage caused by E2 resulted in CA induction but not DNA migration or SCE induction, except for speculating that the DNA damage is different from that caused by DES and the estrogen-like chemicals. Our findings also suggest that E2, DES and BPA have aneuploidogenic properties, and that the former two of chemicals also are polyploidy-inducing agents. PMID:17913570

  7. The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

    PubMed

    Sayer, A N; Hu, Q; Bourdelais, A J; Baden, D G; Gibson, J E

    2006-07-01

    Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal. PMID:16487644

  8. The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro

    PubMed Central

    Sayer, A.N.; Hu, Q.; Bourdelais, A.J.; Baden, D.G.; Gibson, J.E.

    2009-01-01

    Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal. PMID:16487644

  9. Cytotoxic effects of zearalenone and its metabolites and antioxidant cell defense in CHO-K1 cells.

    PubMed

    Tatay, Elena; Font, Guillermina; Ruiz, Maria-Jose

    2016-10-01

    Zearalenone (ZEA) and its metabolites (α-zearalenol; α-ZOL, β-zearalenol; β-ZOL) are secondary metabolites of Fusarium fungi that produce cell injury. The present study explores mycotoxin-induced cell damage and cellular protection mechanisms in CHO-K1 cells. Cytotoxicity has been determined by reactive oxygen species (ROS) production and DNA damage. ROS production was determined using the fluorescein assay and DNA strand breakage by comet assay. Intracellular protection systems were glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The results demonstrated that all mycotoxins increased the ROS levels up to 5.3-fold the control levels in CHO-K1 cells. Zearalenone metabolites, but not ZEA, increased DNA damage 43% (α-ZOL) and 28% (β-ZOL) compared to control cells. The GSH levels decreased from 18% to 36%. The GPx and SOD activities respectively increased from 26% to 62% and from 23% to 69% in CHO-K1 cells, whereas CAT activity decreased from 14% to 52%. In addition, intracellular ROS production was induced by ZEA and its metabolites. The endogenous antioxidant system components GSH, GPx and SOD were activated against ZEA and its metabolites. These antioxidant system components thus could contribute to decrease cell injury by ZEA and its metabolites. PMID:27465603

  10. StARD13(Dlc-2) RhoGap mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells.

    PubMed

    Hatch, Grant M; Gu, Yuan; Xu, Fred Y; Cizeau, Jeannick; Neumann, Shannon; Park, Ji-Seon; Loewen, Shauna; Mowat, Michael R A

    2008-03-01

    To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression. PMID:18162584

  11. Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells.

    PubMed

    Vishwanathan, Nandita; Le, Huong; Jacob, Nitya M; Tsao, Yung-Shyeng; Ng, Sze-Wai; Loo, Bernard; Liu, Zhong; Kantardjieff, Anne; Hu, Wei-Shou

    2014-03-01

    Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples. PMID:24108600

  12. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

    PubMed

    Montejo-López, Wilber; Rivera-Ramírez, Nayeli; Escamilla-Sánchez, Juan; García-Hernández, Ubaldo; Arias-Montaño, José-Antonio

    2016-09-01

    Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation. PMID:27350581

  13. Expression of FSH receptor in the hamster ovary during perinatal development

    PubMed Central

    Chakraborty, Prabuddha; Roy, Shyamal K.

    2014-01-01

    FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

  14. Inhibition of topoisomerase II{alpha} activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

    SciTech Connect

    Grdina, D.J. |; Constantinou, A.; Shigematsu, N.; Murley, J.S.

    1993-06-01

    The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector and antimutagenic agent when it is administered 30 min prior to radiation exposure to Chinese hamster ovary Kl cells at a concentration of 4 mM. Under these exposure conditions, topoisomerase (topo) I and II activities and associated protein contents were measured in the K1 cell line using the DNA relaxation assay, the P4 unknotting assay, and immunoblotting, respectively. WR-1065 was ineffective in modifying topo I activity, but it did reduce topo IIa activity by an average of 50 percent. The magnitude of topo IIa protein content, however, was not affected by these exposure conditions. Cell cycle effects were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for a period of up to 6 h resulted in a buildup of cells in the G2 compartment. However, in contrast to topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotector agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of radiation protection attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by 2-[(aminopropyl)amino]ethanethiol on type II topoisomerase, which is involved in DNA synthesis.

  15. Radioresistance secondary to low pH in human glial cells and Chinese hamster ovary cells

    SciTech Connect

    Rottinger, E.M.; Mendonca, M.

    1982-08-01

    The influence of the extracellular pH on the radiosensitivity of human glial cells and Chinese hamster ovary cells was examined. The period of low pH varied from 0 to 96 hours in glial cells and from 0 to 48 hours in Chinese hamster cells. Maintenance of low pH after a dose of 10 Gy for at least 24 hours for glial cells and at least 6 hours for Chinese hamster cells improved survival by more than one order of magnitude at pH 6.4. Cellular inactivation by irradiation may be impaired by an extracellular pH at or below pH 6.7.

  16. Effects of lunar and mars dust on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Brix, Klaudia; Slenzka, Klaus; Rehders, Maren; Sadhukhan, Annapurna; Mistry, Rima; Duenne, Matthias; Kempf, Juergen

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respira-tory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of lunar dust on human health is required to best support future missions to moon. In this study, we used different methods to assess the specific effects of lunar dust onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and since a well orchestrated program ensures proper repair in cases of wounding. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology, metabolic state, survival and proliferation of the cells were determined. Cytotoxi-city and proliferation were measured using the MTT assay, metabolic activity was analyzed by vital staining of mitochondria, and phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells. It was found that the effects of the two types of soils on the different features of both cell lines varied to considerable extent, and that lunar and mars dust were specific in their effects. The obtained results will facilitate detailed inves-tigations of dust exposure during wound healing and will ease risk assessment studies for e.g. lunar lander approaches. The investigations will help to assess the risks and to determine safety measures to be taken during extraterrestrial expeditions in order to minimize risks to human health associated with exposure of human skin to dust contaminants.

  17. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    NASA Astrophysics Data System (ADS)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  18. Investigation of superparamagnetic (Fe3O4) nanoparticles and magnetic field exposures on CHO-K1 cell line

    NASA Astrophysics Data System (ADS)

    Coker, Zachary; Estlack, Larry; Hussain, Saber; Choi, Tae-Youl; Ibey, Bennett L.

    2016-03-01

    Rapid development in nanomaterial synthesis and functionalization has led to advanced studies in actuation and manipulation of cellular functions for biomedical applications. Often these actuation techniques employ externally applied magnetic fields to manipulate magnetic nanomaterials inside cell bodies in order to drive or trigger desired effects. While cellular interactions with low-frequency magnetic fields and nanoparticles have been extensively studied, the fundamental mechanisms behind these interactions remain poorly understood. Additionally, modern investigations on these concurrent exposure conditions have been limited in scope, and difficult to reproduce. This study presents an easily reproducible method of investigating the biological impact of concurrent magnetic field and nanoparticle exposure conditions using an in-vitro CHO-K1 cell line model, with the purpose of establishing grounds for in-depth fundamental studies of the mechanisms driving cellular-level interactions. Cells were cultured under various nanoparticle and magnetic field exposure conditions from 0 to 500 μg/ml nanoparticle concentrations, and DC, 50 Hz, or 100 Hz magnetic fields with 2.0 mT flux density. Cells were then observed by confocal fluorescence microscopy, and subject to biological assays to determine the effects of concurrent extreme-low frequency magnetic field and nanoparticle exposures on cellnanoparticle interactions, such as particle uptake and cell viability by MTT assay. Current results indicate little to no variation in effect on cell cultures based on magnetic field parameters alone; however, it is clear that deleterious synergistic effects of concurrent exposure conditions exist based on a significant decrease in cell viability when exposed to high concentrations of nanoparticles and concurrent magnetic field.

  19. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed Central

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-01-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. Images PMID:7966574

  20. Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses.

    PubMed

    Lie, Y S; Penuel, E M; Low, M A; Nguyen, T P; Mangahas, J O; Anderson, K P; Petropoulos, C J

    1994-12-01

    We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons. PMID:7966574

  1. DNA-DSB in CHO-K1 cells induced by heavy-ions: Break rejoining and residual damage (GSI)

    NASA Technical Reports Server (NTRS)

    Taucher-Scholz, G.; Heilmann, J.; Becher, G.; Kraft, G.

    1994-01-01

    DNA double strand breaks (DSB's) are the critical lesions involved in cellular effects of ionizing radiation. Therefore, the evaluation of DSB induction in mammalian cells after heavy ion irradiation is an essential task for the assessment of high-LET radiation risk in space. Of particular interest has been the question of how the biological efficiency for the cellular inactivation endpoint relates to the initial lesions (DSBs) at varying LETs. For cell killing, an increased Relative Biological Efficiency (RBE) has been determined for highLET radiation around 100-200 keV/mu m. At higher LET, the RBE's decrease again to values below one for the very heavy particles. At GSI, DSB-induction was measured in CHO-K1 cells following irradiation with accelerated particles covering a wide LET range. The electrophoretic elution of fragmented DNA out of agarose plugs in a constant electrical field was applied for the detection of DSB's. The fraction of DNA retained was determined considering the relative intensities of ethidium bromide fluorescence in the well and in the gel lane. Dose-effect curves were established, from which the RBE for DSB induction was calculated at a fraction of 0.7 of DNA retained In summary, these rejoining studies are in line with an enhanced severity of the DNA DSB's at higher LET's, resulting in a decreased repairability of the induced lesions. However, no information concerning the fidelity of strand breaks rejoining is provided in these studies. To assess correct rejoining of DNA fragments an experimental system involving individual DNA hybridization bands has been set up. In preliminary experiments Sal I generated DNA fragments of 0.9 Mbp were irradiated with xrays and incubated for repair However, restitution of the original signals was not observed, probably due to the high radiation dose necessary for breakage of a fragment of this size. A banding pattern with NotI hybridization signals in a higher MW range (3Mbp) has been obtained by varying

  2. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing

    PubMed Central

    Zhang, Siyan; Hughes, Jason D.; Murgolo, Nicholas; Levitan, Diane; Chen, Janice; Liu, Zhong

    2016-01-01

    Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. PMID:27088091

  3. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    SciTech Connect

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  4. Isolation of Chinese hamster ovary cells with reduced unscheduled DNA synthesis after UV irradiation

    SciTech Connect

    Stefanini, M.; Reuser, A.; Bootsma, D.

    1982-09-01

    A simple procedure has been worked out to obtain UV-sensitive mutants of Chinese hamster ovary (CHO) cells. In this procedure, conventional mutagenesis is followed by BrdU--light treatment to enrich the population for UV-sensitive cells. Colonies that are allowed to form subsequently are duplicated by replica plating and screened on the master plate for their UV sensitivity and their capacity to carry out UV-induced DNA repair synthesis. Putative mutants are isolated from the replica. With this combination of methods, we succeeded in isolating CHO mutants with an 85-95% reduced level of UV-induced DNA synthesis in combination with an increased UV sensitivity.

  5. Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

    SciTech Connect

    Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

    1987-01-01

    The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

  6. The mechanisms of cytotoxicity of urethane dimethacrylate to Chinese hamster ovary cells.

    PubMed

    Chang, Hsiao-Hua; Chang, Mei-Chi; Lin, Li-Deh; Lee, Jang-Jaer; Wang, Tong-Mei; Huang, Chun-Hsun; Yang, Ting-Ting; Lin, Hsueh-Jen; Jeng, Jiiang-Huei

    2010-09-01

    Monomers released from resin-containing products may cause various adverse effects. Urethane dimethacrylate (UDMA) is a principal resin monomer and also a major component released from various dental resin materials. Thus the toxic effects and mechanisms should be elucidated for improving of its safety use. Here we investigated the effects of UDMA on the growth, cell cycle progression, reactive oxygen species (ROS) production and glutathione (GSH) alteration in CHO-K1 cells, and the preventive effects by antioxidants (NAC and catalase) were also evaluated. UDMA elicited growth inhibition (>0.025 mm) of CHO-K1 cells in a clearly dose-dependent manner. Cell cycle perturbation and ROS overproduction were also observed. A 0.1 mm UDMA-induced S-phase cell cycle arrest and ROS accumulation. Cell apoptosis and necrosis became significant when UDMA concentration was 0.25 mm. GSH depletion occurred at cells treated with 0.25 mm UDMA, a highly cytotoxic concentration at which point myriad cells were under apoptosis or necrosis. Thus GSH depletion can be crucial for the death of CHO-K1 cells. Furthermore NAC (0.5-10 mm) and catalase (250-1000 U/ml) obviously attenuated the UDMA-induced toxicity by reducing ROS generation and cell cycle disturbance, and the effects were dose-related. These results suggest that UDMA toxicity is associated with ROS production, GSH depletion, cell cycle disturbance and cell apoptosis/necrosis. PMID:20579731

  7. CELLULAR TOXICITY IN CHINESE HAMSTER OVARY CELL CULTURES. 1. ANALYSIS OF CYTOTOXICITY ENDPOINTS FOR TWENTY-NINE PRIORITY POLLUTANTS

    EPA Science Inventory

    Chinese hamster ovary cells were exposed to 29 toxic chemical substances which were representative of several classes of compounds listed by the Natural Resources Defense Council Consent Decree as priority toxic pollutants. After cell cultures were exposed to the test substance, ...

  8. MULTIPLE-ENDPOINT MUTAGENESIS WITH CHINESE HAMSTER OVARY (CHO) CELLS: EVALUATION WITH EIGHT CARCINOGENIC AND NON-CARCINOGENIC COMPOUNDS

    EPA Science Inventory

    Using Chinese hamster ovary (CHO) cells in culture, the authors have defined an assay, CHO/HGPRT, to quantify mutagen-induced cytotoxicity and mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. This assay permits elucidation of the structure-activity r...

  9. The RNA polymerase II of an alpha-amanitin-resistant Chinese hamster ovary cell line.

    PubMed

    Lobban, P E; Siminovitch, L; Ingles, C J

    1976-05-01

    Amal, an alpha-amanitin-resistant mutant of the Chinese hamster ovary cell line, contains an RNA polymerase activity which elutes from DEAE-Sephadex at a salt concentration characteristic of an RNA polymerase II, but which is not sensitive to alpha-amanitin at levels where the polymerase II of wild-type cells is strongly inhibited. This result suggests that Amal owes its amanitin-resistant phenotype to a mutation affecting one of its genes for RNA polymerase II. To test this hypothesis, we purified the enzyme from Amal and then compared its properties with those of the wild-type enzyme. The mutant enzyme is indeed a polymerase II, and is over 600 times less sensitive to alpha-amanitin and more thermolabile than the wild-type enzyme. PMID:954093

  10. Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells

    SciTech Connect

    Flintoff, W.F.; Weber, M.K.; Nagainis, C.R.; Essani, A.K.; Robertson, D.; Salser, W.

    1982-03-01

    Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parental line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

  11. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  12. Stable Expression of the Motor Protein Prestin in Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Iida, Koji; Konno, Kazuaki; Oshima, Takeshi; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

    Mammalian hearing sensitivity relies on a mechanical amplification mechanism involving the outer hair cells (OHCs), which rapidly alter their longitudinal length in response to changes in their membrane potential. The molecular basis of this mechanism is thought to be a motor protein embedded in the lateral membrane of the OHCs. Recently, this motor protein was identified and termed prestin. Since then, prestin has been researched intensively to elucidate the behavior of the OHCs. However, little progress in the study of prestin at the molecular level has been made because no method of obtaining an adequate amount of prestin has been established. In this study, therefore, an attempt was made to construct a stable expression system of prestin using Chinese hamster ovary (CHO) cells. The expression of prestin in the transfected CHO cells and the activity of prestin on CHO cells were confirmed by immunofluorescence and whole-cell patch-clamp measurements, respectively.

  13. In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.

    PubMed Central

    Moehring, T J; Danley, D E; Moehring, J M

    1984-01-01

    Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980). Images PMID:6717439

  14. Dielectric model for Chinese hamster ovary cells obtained by dielectrophoresis cytometry.

    PubMed

    Salimi, E; Braasch, K; Butler, M; Thomson, D J; Bridges, G E

    2016-01-01

    We present a dielectric model and its parameters for Chinese hamster ovary (CHO) cells based on a double-shell structure which includes the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm. Employing a dielectrophoresis (DEP) based technique and a microfluidic system, the DEP response of many single CHO cells is measured and the spectrum of the Clausius-Mossotti factor is obtained. The dielectric parameters of the model are then extracted by curve-fitting to the measured spectral data. Using this approach over the 0.6-10 MHz frequency range, we report the values for CHO cells' membrane permittivity, membrane thickness, cytoplasm conductivity, nuclear envelope permittivity, and nucleoplasm conductivity. The size of the cell and its nuclei are obtained using optical techniques. PMID:26858823

  15. Inhibition of topoisomerase II activity in repair-proficient CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

    SciTech Connect

    Grdina, D.J.; Constantinou, A.; Shigematsu, N.

    1992-09-01

    The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector under in vitro conditions when it is administered 30 min prior to radiation exposure at a concentration of 4 mM to repair-proficient Chinese hamster ovary Kl cells (i.e., a dose modification factor of 1.4). In contrast, the DNA double-strand break, repair-deficient Chinese hamster ovary xrs-5 cell line is not protected under these conditions (i.e., a dose modification factor of 1.0). Topoisomerase (topo) I and II activities and protein contents were measured in both Kl and xrs-5 cell lines and were found to be similar in magnitude. Neither exposure to radiation, to WR-1065, or to both affected these variables in xrs-5 cells. WR 1065 was effective, however, in reducing topo 11 activity by a factor of 2 in the repair-proficient Kl cell line. Topo II protein content, however, was not affected by these exposure conditions. One of several mechanisms of radiation protection attributed to aminothiol compounds has been their ability to affect enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results demonstrate a modifying effect by 2-[(aminopropyl)amino]ethanethiol on a specific nuclear enzyme (i.e., type H topoisomerase), which is involved in DNA synthesis. These results also suggest that differences do exist between the topo 11 enzymes isolated from the parent repair-proficient Kl and the DNA double-strand break, repair-deficient xrs-5 mutant cell lines.

  16. Induction of chromosomal damage in CHO-K1 cells and their repair-deficient mutant XRS5 by x-ray and particle irradiation

    NASA Astrophysics Data System (ADS)

    Nasonova, E.; Ritter, S.; Fomenkova, T.; Kraft, G.

    The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G_1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/mum) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.

  17. G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary

    PubMed Central

    Wang, Cheng; Prossnitz, Eric R.; Roy, Shyamal K.

    2008-01-01

    Estradiol-17β (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation. PMID:18499747

  18. Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.

    PubMed

    Morrison, E; Tomasec, P; Linton, E A; Lowry, P J; Lowenstein, P R; Castro, M G

    1995-04-01

    Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior

  19. Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor

    PubMed Central

    Zhang, Ye; Chotteau, Véronique

    2015-01-01

    This data article shows how the recombinant Chinese Hamster Ovary (CHO) cells are located in the interstices of the matrix fibers of a CellTank bioreactor after completion of a perfusion culture, supporting the article entitled “Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor” by Zhang et al. [1]. It provides a visualization of the cell distribution in the non-woven fiber matrix in a deeper view. PMID:26958613

  20. Landfill leachate sludge use as soil additive prior and after electrocoagulation treatment: A cytological assessment using CHO-k1 cells.

    PubMed

    Morozesk, M; Bonomo, M M; Rocha, L D; Duarte, I D; Zanezi, E R L; Jesus, H C; Fernandes, M N; Matsumoto, S T

    2016-09-01

    Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application. PMID:27243586

  1. Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

    PubMed

    Lee, Dong-Hyun; Kim, Tae-Ho; Lee, Sun-Young; Kim, Hyun-Jo; Rhee, Seung Keun; Yoon, ByoungSu; Pfeifer, Gerd P; Lee, Chong-Soon

    2002-12-31

    Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions. PMID:12521305

  2. Effect of dihydroxyanthraquinone (DHAQ) and radiation on the survival of cultured Chinese hamster ovary cells

    SciTech Connect

    Kimler, B.F.

    1983-02-01

    Dihydroxyanthraquinone (DHAQ) is currently being tested as a cancer chemotherapeutic agent because of its structural similarity to Adriamycin (ADR) and other DNA-intercalating antibiotics. The interaction of DHAQ and ionizing radiation on the induction of cell lethality was investigated in Chinese hamster ovary cells in culture. In asynchronous populations of cells, DHAQ produced a slight enhancement of radiation-induced cell lethality as evidenced by changes in both shoulder and slope of the radiation dose-survival curves. However, DHAQ had no effect on either the extent or time course of recovery from sublethal radiation damage. In synchronous populations of cells treated at various times before or after selection in mitosis, the combination of DHAQ and radiation produced greater cell killing than that predicted based on simple additivity of effect, with a decided enhancement for cells treated during S phase. These results indicate that DHAQ is similar to other DNA-intercalating antibiotics in regard to the interaction with ionizing radiation to produce cell lethality.

  3. Endogenous TRPM4-like channel in Chinese hamster ovary (CHO) cells

    SciTech Connect

    Yarishkin, Oleg V.; Hwang, Eun-Mi; Park, Jae-Yong; Kang, Dawon; Han, Jaehee; Hong, Seong-Geun

    2008-05-02

    Chinese hamster ovary (CHO) cells used in many transfection studies have been found to endogenously express channels permeable to monovalent cations, but not to divalent cations. In the presence of intracellular Ca{sup 2+}, 23-pS channel with a linear current-voltage (I-V) relationship could be frequently observed in inside-out patches but not in cell-attached patches. The open probability was voltage-dependent, which is higher at positive potentials. The channel was dose-dependently activated by relatively high level of Ca{sup 2+} (EC{sub 50} = 1.04 {+-} 0.08 mM), and sensitively inhibited by 100 {mu}M ATP, ADP, AMP, and 1 mM spermine. However, ruthenium red (2 {mu}M) had no effect. Reverse transcript polymerase chain reaction (RT-PCR) supported the presence of mRNA encoding TRPM4b channel protein. Western blot assay finally confirmed the presence of this channel protein in membrane fraction of CHO cells. These results provide evidence that CHO cells express an endogenous TRPM4b-like channel, and thereby can be used as a tool to study de novo regulation/modulation of TRPM4 channel.

  4. Trehalose Transporter from African Chironomid Larvae Improves Desiccation Tolerance of Chinese Hamster Ovary Cells

    PubMed Central

    Chakraborty, Nilay; Menze, Michael A.; Elmoazzen, Heidi; Vu, Halong; Yarmush, Martin L.; Hand, Steven C.; Toner, Mehmet

    2011-01-01

    Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid P. vanderplanki [21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese Hamster Ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4 M trehalose for 4 h at 37 °C, a seven-fold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14 % relative humidity. After desiccation to 2.60 g of water per gram dry weight, a 170 % increase in viability and a 400 % increase in growth (after 7 days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation. PMID:22155480

  5. Rapid amplification system for recombinant protein production in Chinese Hamster Ovary (CHO) Cells.

    PubMed

    Metta, M K; Kunaparaju, R K; Tantravahi, S

    2016-01-01

    Recombinant therapeutic proteins have changed the face of modern medicine in the present trend and they continue to provide innovative therapies for deadly diseases. This study describes the development of a novel stable expression system for rapid amplification of genes in Chinese Hamster Ovary (CHO) cells. The expression system consists of a host CHO cell line and an expression vector (pUB-PyOri-D-C) which encodes for Polyomavirus (Py) Origin of Replication (PyOri) for amplification of integrated genes in the presence of Py Large T Antigen (PyLT) and Dihydrofolate Reductase (DHFR) selectable marker gene for selection in the presence of Methotrexate (MTX). Use of both PyOri/PyLT and DHFR can reduce the number of rounds of selection and amplification required for isolation of high producing clones. The efficiency of pUB-PyOri-D-C was compared with that of pUB-D-C plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-D-C-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-D-C-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-D-C-EPO gave a productivity of 119 mg/L in shake flask. PMID:26950459

  6. Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells.

    PubMed

    Braun, Nicole A; Mohler, Peter J; Weisgraber, Karl H; Hasty, Alyssa H; Linton, MacRae F; Yancey, Patricia G; Su, Yan Ru; Fazio, Sergio; Swift, Larry L

    2006-06-01

    We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis. PMID:16534141

  7. Effect of glutamine limitation on the death of attached Chinese hamster ovary cells

    SciTech Connect

    Sanfeliu, A.; Stephanopoulos, G. )

    1999-07-05

    The effect of glutamine depletion on the death of attached Chinese hamster ovary (CHO) cells was investigated. Experiments were performed using an anchorage dependent CHO cell line expressing [gamma]-IFN and a second cell line obtained by transfection of that cell line with the human bcl-2 (hbcl-2). Either cell line could grow in media devoid of glutamine with minimal cell death due to endogenous glutamine synthetase activity that allowed cells to synthesize glutamine from glutamic acid in the medium. However, compared to control cultures in glutamine-containing media, the cell growth rate in glutamine-free media was slower with an increased fraction of cells distributed in the G[sub 0]/G[sub 1] phase. The slower rate of cell cycling apparently protected the cells from entering apoptosis when they were stimulated to proliferate in an environment devoid of other protective factors, such as serum or over-expressed hbcl-2. The depletion of both glutamine and glutamic acid did cause cell death, which could be mitigated by hbcl-2 over-expression.

  8. Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells.

    PubMed

    Gludovacz, Elisabeth; Maresch, Daniel; Bonta, Maximilian; Szöllösi, Helen; Furtmüller, Paul G; Weik, Robert; Altmann, Friedrich; Limbeck, Andreas; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-06-10

    Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions. PMID:27063138

  9. Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells.

    PubMed

    Kishishita, Shohei; Katayama, Satoshi; Kodaira, Kunihiko; Takagi, Yoshinori; Matsuda, Hiroki; Okamoto, Hiroshi; Takuma, Shinya; Hirashima, Chikashi; Aoyagi, Hideki

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian host for large-scale commercial production of therapeutic monoclonal antibodies (mAbs). Chemically defined media are currently used for CHO cell-based mAb production. An adequate supply of nutrients, especially specific amino acids, is required for cell growth and mAb production, and chemically defined fed-batch processes that support rapid cell growth, high cell density, and high levels of mAb production is still challenging. Many studies have highlighted the benefits of various media designs, supplements, and feed addition strategies in cell cultures. In the present study, we used a strategy involving optimization of a chemically defined feed medium to improve mAb production. Amino acids that were consumed in substantial amounts during a control culture were added to the feed medium as supplements. Supplementation was controlled to minimize accumulation of waste products such as lactate and ammonia. In addition, we evaluated supplementation with tyrosine, which has poor solubility, in the form of a dipeptide or tripeptide to improve its solubility. Supplementation with serine, cysteine, and tyrosine enhanced mAb production, cell viability, and metabolic profiles. A cysteine-tyrosine-serine tripeptide showed high solubility and produced beneficial effects similar to those observed with the free amino acids and with a dipeptide in improving mAb titers and metabolic profiles. PMID:25678240

  10. Overexpression of microRNAs enhances recombinant protein production in Chinese hamster ovary cells.

    PubMed

    Loh, Wan Ping; Loo, Bernard; Zhou, Lihan; Zhang, Peiqing; Lee, Dong-Yup; Yang, Yuansheng; Lam, Kong Peng

    2014-09-01

    MicroRNAs (miRNAs) are short, non-coding RNAs that can negatively regulate expression of multiple genes at post-transcriptional levels. Using miRNAs to target multiple genes and pathways is a promising cell-engineering strategy to increase recombinant protein production in mammalian cells. Here, we identified miRs-17, -19b, -20a, and -92a to be differentially expressed between high- and low- monoclonal antibody-producing Chinese hamster ovary (CHO) cell clones using next-generation sequencing and quantitative real-time PCR. These miRNAs were stably overexpressed individually and in combination in a high-producing clone to assess their effects on CHO cell growth, recombinant protein productivity and product quality. Stably transfected pools demonstrated 24-34% increases in specific productivity (qP) and 21-31% increases in titer relative to the parental clone, without significant alterations in proliferation rates. The highest protein-producing clones isolated from these pools exhibited 130-140% increases in qP and titer compared to the parental clone, without major changes in product aggregation and N-glycosylation profile. From our clonal data, correlations between enhanced qP/titer and increased levels of miRs-17, -19b, and -92a were observed. Our results demonstrate the potential of miRs-17, -19b, and -92a as cell-engineering targets to increase recombinant protein production in mammalian cells. PMID:24819042

  11. Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

    SciTech Connect

    Schwartz, J.L.; Morgan, W.F.; Kapp, L.N.; Wolff, S.

    1983-01-01

    3-Aminobenzamide (3AB), in inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30-120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth. 25 references, 3 figures, 2 tables.

  12. [Stable expression of recombinant human podoplanin in Chinese hamster ovary (CHO) cells].

    PubMed

    Qu, Le; Zhao, Xingpeng; Fu, Jianxin; Xia, Lijun; Dai, Lan; Ruan, Changgeng; Zhao, Yiming

    2016-01-01

    Objective To construct podoplanin (PDPN) eukaryotic expression plasmid PDPN-pEGFP-N1, establish Chinese hamster ovary (CHO) cell line stably expressing recombinant human PDPN and investigate its biological activity. Methods PDPN cDNA was cloned from HEK293 cells by reverse transcription PCR and recombinant DNA technology and inserted into plasmid pEGFP-N1 labeled by enhanced green fluorescent protein (EGFP). The recombinant vector was identified by PCR, restriction enzyme digestion and DNA sequencing, and then transfected into CHO cells. Recombinant PDPN-EGFP was observed by fluorescent microscopy and CHO cell line with the high expression of PDPN-EGFP was selected by flow cytometry. Recombinant PDPN was detected by Western blotting and the biological activity of the cell line was determined by platelet aggregation assay. Results DNA sequencing and restriction enzyme digestion proved that the gene of PDPN was inserted successfully into pEGFP-N1 plasmid. After stable transfection of the recombinant plasmid into CHO cells, CHO with EGFP could be seen under a fluorescent microscope. The CHO cell line with the high expression of recombinant PDPN-EGFP was obtained after sorting by flow cytometry. Western blotting showed that the recombinant PDPN was expressed on the cell surface. The over-expressing PDPN-EGFP CHO cells were able to induce human platelet aggregation. Conclusion The CHO cell line with the stable and high expression of recombinant PDPN-EGFP has been constructed successfully, and it could induce platelet aggregation. PMID:26728373

  13. Cytogenetic response to coffee in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes.

    PubMed

    Tucker, J D; Taylor, R T; Christensen, M L; Strout, C L; Hanna, M L

    1989-09-01

    We have investigated the genotoxic effects of three different brands and three types of coffee (freshly brewed regular, instant regular and freshly brewed decaffeinated) in two mammalian systems: the Chinese hamster ovary (CHO) AUXB1 cell line and human peripheral lymphocytes. Sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) were used as the endpoints. Coffee was prepared according to the manufacturer's suggestions, and after cooling, administered to cultured cells at dilutions ranging up to 11% that of full-strength coffee. Each brand and type of coffee induced significant levels of SCEs and ERCs in AUXB1 cells. SCEs, but not ERCs, were induced in human peripheral lymphocytes. Bisulfite, which complexes with carbonyls and reduces their genotoxicity, significantly diminished the number of SCEs and ERCs found after administration of coffee. Catalase and peroxidase, enzymes that destroy hydrogen peroxide activity, had no significant effect upon the SCE and ERC frequencies in AUXB1 cultures treated with freshly brewed regular coffee. These experiments indicate that different brands and types of coffee have sufficient genotoxic activity to increase SCEs and ERCs at levels only a fraction of those normally consumed. 1,2-Dicarbonyls alone and peroxides alone do not appear to be responsible for the majority of SCEs and ERCs that were observed to be induced by dilute coffee. PMID:2687627

  14. Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells

    PubMed Central

    Hansen, Henning Gram; Nilsson, Claes Nymand; Lund, Anne Mathilde; Kol, Stefan; Grav, Lise Marie; Lundqvist, Magnus; Rockberg, Johan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

    2015-01-01

    Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells. PMID:26657798

  15. Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells.

    PubMed Central

    Souès, S.; Laval, F.; Charcosset, J. Y.

    1995-01-01

    We have isolated from Chinese hamster ovary cells, 30 sublines resistant to vincristine, doxorubicin or etoposide and 43 sublines evading treatment with a pair of these drugs. Isolated in one step and under low selective pressure, sublines were 3- to 25-fold more resistant to their selecting drug(s) than the parental cells. Possible P-glycoprotein-associated multidrug resistance was investigated through pgp gene copy number and mRNA expression level. DNA topoisomerase II alteration was evaluated from the ability of nuclear extracts to form cleavable complexes. Vincristine (all sublines) and doxorubicin (6/7 sublines) preferentially selected for pgp gene amplification and mRNA overexpression, whereas selection with etoposide resulted in a decrease of cleavable complex formation in 11 out of 13 sublines. A common pgp gene-mediated resistance was found in the 13 doxorubicin plus vincristine-selected sublines, whereas all but one of the 12 etoposide plus vincristine-resistant sublines displayed both pgp mRNA overexpression and decreased ability to form cleavable complexes. Among the 18 doxorubicin plus etoposide selected sublines, five exhibited a decreased ability to form cleavable complexes only, six exhibited pgp mRNA overexpression only and six exhibited both alterations. Overall, drug resistance could not be attributed to either mechanism in three of the 73 sublines. We conclude that under low selective pressure it is possible to find a combination of drugs which require simultaneous selection of more than one resistance mechanism; such cells emerge with very low frequency. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7880729

  16. Examination of Inertial Cavitation of Optison™ in Producing Sonoporation of Chinese Hamster Ovary Cells

    PubMed Central

    Forbes, Monica M.; Steinberg, Ryan L.; O’Brien, William D.

    2008-01-01

    The objective of this project is to elucidate the relationship between ultrasound contrast agents (UCAs) and sonoporation. Sonoporation is an ultrasound-induced, transient cell membrane permeability change, which allows for the uptake of normally impermeable macromolecules. Specifically, this study will determine the role that inertial cavitation plays in eliciting sonoporation. The inertial cavitation thresholds of the UCA, Optison™, are directly compared to the results of sonoporation in order to determine the involvement of inertial cavitation in sonoporation. Chinese Hamster Ovary (CHO) cells were exposed as a monolayer, in a solution of Optison™, 500,000 Da Fluorescein isothiocyanate-dextran (FITC-dextran), and Phosphate Buffered Saline (PBS) to 30 seconds of pulsed ultrasound (US) at 3.15-MHz center frequency, 5-cycle pulse duration, and 10-Hz pulse repetition frequency. The peak rarefactional pressure (Pr) was varied over a range from 120 kPa to 3.5 MPa, and five independent replicates were performed at each pressure. As the Pr was increased, from 120 kPa to 3.5 MPa, the fraction of sonoporated cells among the total viable population increased from 0.63% to 10.21%, with the maximum occurring at 2.4 MPa. The inertial cavitation threshold for Optison™ at these exposure conditions has previously been shown to be in the range 0.77–0.83 MPa, at which sonoporation activity was found to be 50% of its maximum level. Furthermore, significant sonoporation activity was observed at pressure levels below the threshold for inertial cavitation of Optison™. Above 2.4 MPa, a significant drop in sonoporation activity occurred, corresponding to pressures where >95% of the Optison™ was collapsing. These results demonstrate that sonoporation is not directly due to inertial cavitation of the UCA, rather that the effect was related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the inertial cavitation threshold. PMID:18692296

  17. Effects of selenocystine on lead-exposed Chinese hamster ovary (CHO) and PC-12 cells

    SciTech Connect

    Aykin-Burns, Nukhet; Ercal, Nuran . E-mail: nercal@umr.edu

    2006-07-15

    Lead is a pervasive environmental toxin that affects multiple organ systems, including the nervous, renal, reproductive, and hematological systems. Even though it is probably the most studied toxic metal, some of the symptoms of lead toxicity still cannot be explained by known molecular mechanisms. Therefore, lead-induced oxidative stress has recently started to gain attention. This in vitro study confirms the existence of oxidative stress due to lead exposure. Administration of lead acetate (PbA) to cultures of Chinese hamster ovary cells (CHO) had a concentration-dependent inhibitory effect on colony formation and cell proliferation. This inhibition was eliminated by 5 {mu}M selenocystine (SeCys). In order to evaluate the nature of SeCys's effect, we measured glutathione (GSH), its oxidized form glutathione disulfide (GSSG), malondialdehyde (MDA), catalase, and GSH peroxidase (GPx) activities in lead-exposed CHO cells both in the presence and absence of SeCys. Increases in MDA, catalase, and GPx activities were observed in cultures that received only PbA, but supplementation with SeCys returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in SeCys-enhanced media but declined in cultures treated with PbA only. In order to determine whether SeCys also reverses lead-induced neurotoxicity, a neuronal cell line, PC-12 cells, was used. Lead's inhibition on neurite formation was significantly eliminated by SeCys in PC-12 cells. Our results suggest that SeCys can confer protection against lead-induced toxicity in CHO cells and neurotoxicity in PC-12 cells.

  18. Characterization of a Chinese hamster ovary cell line resistant to uncouplers.

    PubMed

    Freeman, K B; Yatscoff, R W; Mason, J R; Patel, H V; Buckle, M

    1983-08-01

    The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered. PMID:6223814

  19. Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-06-28

    DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia. PMID:8687482

  20. DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells

    SciTech Connect

    Schwartz, J.L. |; Mustafi, R.; Hughes, A.; DeSombre, E.R.

    1996-08-01

    The effects of the Auger electron-emitting isotope {sup 123}I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with {sup 60}Co {gamma} rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-labeled estrogen. The {sup 123}I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 {+-} 1.7, which is similar to that seen with high-LET radiations. Our results suggest that {sup 123}I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by {sup 123}I bound to estrogen is very like damage induced by high-LET radiation, and the {sup 123}I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA. 40 refs., 7 figs.

  1. Metabolomics profiling of extracellular metabolites in recombinant Chinese Hamster Ovary fed-batch culture.

    PubMed

    Chong, William P K; Goh, Lin Tang; Reddy, Satty G; Yusufi, Faraaz N K; Lee, Dong Yup; Wong, Niki S C; Heng, Chew Kiat; Yap, Miranda G S; Ho, Ying Swan

    2009-12-01

    A metabolomics-based approach was used to time profile extracellular metabolites in duplicate fed-batch bioreactor cultures of recombinant Chinese Hamster Ovary (CHO) cells producing monoclonal IgG antibody. Culture medium was collected and analysed using a high-performance liquid chromatography (HPLC) system in tandem with an LTQ-Orbitrap mass spectrometer. An in-house software was developed to pre-process the LC/MS data in terms of filtering and peak detection. This was followed by principal component analysis (PCA) to assess variance amongst the samples, and hierarchical clustering to categorize mass peaks by their time profiles. Finally, LC/MS2 experiments using the LTQ-Orbitrap (where standard was available) and SYNAPT HDMS (where standard was unavailable) were performed to confirm the identities of the metabolites. Two groups of identified metabolites were of particular interest; the first consisted of metabolites that began to accumulate when the culture entered stationary phase. The majority of them were amino acid derivatives and they were likely to be derived from the amino acids in the feed media. Examples included acetylphenylalanine and dimethylarginine which are known to be detrimental to cell growth. The second group of metabolites showed a downward trend as the culture progressed. Two of them were medium components--tryptophan and choline, and these became depleted midway into the culture despite the addition of feed media. The findings demonstrated the potential of utilizing metabolomics to guide medium design for fed-batch culture to potentially improve cell growth and product titer. PMID:19902412

  2. Microphysiometric analysis of human α1a-adrenoceptor expressed in Chinese hamster ovary cells

    PubMed Central

    Taniguchi, Takanobu; Inagaki, Rika; Murata, Satoshi; Akiba, Isamu; Muramatsu, Ikunobu

    1999-01-01

    The human recombinant α1a-adrenoceptor (AR) has been stably expressed in Chinese hamster ovary cells. Four stable clones, aH4, aH5, aH6 and aH7, expressing 30, 370, 940 and 2900 fmol AR mg−1 protein, respectively, have been employed to characterize this AR subtype using radioligand binding and microphysiometry to measure extracellular acidification rates.Noradrenaline (NA) gave concentration-dependent responses in microphysiometry with increasing extracellular acidification rates. The potency of NA increased as the receptor density increased; pEC50 values of NA for the clones aH4, aH5, aH6 and aH7 were 6.9, 7.5, 7.8 and 8.1, respectively. This increase of potency according to receptor density indicates the presence of spare receptor for NA. Methoxamine, phenylephrine, oxymetazoline and clonidine also gave concentration-dependent responses with various intrinsic activities.Antagonists shifted concentration-response curves for NA rightward in a concentration-dependent manner. Schild analysis revealed that the affinity profile of this AR subtype to antagonists in the clone aH7 had a typical pattern for the α1a-AR; high affinity for prazosin and WB 4101, and low affinity for BMY7378 (pA2=9.5, 9.8 and 7.3, respectively). This profile is similar in the case of the clone aH4. These affinities were in good agreement with those obtained in binding experiments.These results have demonstrated that (1) classical receptor theory can be applied in microphysiometry, and (2) microphysiometry is a useful tool to investigate the pharmacological characterization of α1a-AR. PMID:10433504

  3. Proteomic profiling of a high-producing Chinese hamster ovary cell culture.

    PubMed

    Carlage, Tyler; Hincapie, Marina; Zang, Li; Lyubarskaya, Yelena; Madden, Helena; Mhatre, Rohin; Hancock, William S

    2009-09-01

    The productivity of mammalian cell culture expression systems is critically important to the production of biopharmaceuticals. In this study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-X(L) gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomarker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene that is known to have growth regulating properties in human cells. A total of 392 proteins were identified in this study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were downregulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-X(L). The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures. PMID:19663468

  4. Metabolism of 2-acetylaminofluorene in the Chinese hamster ovary cell mutation assay

    SciTech Connect

    Heflich, R.H.; Djuric, Z.; Zhuo, Z.; Fullerton, N.F.; Casciano, D.A.; Beland, F.A.

    1988-01-01

    Chinese hamster ovary (CHO) cells were exposed to 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF), and several of their N-oxidized metabolites in order to study the mechanisms by which arylamides and arylamines produce mutations in mammalian cells. The number of mutations induced at the hypoxanthine-guanine phosphoribosyl transferase locus by each compound was estimated to be: N-acetoxy-2-AAF, 310; N-hydroxy-2-AF, 3; N-hydroxy-2-AAF, 0.7; 2-AAF, 0.1; and 2-AF, 0.09. With each compound, DNA adducts were also identified and quantified, and in all cases the major adduct was N-(deoxyguanosin-8-yl)-2-AF. The relationship between mutation induction and adduct formation for each of the derivatives was similar to that previously reported for N-hydroxy-2-AF. Inclusion of the deacetylase inhibitor, paraoxon, reduced the mutagenicity of 2-AFF, N-hydroxy-2-AAF and N-acetoxy-2-AAF, and the DNA adducts produced by N-acetoxy-2-AAF to background levels. Taken together, these data indicate that CHO cells metabolized N-acetoxy-2-AAF to a reactive derivative by N-deacetylation to N-acetoxy-2-AF, while N-hydroxy-2-AF reacted directly with DNA. The major pathway of N-hydroxy-2-AAF activation appeared to be an initial O-acetylation to N-acetoxy-2-AAF and this occurred to only a limited extent in the CHO cells. The initial step in the activation of 2-AAF and 2-AF was an N-oxidation to N-hydroxy-2-AAF and N-hydroxy-2-AF, respectively. The limited O-acetylase activity in CHO cells appeared to contribute to the low sensitivity of these cells toward mutation induction by arylamines and arylamides.

  5. Diversity in host clone performance within a Chinese hamster ovary cell line.

    PubMed

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. PMID:25918883

  6. Taenia solium Oncosphere Adhesion to Intestinal Epithelial and Chinese Hamster Ovary Cells In Vitro▿

    PubMed Central

    Verastegui, Manuela; Gilman, Robert H.; Arana, Yanina; Barber, Dylan; Velásquez, Jeanette; Farfán, Marilu; Chile, Nancy; Kosek, Jon C.; Kosek, Margaret; Garcia, Hector H.; Gonzalez, Armando

    2007-01-01

    The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4°C compared with the adherence at 37°C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes. PMID:17698575

  7. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  8. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

    SciTech Connect

    Matzuk, M.M.; Krieger, M.; Corless, C.L.; Boime, I.

    1987-09-01

    Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results reveal that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.

  9. UTILIZATION OF THE RABBIT ALVEOLAR MACROPHAGE AND CHINESE HAMSTER OVARY CELL FOR EVALUATION OF THE TOXICITY OF PARTICULATE MATERIALS. II. PARTICLES FROM COAL-RELATED PROCESSES

    EPA Science Inventory

    Rabbit alveolar macrophage (RAM) and Chinese hamster ovary (CHO) cells were used in vitro tests to evaluate the toxicity of particulate effluents from coal gasification, fluidized-bed combustion, and conventional coal combustion. Evaluation of the cytotoxicity of nine samples fro...

  10. COMPARISON OF THE TOXICITY OF ACRYLAMIDE, CYCLOPHOSPHAMIDE, CHLRODECONE, AND DIETHYLSTILBESTROL IN CHINESE HAMSTER OVARY (CHO) CELLS WITH THEIR TOXICITY IN VIVO

    EPA Science Inventory

    In order to compare in vitro toxicity with in vivo toxicity, four chemicals that have been tested in the in vivo/in vitro toxicological screen proposed by the Health Effects Research Laboratory, EPA were tested in a Chinese Hamster Ovary (CHO) cytotoxicity assay. Viability index,...

  11. Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

    PubMed

    Pany, Satyabrata; Majhi, Anjoy; Das, Joydip

    2016-04-12

    Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state. PMID

  12. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells

    SciTech Connect

    Pendse, G.J.; Bailey, J.E. . Dept. of Chemical Engineering)

    1994-12-01

    Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA cells show a reduced specific growth rate in the VHb-expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture.

  13. Sister chromatid exchanges in Chinese hamster ovary cells exposed to high intensity pulsed ultrasound: inability to confirm previous positive results.

    PubMed

    Miller, M W; Azadniv, M; Pettit, S E; Church, C C; Carstensen, E L; Hoffman, D

    1989-01-01

    This study was undertaken in an attempt to determine a physical mechanism of action for a recently published report of a small but statistically significant increase in sister chromatid exchanges (SCEs) in Chinese hamster ovary cells exposed to high-intensity pulsed ultrasound. The "positive" report's protocol involved a sizeable chance of ultrasound beam impingement on the side wall of the cell exposure chamber. Ten experiments per regimen were conducted; the regimens included exposures of (a) chamber center, (b) chamber wall, (c) nine grid sites, 0.5 mm between sites, and (d) nine grid sites, 1.5 mm between sites. The last was an exact replication of the conditions previously reported to induce the small SCE effect. The results did not support the postulate of an increase in SCEs with the ultrasound exposures. PMID:2741252

  14. An inhibitory factor for cell-free protein synthesis from Salmonella enteritidis exhibits cytopathic activity against Chinese hamster ovary cells.

    PubMed

    Iwamaru, Y; Miyake, M; Arii, J; Tanabe, Y; Noda, M

    2001-12-01

    A factor inhibiting cell-free protein synthesis was purified from Salmonella enteritidis cell lysate by sequential ammonium sulfate precipitation, chromatography on anion exchange and hydrophobic interaction columns, and polyacrylamide disc gel electrophoresis. The purified factor, which was named SIPS (Salmonella inhibitor of protein synthesis), inhibited in vitro protein synthesis in rabbit reticulocyte lysate and had a molecular mass of 38 kDa, estimated by PAGE under denaturing conditions. SIPS was also cytopathic for Chinese hamster ovary cells. The N-terminal amino acid sequence (20 residues) of SIPS was found to be identical to that of mature L-asparaginase II of Escherichia coli. Indeed, the purified SIPS exhibited asparaginase activity, E. coli L-asparaginase II had cytopathic activity and inhibited in vitro protein synthesis. The results suggest that at least a part of cytotoxicity and inhibition of cell-free protein synthesis caused by S. enteritidis is a property of the bacterial L-asparaginase. PMID:11747376

  15. Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.

    1984-01-01

    Seven promutagens belonging to two chemical classes - polycyclic aromatic hydrocarbons (PAHs) and alkyl nitrosamines - were studied in Chinese hamster ovary (CHO) cells. Findings of practical importance in the use of Aroclor 1254-induced rat liver homogenate (S9) in the CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay were made. Novel findings are (1) the inclusion of CaCl/sub 2/ in the S9 cofactor mixture dramatically decreased the cytotoxicity of S9, and (2) different S9 optimum concentrations were observed for struturally similar promutagens. The inclusion of CaCl/sub 2/ in the S9 cofactor mixture and the testing of each chemical of unknown S9 requirement at several S9 concentrations are therefore recommended for assaying promutagens in the CHO/HGPRT mutation assay.

  16. Inhibition of DNA excision repair by methotrexate in Chinese hamster ovary cells following exposure to ultraviolet irradiation or ethylmethanesulfonate

    SciTech Connect

    Borchers, A.H.; Kennedy, K.A.; Straw, J.A. )

    1990-03-15

    Previous results have suggested that methotrexate (MTX) could interfere with the repair of spontaneous DNA damage. To determine its effects on induced DNA damage, MTX was compared to hydroxyurea and arabinofuranosylcytosine (H/A), a drug combination known to block the DNA polymerase step of excision repair, for its ability to cause the accumulation of single-strand breaks (SSB) following exposure to either UV light or the alkylating agent ethylmethanesulfonate in Chinese hamster ovary cells. SSB were measured by alkaline elution 1, 2, and 6 h after exposure to either 1.8 mg/ml of ethylmethanesulfonate or 10 J/m2 of UV in cells pretreated with MTX or H/A. Following exposure to ethylmethanesulfonate, significant accumulation of SSB occurred in cells pretreated with either H/A or MTX. Coadministration of hypoxanthine and thymidine in MTX-treated cells prevented SSB accumulation, indicating that nucleotide depletion by MTX had inhibited repair synthesis. After UV irradiation, SSB accumulation was much less in MTX- than in H/A-treated cells. MTX was found to have no effect on the incision of UV damage. These results indicate that nucleotide depletion by MTX can affect the repair of DNA damage by exogenous agents, and that the extent of inhibition is dependent on the type of damage induced.

  17. Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

    PubMed

    Wang, X Y; Zhang, J H; Sun, Q L; Yao, Z Y; Deng, B G; Guo, W Y; Wang, L; Dong, W H; Wang, F; Zhao, C P; Wang, T Y

    2015-01-01

    Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression. PMID:26345852

  18. /sup 31/P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells

    SciTech Connect

    Lopez, A.; Rols, M.P.; Teissie, J.

    1988-02-23

    Chinese hamster ovary (CHO) cells were reversibly permeabilized by submitting them to short, high-intensity, square wave pulses (1.8 kV/cm, 100 ..mu..s). The cells remained in a permeable state without loss of viability for several hours at 4/sup 0/C. A new anisotropic peak with respect to control cells was observed on /sup 31/P NMR spectroscopic analysis of the phospholipid components. This peak is only present when the cells are permeable, and normal anisotropy is recovered after resealing. Taking into account the fusogenicity of electropermeabilized cells, comparative studies were performed on 5% poly(ethylene glycol) treated cells. The /sup 31/P NMR spectra of the phospholipids displayed the same anisotropic peak as in the case of the electropermeabilized cells. In the two cases, this anisotropic peak was located downfield from the main peak associated to the phospholipids when organized in bilayers. The localization of this anisotropic peak is very different from the one of a hexagonal phase. The authors proposed a reorganization of the polar head group region leading to a weakening of the hydration layer to account for these observations. This was also thought to explain the electric field induced fusogenicity of these cells.

  19. Artichoke induces genetic toxicity and decreases ethyl methanesulfonate-related DNA damage in Chinese hamster ovary cells.

    PubMed

    Jacociunas, Laura Vicedo; de Andrade, Heloisa Helena Rodrigues; Lehmann, Mauricio; de Abreu, Bianca Regina Ribas; Ferraz, Alexandre de Barros Falcão; da Silva, Juliana; Dihl, Rafael Rodrigues

    2012-10-01

    Cynara scolymus L. (Asteraceae), popularly known as artichoke, has been widely used in herbal medicine for the treatment of hepatic diseases. The genotoxicity of C. scolymus L. leaf extract (LE) and the ability to modulate the genetic toxicity of the alkylating agent ethyl methanesulfonate (EMS) were assessed using the comet assay on Chinese hamster ovary cells. Genotoxicity was evaluated after 1- and 24-h treatments using four different LE concentrations: 0.62, 1.25, 2.5, and 5.0 mg/mL. Antigenotoxicity was assessed for pretreatment, simultaneous treatment, and post-treatment. All doses used led to a significant increase in the frequency of DNA damage, after exposure for 1 and 24 h. In the antigenotoxicity experiments, LE reduced the frequency of DNA damage induced by EMS in the simultaneous treatment only. However, the lowest dose was more protective than higher concentrations. Flavonoids and phenolic compounds are, probably, the C. scolymus constituents responsible for its genotoxic and antigenotoxic effects. PMID:22892059

  20. Quantitative feature extraction from the Chinese hamster ovary bioprocess bibliome using a novel meta-analysis workflow.

    PubMed

    Golabgir, Aydin; Gutierrez, Jahir M; Hefzi, Hooman; Li, Shangzhong; Palsson, Bernhard O; Herwig, Christoph; Lewis, Nathan E

    2016-01-01

    The scientific literature concerning Chinese hamster ovary (CHO) cells grows annually due to the importance of CHO cells in industrial bioprocessing of therapeutics. In an effort to start to catalogue the breadth of CHO phenotypes, or phenome, we present the CHO bibliome. This bibliographic compilation covers all published CHO cell studies from 1995 to 2015, and each study is classified by the types of phenotypic and bioprocess data contained therein. Using data from selected studies, we also present a quantitative meta-analysis of bioprocess characteristics across diverse culture conditions, yielding novel insights and addressing the validity of long held assumptions. Specifically, we show that bioprocess titers can be predicted using indicator variables derived from viable cell density, viability, and culture duration. We further identified a positive correlation between the cumulative viable cell density (VCD) and final titer, irrespective of cell line, media, and other bioprocess parameters. In addition, growth rate was negatively correlated with performance attributes, such as VCD and titer. In summary, despite assumptions that technical diversity among studies and opaque publication practices can limit research re-use in this field, we show that the statistical analysis of diverse legacy bioprocess data can provide insight into bioprocessing capabilities of CHO cell lines used in industry. The CHO bibliome can be accessed at http://lewislab.ucsd.edu/cho-bibliome/. PMID:26948029

  1. Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression

    PubMed Central

    Rahimpour, Azam; Ahani, Roshanak; Najaei, Azita; Adeli, Ahmad; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2016-01-01

    Background Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). Methods CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. Results Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. Conclusion The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression. PMID:27547109

  2. Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD sup + oxidoreductase activity

    SciTech Connect

    James, P.F.; Lee, J. ); Rizzo, W.B.; Zoeller, R.A. )

    1990-08-01

    The authors have isolated a mutant Chinese hamster ovary cell line that is defective in long-chain fatty alcohol oxidation. The ability of the mutant cells to convert labeled hexadecanol to the corresponding fatty acid in vivo was reduced to 5% of the parent strain. Whole-cell homogenates from the mutant strain, FAA.1, were deficient in long-chain fatty alcohol:NAD{sup +} oxidoreductase activity, which catalyzes the oxidation of hexadecanol to hexadecanoic acid, although the intermediate fatty aldehyde was formed normally. A direct measurement of fatty aldehyde dehydrogenase showed that the FAA.1, strain was defective in this component of FAO activity. FAA.1 is a two-stage mutant that was selected from a previously described parent strain, ZR-82, which is defective in ether lipid biosynthesis and peroxisome assembly. Because of combined defects in ether lipid biosynthesis and fatty alcohol oxidation, the ability of the FAA.1 cells to incorporate hexadecanol into complex lipids was greatly impaired, resulting in a 60-fold increase in cellular fatty alcohol levels. As the FAO deficiency in FAA.1 cells appears to be identical to the defect associated with the human genetic disorder Sjoegren-Larsson syndrome, the FAA.1 cell line may be useful in studying this disease.

  3. Development of a method to quantify gene expression levels for glycosylation pathway genes in chinese hamster ovary cell cultures.

    PubMed

    Clark, Kevin J-R; Harcum, Sarah W; Griffiths, Jennifer; Bailey, Kevin M

    2005-06-01

    Changes in protein glycosylation owing to changes in environmental conditions are not well understood. To better understand these relationships, methods to quantify controlling factors are needed. Because enzymes are translated from genes, the ability to quantify gene expression levels for glycosylation-related enzymes would be advantageous. We developed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays to monitor gene expression in Chinese hamster ovary (CHO) cells for five terminal glycosylation genes. The five enzymes were sialidase, a putative alpha2,3-sialyltransferase, beta1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the alpha2,3-sialyltransferase gene for Cricetulus griseus (CHO cell species) was not available and, therefore, was sequenced as a part of this work. The qRT-PCR primers and probes (based on the TaqMan chemistry) were designed and validated for these five genes. The gene expression profiles were obtained for CHO cells producing the recombinant interleukin-4/13 cytokine trap molecule in batch reactors. PMID:15917580

  4. Contribution of chlorination to the mutagenic activity of drinking water extracts in Salmonella and Chinese hamster ovary cells

    SciTech Connect

    Douglas, G.R.; Nestmann, E.R.; Lebel, G.

    1986-11-01

    The production of chlorinated by-products through chlorine disinfection of drinking water has been well documented. Natural organic precursors for these chemicals include fulvic and humic acids, the chlorination of which leads to the production of mutagenic compounds. Comparisons of extracts of raw versus treated waters have confirmed that clorination during water treatment produces mutagenic activity in the Salmonella (Ames) test. Present work on XAD-2 extracts of raw and chlorinated water from six municipalities in the Great Lakes region of Canada has involved a battery of mutagenicity assays for various genetic endpoints: the Salmonella test, the sister-chromatid exchange (SCE) and the micronucleus (MN) induction in Chinese hamster ovary (CHO) cells. All extracts of treated (chlorinated), but none of untreated, water were mutagenic in the Salmonella assay. On the other hand, extracts of both treated and untreated water samples showed activity in the SCE and MN assays, but no consistent pattern of response with regard to treatment (chlorination) was evident. These data show that chlorination contributes mutagens to drinking water and suggest that mammalian in vitro assays may be more sensitive for detecting mutagenicity in water samples than the Salmonella test.

  5. TOXICOLOGY STUDIES OF LEWISITE AND SULFUR MUSTARD AGENTS:GENETIC TOXICITY OF LEWISITE (L) IN CHINESE HAMSTER OVARY CELLS

    SciTech Connect

    Jostes,R.F. Jr.; Sasser, LB; Rausch, R.J.

    1989-05-31

    The cytotoxic clastogenic and mutagenic effects of the arsenic containing vesicant, Lewisite (L) [dichloro(2-chlorovinyl) arsine], have been investigated using Chinese hamster ovary cells. One hour exposures to Lewisite were cytotoxic in uM amounts. The cell survival response yields a D37 of 0.6 uM and an extrapolation number of 2.5. The mutagenic response at the hypoxantnine-guanine phosporibosyl transferase (HGPRT) locus was sporadic and not significantly greater than control values when cells were exposed over a range of 0.125 to2.0 uM. Sister chromatid exchange (SCE) induction, a measure of chromosomal rearrangement, was weakly positive over a range of 0.25 to 1.0 uM but the values were not significantly greater than the control response. Chromosomal aberrations were induced at 0.75 and 1.0 UMin one experiment and 0.5 and 0.75 uM in another experiment. The Induced values were significantly greater than the control values. Lewisite appears to be cytotoxic and clastogenic in our investigations but SCE and mutation at the HGPRT locus are not significantly greater than control values. Lewisita toxicity was in some ways similar to radiomimetic chemicals such as bleomycin.

  6. Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro III: Results with 27 chemicals

    SciTech Connect

    Gulati, D.K. ); Witt, K.; Anderson, B.; Zeiger, E.; Shelby, M.D. )

    1989-01-01

    Twenty-seven chemicals previously tested in rodent carcinogenicity assays were tested for induction of chromosomal aberrations (ABS) and sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells as part of a larger analysis of the correlation between results of in vitro genetic toxicity assays and carcinogenicity bioassays. Chemicals were tested up to toxic doses with and without exogenous metabolic activation. Seventeen of the chemicals tested were carcinogens; only two of these were negative for both ABS and SCE. Of the eight noncarcinogens tested, four were negative for both endpoints and four gave a positive response for at least one endpoint. Of the remaining two chemicals, one, diallylphthalate, gave an equivocal response in the bioassay and a positive response in these CHO cell cytogenetics tests. The other chemical, 2,4-toluene diisocyanate, was tested for carcinogenicity as a mixture with the 2,6-isomer; the mixture was carinogenic, but the cytogenetic test results for the 2,4-isomer were negative. Experiments with unsynchronized CHO cells demonstrated that mean SCE frequency increased with increasing culture time, and this may have been a factor in the positive results obtained for five chemicals in the SCE test under conditions of delayed harvest.

  7. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    PubMed

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. PMID:26850366

  8. Comparative mutagenicity of a coal combustion fly ash extract in Salmonella typhimurium and Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.; Clark, C.R.; Hanson, R.L.; Henderson, T.R.; Hobbs, C.H.

    1983-01-01

    The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreductase deficient strains TA98NR and TA98/1,8DNP/sub 6/. The mutagenicity observed in TA98NR and TA98/1,8DNP/sub 6/ was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na/sup +/-K/sup +/-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.

  9. Comparative mutagenicity of a coal combustion fly ash extract in Salmonella typhimurium and chinese hamster ovary cells

    SciTech Connect

    Li, A.P.; Clark, C.R.; Hanson, R.L.; Henderson, T.R.; Hobbs, C.H.

    1983-01-01

    The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreducatase deficient strains TA98NR and TA98/1,8DNP/sub 6/. The mutagenicity observed in TA98NR and TA98/1,8DNP/sub 6/ was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na/sup +/ -K/sup +/ -ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.

  10. Expression of secreted recombinant human insulin-like growth factor-II (IGF-II) in Chinese hamster ovary cells.

    PubMed

    Bekkari, H; Sekkat, D; Straczek, J; Hess, K; Belleville-Nabet, F; Nabet, P

    1994-07-29

    Chinese hamster ovary (CHO-KI) cells were cotransfected with a plasmid pcDNAI containing the human preproinsulin-like growth factor II cDNA linked downstream to the human cytomegalovirus promoter and with a plasmid containing the neomycin resistance gene (pMAM-neo). CHO neo+ were selected by growth in medium supplemented with G418 geneticin. After amplification, the neomycin-resistant clones were screened for IGF-II production. IGF-II produced was identified by dot blot and quantified by ELISA. The clones C24, C40 and C94 secreted IGF-II at about 350-400 ng per 10(6) cells per day. DNA analysis of C24 and C40 CHO cells by PCR demonstrated the presence of the IGF-II construct in the transfected cells, presumably integrated into the chromosomal DNA. IGF-II produced by CHO cells and purified by RP-HPLC was a mitogen for MCF-7 stimulating mitosis 2-fold. PMID:7765161

  11. N-acetylcysteine protects Chinese Hamster ovary cells from oxidative injury and apoptosis induced by microcystin-LR

    PubMed Central

    Xue, Lijian; Li, Jinhui; Li, Yang; Chu, Chu; Xie, Guantao; Qin, Jin; Yang, Mingfeng; Zhuang, Donggang; Cui, Liuxin; Zhang, Huizhen; Fu, Xiaoli

    2015-01-01

    This study aimed to investigate the MC-LR induced oxidative injury and apoptosis in Chinese hamster ovary (CHO) cells, and the protective effects of N-acetylcysteine (NAC) on these cells. Cell viability was determined by MTT assay after exposure to NAC at various concentrations (0, 1, 5, 10, 20, 30, 40, 50, 60 and 80 mmol/L) alone, or NAC (0, 1 and 5 mmol/L) plus MC-LR (0, 2.5, 5 and 10 μg/ml) for 24 h. The reactive oxygen species (ROS) in CHO cells were measured by DCFH-DA, mitochondrial membrane potential (MMP) by fluorescence probe JC-1 staining, and apoptosis index determined by Annexin V-PI staining. Results showed, following exposure to NAC alone for 24 h, cell viability remains higher than 80% at 1 and 5 mmol/L. After exposure to NAC at different concentrations plus MC-LR, cell viability increased, ROS decreased, MMP elevated, and apoptosis index reduced to a certain extent. In conclusion, MC-LR may induce the apoptosis of CHO cells by inducing ROS production which is protected by NAC. PMID:26131064

  12. A hybrid approach identifies metabolic signatures of high-producers for chinese hamster ovary clone selection and process optimization.

    PubMed

    Popp, Oliver; Müller, Dirk; Didzus, Katharina; Paul, Wolfgang; Lipsmeier, Florian; Kirchner, Florian; Niklas, Jens; Mauch, Klaus; Beaucamp, Nicola

    2016-09-01

    In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. This requires applying appropriate methods during bioprocess development to enable meaningful characterization of CHO clones and processes. Here, we present a novel hybrid approach for supporting comprehensive characterization of metabolic clone performance. The approach combines metabolite profiling with multivariate data analysis and fluxomics to enable a data-driven mechanistic analysis of key metabolic traits associated with desired cell phenotypes. We applied the methodology to quantify and compare metabolic performance in a set of 10 recombinant CHO-K1 producer clones and a host cell line. The comprehensive characterization enabled us to derive an extended set of clone performance criteria that not only captured growth and product formation, but also incorporated information on intracellular clone physiology and on metabolic changes during the process. These criteria served to establish a quantitative clone ranking and allowed us to identify metabolic differences between high-producing CHO-K1 clones yielding comparably high product titers. Through multivariate data analysis of the combined metabolite and flux data we uncovered common metabolic traits characteristic of high-producer clones in the screening setup. This included high intracellular rates of glutamine synthesis, low cysteine uptake, reduced excretion of aspartate and glutamate, and low intracellular degradation rates of branched-chain amino acids and of histidine. Finally, the above approach was integrated into a workflow that enables standardized high-content selection of CHO producer clones in a high-throughput fashion. In conclusion, the combination of quantitative metabolite profiling, multivariate data analysis, and mechanistic network model simulations can identify metabolic

  13. Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

    PubMed Central

    Potvin, B; Stanley, P

    1991-01-01

    Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products. PMID:1724918

  14. Oxygen and exposure kinetics as factors influencing the cytotoxicity of porfiromycin, a mitomycin C analogue, in Chinese hamster ovary cells.

    PubMed

    Marshall, R S; Rauth, A M

    1988-10-15

    Some factors affecting the cytotoxicity of porfiromycin (PM), an analogue of mitomycin C (MMC), were investigated in suspension cultures of wild-type (AA8-4) and repair-deficient (UV-20) Chinese hamster ovary cells. Oxygen was an important modulator of PM toxicity in AA8-4 cells. The aerobic toxicity was significantly less, and toxicity under extremely hypoxic conditions was significantly greater for PM than MMC. Porfiromycin cytotoxicity at intermediate O2 levels was similar to that observed previously for MMC. While the aerobic/hypoxic ratio was greater for PM than MMC, survival at intermediate oxygen concentrations could limit the therapeutic utility of these drugs as adjuncts to radiotherapy. Ascorbic acid was found to increase the aerobic, but not hypoxic, cytotoxicity of PM in AA8-4 cells, as was observed previously for MMC. Investigation of various exposure times and drug concentrations revealed that drug toxicity for both aerobic and hypoxic cells was dependent on the product of drug concentration and time, and that the aerobic/hypoxic differential observed in AA8-4 cells was constant over a broad range of exposure conditions. The sensitivity of UV-20 cells was also a linear function of concentration and time, but no aerobic/hypoxic differential was observed in these cells. It is suggested that the sensitivity of UV-20 to PM and MMC, and its lack of an hypoxic/aerobic differential could result from lethality being due to a different lesion than in wild-type cells. PMID:3167822

  15. Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

    PubMed

    Fu, Tuo; Zhang, Cunchao; Jing, Yu; Jiang, Cheng; Li, Zhenhua; Wang, Shengyu; Ma, Kai; Zhang, Dapeng; Hou, Sheng; Dai, Jianxin; Kou, Geng; Wang, Hao

    2016-06-01

    Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins. PMID:26841889

  16. Isolation and characterization of a Chinese hamster ovary (CHO) mutant defective in the second step of glycosylphosphatidylinositol biosynthesis.

    PubMed Central

    Stevens, V L; Zhang, H; Harreman, M

    1996-01-01

    Mutant cell lines defective in the biosynthesis of glycosylphosphatidylinositol (GPI) described to date were isolated by selecting cells which no longer expressed one or more endogenous GPI-anchored proteins on their surface. In this study, a new mutant in this pathway was isolated from ethylmethane-sulphonate-mutagenized Chinese hamster ovary cells stably transfected with human placental alkaline phosphatase (PLAP) as a marker of GPI-anchored proteins. A three-step protocol was employed. In the first step, cells with decreased surface expression of PLAP were selected by four rounds of complement-mediated lysis with an anti-(alkaline phosphatase) antibody. The surviving cells were cloned by limiting dilution and those with low levels of total alkaline phosphatase activity were selected in the second step. Finally, the ability of each clone to synthesize the first three intermediates in GPI biosynthesis in vitro was assessed to determine which cells with low alkaline phosphatase activity harboured a defect in one of these reactions. Of 230 potential mutants, one was defective in the second step of GPI biosynthesis. Microsomes from this mutant, designated G9PLAP.85, were completely unable to deacetylate either endogenous GlcNAc-phosphatidylinositol (PI) synthesized from UDP[6-3H]GlcNAc or exogenous GlcNAc-PI added directly to the membranes. Complementation analysis with the Thy-1-deficient murine lymphoma cells demonstrated that G9PLAP.85 has a molecular defect distinct from these previously described mutants. Therefore, these results suggest that mutants in GPI biosynthesis could be selected from almost any cell line expressing a GPI-anchored marker protein. PMID:8546692

  17. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    PubMed

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  18. Folk medicine Terminalia catappa and its major tannin component, punicalagin, are effective against bleomycin-induced genotoxicity in Chinese hamster ovary cells.

    PubMed

    Chen, P S; Li, J H; Liu, T Y; Lin, T C

    2000-05-01

    Terminalia catappa L. is a popular folk medicine for preventing hepatoma and treating hepatitis in Taiwan. In this paper, we examined the protective effects of T. catappa leaf water extract (TCE) and its major tannin component, punicalagin, on bleomycin-induced genotoxicity in cultured Chinese hamster ovary cells. Pre-treatment with TCE or punicalagin prevented bleomycin-induced hgprt gene mutations and DNA strand breaks. TCE and punicalagin suppressed the generation of bleomycin-induced intracellular free radicals, identified as superoxides and hydrogen peroxides. The effectiveness of TCE and punicalagin against bleomycin-induced genotoxicity could be, at least in part, due to their antioxidative potentials. PMID:10773401

  19. Quantifaction of mutagens at the Na/sup +/-K/sup +/-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.

    1982-01-01

    The Chinese hamster ovary (CHO) cell/hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutagen assay developed by Hsie et al., was simplified by culturing the cells as unattached cultures, and also modified to include mutation at the Na/sup +/-K/sup +/ ATPase (ouabain resistance) gene locus. The cost and time involved were decreased by culturing the CHO cells unattached on nontissue culture plates during the expression period. The inclusion of a second gene locus ensures that mutagenicities observed were not due to the peculiar properties of a specific gene locus. These procedures are now used in our laboratory for routine testing of environmental chemicals and complex mixtures.

  20. Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

    PubMed Central

    Bair, C H; Chung, C S; Vasilevskaya, I A; Chang, W

    1996-01-01

    The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and

  1. Frequency-Dependent Evaluation of the Role of Definity in Producing Sonoporation of Chinese Hamster Ovary Cells

    PubMed Central

    Forbes, Monica M.; Steinberg, Ryan L.; O'Brien, William D.

    2011-01-01

    Objectives Sonoporation uses ultrasound (US) and ultrasound contrast agents (UCAs) to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds non-invasively into specific target cells. The objective of this study was to elucidate the biophysical mechanism of sonoporation, specifically the role of UCAs as well as exposure frequency. The inertial cavitation (IC) thresholds of the lipid-shelled octafluoropropane UCA were directly compared to the levels of generated sonoporation to determine the involvement of UCAs in producing sonoporation. Methods Chinese hamster ovary cells were exposed as a monolayer in a solution of the UCA, 500,000-Da fluorescein isothiocyanate-dextran, and phosphate-buffered saline to 30 seconds of pulsed US (pulse duration, 5 cycles; pulse repetition frequency, 10 Hz) at 3 frequencies (0.92, 3.2, and 5.6 MHz). The peak rarefactional pressure (Pr) was varied over a range from 4 kPa to 4.1 MPa, and 5 to 7 independent replicates were performed at each pressure. Results The experimental observations demonstrated that IC was likely not the physical mechanism for sonoporation. Sonoporation activity was observed at pressure levels below the threshold for IC of the UCA (1.27 ± 0.32 MPa at 0.92 MHz, 0.84 ± 0.19 MPa at 3.2 MHz, and 2.57 ± 0.26 MPa at 5.6 MHz) for all 3 frequencies examined. The Pr values at which the peak sonoporation activity occurred were 1.4 MPa at 0.92 MHz, 0.25 MPa at 3.2 MHz, and 2.3 MPa at 5.6 MHz. The UCA collapse thresholds followed a similar trend. A 1-way analysis of variance test confirmed that sonoporation activity differed among the 3 frequencies examined (P = 10−8). Conclusions These results thus suggest that sonoporation is related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the IC threshold. PMID:21193706

  2. Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

    SciTech Connect

    Holahan, E.V.; Highfield, D.P.; Holahan, P.K.; Dewey, W.C.

    1984-01-01

    To quantitatively relate heat killing and heat radiosensitization, asynchronous or G/sub 1/ Chinese hamster ovary (CHO) at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min. later. Since no progression of G/sub 1/cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. Hyperthermic radiosensitizaiton was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D/sub 0/ of the radiation survival curve to that of the D/sub 0/ radiation survival curve for heat plus radiation. The TEF increased continuously with increased of the heat killing at 45.5/sup 0/ C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4/sup 0/ C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (D/sub q/) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (D/sub q/ results). Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5/sup 0/ C than at 45.5 C where thermal tolerance did not develop. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5/sup 0/ C than of 45.5/sup 0/ C.

  3. Localization and functional analysis of CHIP28k water channels in stably transfected Chinese hamster ovary cells.

    PubMed

    Ma, T; Frigeri, A; Tsai, S T; Verbavatz, J M; Verkman, A S

    1993-10-25

    CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proximal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced approximately 8 x 10(6) copies of CHIP28k protein/cell. Plasma membrane osmotic water permeability (Pf), measured by stopped-flow light scattering, was 0.004 cm/s in control (vector-transfected) cells (10 degrees C) and 0.014 cm/s in the CHIP28k-transfected cells. Pf in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgCl2. CHIP28k expression did not affect the transport of protons and the small polar non-electrolytes urea and formamide. CHIP28k immunoreactivity and function was then determined in subcellular fractions. Pf in 6-carboxyfluorescein-labeled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002 cm/s (control cells) and 0.011 cm/s (CHIP28k-transfected cells); Pf in transfected cells was inhibited by HgCl2. Immunoblotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (approximately 5000 monomers/microns 2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies establish a stably transfected somatic cell line that strongly expresses functional CHIP28k water channels. As in the original proximal tubule cells

  4. The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxantine-guanine phosphoribosyl transferase mutational assay

    SciTech Connect

    Bermudez, E.; Couch, D.B.; Tillery, D.

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with chinese hamster ovary (CHO) cells to provide metabolic activation of promutgens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fisher-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B/sub 1/ (AFB/sub 1/) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB/sub 1/ was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating methobolic pathways important in the production and detoxification of genotoxic products in vivo.

  5. Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells.

    PubMed

    Fracasso, P M; Sartorelli, A C

    1986-08-01

    Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under

  6. Degradation or consumption of exogenous thymidine in absence or presence of exogenous deoxycytidine: Effect on DNA chain elongation and (/sup 3/H)thymidine incorporation in control and uv-irradiated CHO-K1 cells

    SciTech Connect

    Newman, C.N.; Hagler, M.

    1986-04-01

    When CHO-K1 cells monolayers are grown in Ham's F-12 culture medium containing 10% fetal calf serum (medium A) exogenous thymidine (dThd) is degraded to thymine by a putative dThd phosphorylase. Thymine is then poorly incorporated into cellular DNA. When 2 mM deoxycytidine (dCyd) is added to medium A (medium B) no degradation of exogenous dThd occurs; rather dThd is consumed in the synthesis of DNA, presumably via a dThd kinase or other nucleoside salvage pathway. Differences in the kinetics of DNA synthesis, measured by (/sup 3/H)dThd pulse-incorporation or by alkaline sucrose velocity sedimentation, are observed in the two media. In comparison to cells in medium A, DNA chain elongation rates of cells in medium B are faster but total DNA synthesis in these cells, measured by incorporation of (/sup 3/H)dThd, appears to be 2- to 3-fold more sensitive to the inhibitory effects of 10 Jm/sup -2/ uv-radiation. 23 refs., 3 figs.

  7. Efficient cleavage of single and clustered AP site lesions within mono-nucleosome templates by CHO-K1 nuclear extract contrasts with retardation of incision by purified APE1

    PubMed Central

    Eccles, Laura J.; Menoni, Hervé; Angelov, Dimitar; Lomax, Martine E.; O’Neill, Peter

    2015-01-01

    Clustered DNA damage is a unique characteristic of radiation-induced DNA damage and the formation of these sites poses a serious challenge to the cell’s repair machinery. Within a cell DNA is compacted, with nucleosomes being the first order of higher level structure. However, few data are reported on the efficiency of clustered-lesion processing within nucleosomal DNA templates. Here, we show retardation of cleavage of a single AP site by purified APE1 when contained in nucleosomal DNA, compared to cleavage of an AP site in non-nucleosomal DNA. This retardation seen in nucleosomal DNA was alleviated by incubation with CHO-K1 nuclear extract. When clustered DNA damage sites containing bistranded AP sites were present in nucleosomal DNA, efficient cleavage of the AP sites was observed after treatment with nuclear extract. The resultant DSB formation led to DNA dissociating from the histone core and nucleosomal dispersion. Clustered damaged sites containing bistranded AP site/8-oxoG residues showed no retardation of cleavage of the AP site but retardation of 8-oxoG excision, compared to isolated lesions, thus DSB formation was not seen. An increased understanding of processing of clustered DNA damage in a nucleosomal environment may lead to new strategies to enhance the cytotoxic effects of radiotherapeutics. PMID:26439176

  8. Toxicology Studies on Lewisite and Sulfur Mustard Agents: Genetic Toxicity of Sulfur Mustard (HD) in Chinese Hamster Ovary Cells Final Report

    SciTech Connect

    Jostes, Jr., R. F.; Sasser, L. B.; Rausch, R. J.

    1989-05-01

    The cytotoxic, clastogenic and mutagenic effects of sulfur nustard in Chinese hamster ovary cells are described in this reoort. The cytotoxicity data indicate that micromolar amounts of HC are highly toxic in microrolar amounts. Chromosone aberration frequencies increased in a dose-dependent manner over a dose range of 0. 5 to 1.0 {micro}m and SCE increased in a dose-dependent fashion in the dose range of 0.0625 to 0.25 {micro}M. Mutation induction at the HGPRT locus was sporadic, but the majority of the exoosures resulted in mutation frequencies which were 1.2 to 4.3 fold higher than the spontaneous frequencies.

  9. Enhanced efflux of (/sup 3/H)vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene

    SciTech Connect

    Hammond, J.R.; Johnstone, R.M.; Gros, P.

    1989-07-15

    Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain (/sup 3/H)vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in (/sup 3/H)vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in (/sup 3/H)vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of (/sup 3/H)vinblastine release. The observation that the mdr1 transfectants show a decreased (/sup 3/H)vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for (/sup 3/H)vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

  10. Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

    PubMed Central

    Wang, Cheng; Roy, Shyamal K.

    2009-01-01

    To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors. PMID:19074578

  11. Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

    PubMed Central

    2014-01-01

    Background Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation. Results Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism. Conclusions The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids. PMID:24773761

  12. Expression of E-Cadherin and N-Cadherin in Perinatal Hamster Ovary: Possible Involvement in Primordial Follicle Formation and Regulation by Follicle-Stimulating Hormone

    PubMed Central

    Wang, Cheng; Roy, Shyamal K.

    2010-01-01

    We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2. PMID:20219978

  13. Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture.

    PubMed

    Kishishita, Shohei; Nishikawa, Tomoko; Shinoda, Yasuharu; Nagashima, Hiroaki; Okamoto, Hiroshi; Takuma, Shinya; Aoyagi, Hideki

    2015-06-01

    During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content. PMID:25466646

  14. Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line

    PubMed Central

    Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

    2012-01-01

    Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting. Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models. PMID:22977370

  15. Effects of radiofrequency radiation and simultaneous exposure with mitomycin C on the frequency of sister chromatid exchanges in Chinese hamster ovary cells

    SciTech Connect

    Ciaravino, V.; Meltz, M.L.; Erwin, D.N.

    1987-01-01

    Chinese hamster ovary (CHO) cells were exposed for 2 hr with and without mitomycin C (MMC) (1 X 10(-8)M) to pulsed wave radiofrequency radiation (RFR) at 2450 MHz. The repetition rate of 25,000 pulses per sec (pps), pulse width of 10 microseconds, and exposure geometry used, resulted in a specific absorption rate (SAR) of 33.8 W/kg. The following exposure regimens were used: a 37 degrees C water bath control; a water bath temperature control (TC) in which the continuously monitored medium temperature closely followed the temperature rise in the RFR-exposed flasks; and the RFR-exposed cells in a water bath set at 37 degrees C prior to exposure. RFR exposure resulted in a maximum cell culture medium temperature of 39.2 degrees C. In the absence of MMC, there was no significant increase in sister chromatid exchange (SCE) in the RFR-exposed or TC groups over that of the 37 degrees C control. When a simultaneous treatment of RFR and MMC occurred there was no statistical difference in SCE frequency from that caused by chemical treatment alone.

  16. Effects of radiofrequency radiation and simultaneous exposure with mitomycin C on the frequency of sister chromatid exchanges in Chinese hamster ovary cells

    SciTech Connect

    Ciaravino, V.; Meltz, M.L.; Erwin, D.N.

    1987-01-01

    Chinese hamster ovary (CHO) cells were exposed for 2 hr with and without mitomycin C (MMC) to pulsed wave radiofrequency radiation (RFR) at 2450 MHz. The repetition rate of 25,000 pulses per sec (pps), and exposure geometry used, resulted in a specific absorption rate (SAR) of 33.8 W/kg. The following exposure regimens were used: 1) a 37 C water bath control; 2) a water bath temperature control (TC) in which the continuously monitored medium temperature closely followed teh temperature rise in the RFR-exposed flasks; and 3) the RFR-exposed cells in a water bath set at 37 C prior to exposure. RFR exposure resulted in a maximum cell culture medium temperature of 39.2 C. In the absence of MMC, there was no significant increase in sister chromatid exchange (SCE) in the RFR-exposed or TC groups over that of teh 37 C control. When a simultaneous treatment of RFR and MMC occurred there was no statistical difference in SCE frequency from that caused by chemical treatment alone.

  17. Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate.

    PubMed

    Lee, Sang Min; Kim, Yeon-Gu; Lee, Eun Gyo; Lee, Gyun Min

    2014-02-10

    To understand the effects of sodium butyrate (NaBu) on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing Fc-fusion glycoprotein were subjected to 3mM NaBu. The addition of NaBu to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of the glycoprotein. Fifty-two N-glycosylation-related gene expressions were also assessed by the NanoString nCounter system, which can provide a direct digital readout using custom-designed color-coded probes. Among them, ten genes (ugp, slc35a2, ganc, man1a, man1c, mgat5a, st3gal5, glb1, neu1, and neu3) were up-regulated and three genes (b4galt2, st3gal3, and neu2) were down-regulated significantly. Altered expression patterns in st3gal3, neu1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the glycoprotein by NaBu. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of NaBu on N-glycosylation in rCHO cells. PMID:24333461

  18. Monitoring utilizations of amino acids and vitamins in culture media and Chinese hamster ovary cells by liquid chromatography tandem mass spectrometry.

    PubMed

    Qiu, Jinshu; Chan, Pik Kay; Bondarenko, Pavel V

    2016-01-01

    Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (<10%) and good accuracy (100±10%) were achieved for nearly all the 30 compounds. Applying this method to CHO cell extracts, statistically significant differences in the metabolite levels were measured between two cell lines originated from the same host, indicating differences in genetic makeup or metabolic activities and nutrient supply levels in the culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures. PMID:26355770

  19. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells.

    PubMed

    Sung, Y H; Lim, S W; Chung, J Y; Lee, G M

    2004-02-01

    To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l(-1) YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 microg ml(-1), which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q(hTPO) (the specific rate of hTPO production). The supplementation of YH in SFM increased q(hTPO) by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells. PMID:12856163

  20. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

    PubMed

    Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. PMID:24561002

  1. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  2. The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1-transfected Chinese hamster ovary cells.

    PubMed

    Seo, Bo-Ra; Lee, Sook-Jeong; Cho, Kyung Sook; Yoon, Young Hee; Koh, Jae-Young

    2015-12-01

    Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate-conjugated Aβ(1-42) was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers. PMID:26453000

  3. The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1998-04-10

    NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity. PMID:9535868

  4. [Analysis of the transcriptional profiling of cell cycle regulatory networks of recombinant Chinese hamster ovary cells in batch and fed-batch cultures].

    PubMed

    Liu, Xingmao; Ye, Lingling; Liu, Hong; Li, Shichong; Wang, Qiwei; Wu, Benchuan; Chen, Zhaolie

    2011-08-01

    In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene. PMID:22097809

  5. Comparison of the kinetics and extent of muscarinic M1-M5receptor internalization, recycling and downregulation in Chinese Hamster Ovary cells

    PubMed Central

    Thangaraju, Arunkumar; Sawyer, Gregory W.

    2010-01-01

    We characterized agonist-induced internalization, recycling and downregulation of each muscarinic receptor subtype (M1 – M5) stably expressed in Chinese hamster ovary (CHO) cells. The radioligands [3H]QNB and [3H]NMS were used to measure the total and plasma membrane populations of muscarinic receptors, respectively. Following carbachol treatment (1 mM), the rank orders for the rate of carbachol-induced internalization of the muscarinic subtypes were M2 > M4 = M5 > M3 = M1, respectively. Unlike the M2 receptor, M1, M3, M4 and M5 receptors recycled back to the plasma membrane after one-hour carbachol treatment. The receptor downregulation elicited to 24-hour carbachol treatment was similar for M2, M3, M4 and M5 receptors, whereas that for the M1 receptor was greater. Our results indicate that there are subtype-specific differences in the rate and extent of agonist-induced muscarinic receptor internalization, recycling and downregulation in CHO cells. PMID:21044619

  6. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

    PubMed

    Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell. PMID:27271046

  7. Retinoids increase transglutaminase activity and inhibit ornithine decarboxylase activity in Chinese hamster ovary cells and in melanoma cells stimulated to differentiate.

    PubMed Central

    Scott, K F; Meyskens, F L; Russell, D H

    1982-01-01

    Transglutaminase (TGase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) and ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase, EC 4.1.1.17) activities were measured after the addition of retinoid analogs to Chinese hamster ovary (CHO) cells released from quiescence and Cloudman S91 (CCL 53.1) mouse melanoma cells stimulated to differentiate with alpha-melanocyte-stimulating hormone (MSH, melanotropin). In both cell culture lines, we detected a biphasic increase in TGase activity and a single peak of ODCase activity within 7 hr after release or stimulation. Retinoid analogs altered the expression of the initial TGase peak in both CHO and melanoma cells. Retinol increased the activity of TGase 1 hr after release in CHO cells, and the activity remained elevated until hr 4. A broad peak of TGase activity also occurred after the addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODCase, and after addition of alpha-difluoromethylornithine plus retinol. In mouse melanoma cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. Retinoic acid alone also increased TGase activity biphasically in these cells without the addition of MSH. These studies suggest that retinoid effects that increase TGase activity may alter the ODCase expression in proliferation and differentiation. PMID:6125941

  8. Use of an antikinetochore antibody and DNA probes to measure aneuploidy induction in interphase human lymphocytes and Chinese hamster ovary cells

    SciTech Connect

    Eastmond, D.A.; Tucker, J.D.; Pinkel, D.

    1988-12-05

    Aneuploidy in germ cells is associated with birth defects, spontaneous abortions, and infertility, whereas in somatic cells aneuploidy may lead to cell death and carcinogenesis. The nonrandom numerical chromosomal changes that are often observed in tumors or transformed cells suggest that aneuploidy induction by chemicals may be involved in carcinogenesis. The identification of aneuploidy inducing agents (aneuploidogens) and studies into the mechanisms by which aneuploidy may be involved in carcinogenesis are currently limited in that standard cytogenetic techniques are time consuming, require highly skilled personnel and are prone to technical artifacts. Recent developments in immunology and molecular biology have resulted in new techniques which may allow simple and rapid identification of aneuploidogens. We report the development of two new approaches to determine the aneuploidy-inducing potential of chemicals. The first approach involves the induction of micronuclei in human lymphocytes and Chinese hamster ovary (CHO) cells and the use of an antikinetochore antibody to determine whether micronuclei contain centromeres---a condition indicating potential aneuploidy. The second approach involves the use of in situ hybridization with fluorescently labeled chromosome-specific DNA probes and the subsequent counting of the number of copies of that chromosome in the interphase nuclei of human lymphocytes. 8 refs., 1 fig., 1 tab.

  9. Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

    PubMed

    Carlage, Tyler; Kshirsagar, Rashmi; Zang, Li; Janakiraman, Vijay; Hincapie, Marina; Lyubarskaya, Yelena; Weiskopf, Andy; Hancock, William S

    2012-01-01

    Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance. PMID:22556165

  10. Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture.

    PubMed

    Hu, Dongdong; Sun, Yating; Liu, Xuping; Liu, Jintao; Zhang, Xintao; Zhao, Liang; Wang, Haibin; Tan, Wen-Song; Fan, Li

    2015-10-01

    Yeast extract (YE), as a non-animal source additive for mammalian cell culture medium, has been widely used for manufacturing of therapeutic proteins. In the present study, one particular YE was found to have significantly improved the specific productivity (q p) of Fc-fusion protein in recombinant Chinese hamster ovary (rCHO) cell culture. In order to elucidate the intracellular effects of YE on protein productivity, steps of the target protein synthesis process were investigated to unveil their variations caused by YE addition. Stepwise analysis on Fc-fusion protein synthesis process showed that YE enhanced Fc-fusion protein gene transcription with cell cycle arrest at G1 phase; mammalian target of rapamycin (mTOR) signaling pathway was activated to enhance the translation of Fc-fusion protein, and the block in post-translational steps of Fc-fusion protein was alleviated by YE addition as well. Our results revealed the responses of multiple protein production steps to the addition of YE and provided a practical guidance for the separation and application of active compounds from hydrolysates. PMID:26162671

  11. Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system): expression in division-arrested cell cultures

    SciTech Connect

    O'Neill, J.P.; Machanoff, R.; Hsie, A.W.

    1982-01-01

    The phenotypic expression time of ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants was studied with Chinese hamster ovary cells in culture (CHO/HGPRT system). After mutagen treatment of exponential phase cultures, the cells were maintained either in the exponential phase through subculture in medium containing 5% dialyzed fetal bovine serum (FBS) or in a nondividing viable state by use of medium containing 0-1% dialyzed FBS. The time course of expression of the 6-thioguanine-resistant phenotype was similar with both exponential phase division-arrested cultures showing maximum expression by 9 days after mutagen event is fixed, the expression of the mutant phenotype does not require continued cell division since it occurs in division-arrested cultures. These results also suggest that both dilution of pre-existing hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme by cell division and turnover by protein degradation are involved in the phenotypic expression. These results separate genetic damage and phenotypic expression in a temporal sense, and point out the need to consider the mechanisms responsible for each process involved in the induction and expression of mutations.

  12. Cyto-genotoxicity assessment of potential radioprotector, 3,3'-diselenodipropionic acid (DSePA) in Chinese Hamster Ovary (CHO) cells and human peripheral blood lymphocytes.

    PubMed

    Chaurasia, Rajesh Kumar; Balakrishnan, Sreedevi; Kunwar, Amit; Yadav, Usha; Bhat, Nagesh; Anjaria, Kshiti; Nairy, Rajesha; Sapra, Balvinder Kaur; Jain, Vimal Kumar; Priyadarsini, K Indira

    2014-11-01

    Our previous study showed that 3,3'-diselenodipropionic acid (DSePA), a simple, stable, and water-soluble organoselenium exhibiting glutathione peroxidase (GPx)-like activity offered good radioprotection under in vitro and in vivo conditions. Herein, we investigated the anti-genotoxic effect of DSePA in model cellular systems such as Chinese Hamster Ovary (CHO) cell line and human peripheral lymphocytes after exposure to γ-radiation. The measurements on the induction of γ-H2AX foci and micronuclei frequency in the cell nuclei indicated that pretreatment with DSePA significantly prevented the radiation induced DNA damage or genotoxicity and subsequent cytotoxicity without exerting its own toxicity. The maximum protective effect of DSePA was seen at a pre-treatment concentration of 3 μg/ml. The mechanistic investigations in CHO cells revealed that DSePA pretreatment prevented the radiation induced ROS generation, lipid peroxidation and subsequent apoptosis in these cells. Further, it was seen to augment the mRNA expressions of GPx2 significantly and GPx4 marginally without causing much change in the total GPx activity after radiation exposure. These results suggested the roles of GPx2 and GPx4 in DSePA mediated radioprotection. In conclusion our results confirm the nongenotoxic nature of the DSePA and validate its radioprotective efficacy and mechanisms of action in model cellular systems. PMID:25440905

  13. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -Cl-alanine

    SciTech Connect

    Medlock, K.A.; Merrill, A.H. Jr.

    1987-05-01

    Serine palmitoyltransferase (SPT) is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the first committed step of long-chain base (LCB) synthesis. Inhibition of SPT activity and de novo biosynthesis of sphinganine and sphingosine was observed in vitro and in intact Chinese hamster ovary cells (CHO). In vitro studies revealed that inhibition was irreversible and concentration- and time-dependent, which are characteristics of suicide inhibition. Incubation of intact CHO cells with 5 mM ..beta..-Cl-alanine for 15 min completely inhibited SPT activity and LCB synthesis from (/sup 14/C)serine. The concentration dependences of inhibition of SPT activity and LCB formation were identical. There was no loss of viability of recovery of SPT activity over the 2 hour time course of these experiments. The synthesis of several other lipids was not affected by the same treatment. These results establish the association between the activity of SPT and the cellular rate of LCB formation and indicate that ..beta..-Cl-alanine can be used to study alterations in cellular LCB synthesis.

  14. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell. PMID:27271046

  15. Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

    PubMed Central

    Elliott, E M; Henderson, G; Sarangi, F; Ling, V

    1986-01-01

    The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed. PMID:3773896

  16. Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice.

    PubMed

    Hou, Yingping; Guey, Lin T; Wu, Timothy; Gao, Robert; Cogan, Jon; Wang, Xinyi; Hong, Elizabeth; Ning, Weihuang Vivian; Keene, Douglas; Liu, Nan; Huang, Yan; Kaftan, Craig; Tangarone, Bruce; Quinones-Garcia, Igor; Uitto, Jouni; Francone, Omar L; Woodley, David T; Chen, Mei

    2015-12-01

    Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB. PMID:26203639

  17. Fragmentation radioinduite de l'ADN et réparation étudiée par immunomarquage anti poly(ADP-ribose)polymérase (PARP) dans les cellules de hamster chinois (CHO)

    NASA Astrophysics Data System (ADS)

    Bidon, N.; Varlet, P.; Noël, G.; Demurcia, G.; Salamero, J.; Averbeck, D.

    1998-04-01

    The poly(ADP-ribose)polymerase is a nuclear ubiquitous enzyme capable of binding to DNA breaks. Chinese hamster ovary cells were (CHO-K1) cultured on slides and γ-irradiated (137Cs) at a high (12.8 Gy/min) or medium dose rate (5 Gy/min), and immunolabelling against (ADP-ribose) polymers immediatly or three hours after irradiation. Quantification and localisation of γ-ray induced breaks was performed by confocal microscopy. The results show a dose effect relationship, a dose-rate effect and the signal disappearence after 3 hours at 37 °C. The presence of PARP activity appears to reflect γ-rays induced DNA fragmentation. Le poly(ADP-ribose)polymérase est une enzyme nucléaire ubiquitaire capable de se fixer sur les cassures de l'ADN. Sur une lignée sauvage de cellules d'ovaire de hamster chinois (CHO-K1) cultivée sur lame et irradiée aux rayons γ à haut débit de dose (HD) 12,8 Gy/min ou à moyen débit de dose (MD) 5 Gy/min, nous avons réalisé un immunomarquage anti-polymères d'ADP-ribose immédiatement après l'irradiation γ ou après trois heures d'incubation à 37 °C. La quantification et la localisation des lésions radioinduites ont été réalisées par microscopie confocale. Les résultats montrent une relation dose-effet et un effet de débit de dose, ainsi qu'une disparition du signal après 3 heures à 37 °C. La présence de la PARP semble donc bien refléter la fragmentation radioinduite de l'ADN.

  18. Chinese Hamster Ovary (CHO) Host Cell Engineering to Increase Sialylation of Recombinant Therapeutic Proteins by Modulating Sialyltransferase Expression

    PubMed Central

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R.; George, Henry J.; Brooks, Jeanne; Kayser, Kevin J.; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio-better” protein therapeutics and cell culture vaccine production. PMID:25641927

  19. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  20. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  1. Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

    PubMed

    Ha, Tae Kwang; Kim, Yeon-Gu; Lee, Gyun Min

    2015-08-01

    To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells. PMID:25728222

  2. Chemometrics and in-line near infrared spectroscopic monitoring of a biopharmaceutical Chinese hamster ovary cell culture: prediction of multiple cultivation variables.

    PubMed

    Clavaud, Matthieu; Roggo, Yves; Von Daeniken, Ralph; Liebler, André; Schwabe, Jan-Oliver

    2013-07-15

    In the present study near infrared (NIR) spectroscopy was used to monitor the cultivation of mammalian Chinese hamster ovary (CHO) cells producing a monoclonal antibody in a fed-batch cell culture process. A temperature shift was applied during the cultivation. The cells were incubated at 37 °C and 33 °C. The Fourier transform near infrared (FT-NIR) multiplex process analyzer spectroscopy was investigated to monitor cultivation variables of the CHO cell culture from 10 independent batches using two channels of the FT-NIR. The measurements were performed on production scale bioreactors of 12,500 L. The cell cultures were analyzed with the spectrometer coupled to a transflection sterilizable fiber optic probe inserted into the bioreactors. Multivariate data analysis (MVDA) employing unsupervised principal component analysis (PCA) and partial least squares regression methods (PLS) were applied. PCA demonstrated that 96% of the observed variability was explained by the process trajectory and the inter-batch variability. PCA was found to be a significant tool in identifying batch homogeneity between lots and in detecting abnormal fermentation runs. Seven different cell culture parameters such as osmolality, glucose concentration, product titer, packed cell volume (PCV), integrated viable packed cell volume (ivPCV), viable cell density (VCD), and integrated viable cell count (iVCC) were monitored inline and predicted by NIR. NIR spectra and reference analytics data were computed using control charts to evaluate the monitoring abilities. Control charts of each media component were under control by NIR spectroscopy. The PLS calibration plots offered accurate predictive capabilities for each media. This paper underlines the capability for inline prediction of multiple cultivation variables during bioprocess monitoring. PMID:23622522

  3. Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.

    PubMed Central

    Kojima, Hiroyuki; Katsura, Eiji; Takeuchi, Shinji; Niiyama, Kazuhito; Kobayashi, Kunihiko

    2004-01-01

    We tested 200 pesticides, including some of their isomers and metabolites, for agonism and antagonism to two human estrogen receptor (hER) subtypes, hERalpha and hERbeta, and a human androgen receptor (hAR) by highly sensitive transactivation assays using Chinese hamster ovary cells. The test compounds were classified into nine groups: organochlorines, diphenyl ethers, organophosphorus pesticides, pyrethroids, carbamates, acid amides, triazines, ureas, and others. These pesticides were tested at concentrations < 10-5 M. Of the 200 pesticides tested, 47 and 33 showed hER- and hERbeta-mediated estrogenic activities, respectively. Among them, 29 pesticides had both hERalpha and hERbeta agonistic activities, and the effects of the organochlorine insecticides beta-benzene hexachloride (BHC) and delta-BHC and the carbamate insecticide methiocarb were predominantly hERbeta rather than hERalpha agonistic. Weak antagonistic effects toward hERalpha and hERbeta were shown in five and two pesticides, respectively. On the other hand, none of tested pesticides showed hAR-mediated androgenic activity, but 66 of 200 pesticides exhibited inhibitory activity against the transcriptional activity induced by 5alpha-dihydrotestosterone. In particular, the antiandrogenic activities of two diphenyl ether herbicides, chlornitrofen and chlomethoxyfen, were higher than those of vinclozolin and p,p -dichlorodiphenyl dichloroethylene, known AR antagonists. The results of our ER and AR assays show that 34 pesticides possessed both estrogenic and antiandrogenic activities, indicating pleiotropic effects on hER and hAR. We also discussed chemical structures related to these activities. Taken together, our findings suggest that a variety of pesticides have estrogenic and/or antiandrogenic potential via ER and/or AR, and that numerous other manmade chemicals may also possess such estrogenic and antiandrogenic activities. PMID:15064155

  4. Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells

    PubMed Central

    Yu, Chao; Crispin, Max; Sonnen, Andreas F.-P.; Harvey, David J.; Chang, Veronica T.; Evans, Edward J.; Scanlan, Christopher N.; Stuart, David I.; Gilbert, Robert J. C.; Davis, Simon J.

    2011-01-01

    Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3–5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3–4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a = 43.9, b = 51.5, c = 102.9 Å and space group P212121 that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems. PMID:21795794

  5. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture

    PubMed Central

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T.; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A.; Valdez-Cruz, Norma A.

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  6. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

    SciTech Connect

    Nishijima, M.; Kuge, O.; Akamatsu, Y.

    1986-05-05

    The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and /sup 32/Pi, the incorporation of /sup 32/Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. /sup 32/Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of /sup 32/Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using (/sup 3/H)serine-labeled phospholipid. Pulse and pulse-chase experiments with L-(U-/sup 14/C) serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine.

  7. Expression of Human NAD(P)H:Quinone Oxidoreductase (DT-Diaphorase) in Chinese Hamster Ovary Cells: Effect on the Toxicity of Antitumor Quinones

    PubMed Central

    GUSTAFSON, DANIEL L.; BEALL, HOWARD D.; BOLTON, EMIKO M.; ROSS, DAVID; WALDREN, CHARLES A.

    2013-01-01

    SUMMARY Previous studies have indicated that NAD(P)H:quinone oxidoreductase [DT-diaphorase (NQO1)] plays an important role in the bioreductive activation of quinone-containing antitumor agents. Although these studies demonstrated that purified NQO1 can reduce these compounds in vitro, the importance NQO1 in the intracellular activation of quinone-containing antitumor agents remains controversial. In our study, we transfected human NQO1 into Chinese hamster ovary cells that do not normally express NQO1 activity and obtained stable clones that expressed NQO1 activity of 19–3527 nmol of 2,6-dichlorophenolindophenol reduced/min/mg of protein. The level of NQO1 expression correlated with an increased killing by streptonigrin, EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propenol), and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone, but mitomycin C sensitivity was independent of this activity. NQO1 expression also led to a slight decrease in the sensitivity of cells to menadione. Our data demonstrate that compounds that are efficient substrates for NQO1 in vitro are also bioactivated in cultured mammalian cells when they are transfected with human NQO1. These results are consistent with the relative abilities of mitomycin C, streptonigrin, EO9, and 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone to serve as substrates for bioreduction by human NQO1, and show that NQO1 levels are not necessarily predictive in terms of sensitivity to mitomycin C. PMID:8863816

  8. Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.

    PubMed

    Bedoya-López, Andrea; Estrada, Karel; Sanchez-Flores, Alejandro; Ramírez, Octavio T; Altamirano, Claudia; Segovia, Lorenzo; Miranda-Ríos, Juan; Trujillo-Roldán, Mauricio A; Valdez-Cruz, Norma A

    2016-01-01

    Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system. PMID:26991106

  9. Down-regulation of cold-inducible RNA-binding protein does not improve hypothermic growth of Chinese hamster ovary cells producing erythropoietin.

    PubMed

    Hong, Jong Kwang; Kim, Yeon-Gu; Yoon, Sung Kwan; Lee, Gyun Min

    2007-03-01

    Discovery of the cold-inducible RNA-binding protein (CIRP) in mouse fibroblasts suggests that growth suppression at hypothermic conditions is due to an active response by the cell rather than due to passive thermal effects. To determine the effect of down-regulated CIRP expression on cell growth and erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells at low culture temperature, stable CHO cell clones with reduced CIRP expression level were established by transfecting (rCHO) cells with the CIRP siRNA vector with a target sequence of TCGTCCTTCCATGGCTGTA. For comparison of the degree of specific growth rate (micro) reduction at low culture temperature, three CIRP-reduced clones with different mu and three control clones transfected with null vector were cultivated at two different temperatures, 32 degrees C and 37 degrees C. Unlike mouse fibroblasts, alleviation of hypothermic growth arrest of rCHO cells by CIRP down-regulation was insignificant, as shown by statistical analysis using the t-test (P<0.18, n=3). The ratios of mu at 32 degrees C to micro at 37 degrees C of CIRP-reduced clones and control clones were 0.29+/-0.03 and 0.25+/-0.03 on an average, respectively. Furthermore, it was also found that overexpression of CIRP did not inhibit rCHO cell growth significantly at 37 degrees C. Taken together, the data obtained show that down-regulation of only CIRP in rCHO cells, unlike mouse fibroblasts, is not sufficient to recover growth arrest at low-temperature culture (32 degrees C). PMID:17239640

  10. Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells.

    PubMed

    Morton, H C; Atkin, J D; Owens, R J; Woof, J M

    1993-11-01

    Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function. PMID:8409433

  11. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -chloroalanine

    SciTech Connect

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-09-06

    The effects of ..beta..-chloroalanine (..beta..-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with ..beta..-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with ..beta..-Cl-alanine and this treatment inhibited (/sup 14/C)serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of ..beta..-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, (/sup 14/C)serine uptake was not blocked, total lipid biosynthesis from (/sup 14/C)acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and (/sup 3/H)thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of (/sup 3/H)leucine, which was only decreased by 14%. Although ..beta..-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that ..beta..-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

  12. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    PubMed

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology. PMID:21959846

  13. Folate-dependent enzymes in cultured Chinese hamster ovary cells: impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine-auxotroph complementation classes

    SciTech Connect

    Taylor, R.T.; Hanna, M.L.

    1982-09-01

    Two glycine-requiring Chinese hamster ovary (CHO) auxotrophs (GLYB and AUXB2) representative of the Gly/sup -/ mutant classes B and C are shown to have defects in folate metabolism. These defects result in 10-fold lower rates of whole cell L-(U-/sup 14/C)serine-to-(/sup 14/C)glycine conversion relative to the parental CHO lines (2 vs 20 nmol/h/10/sup 6/ cells). This restriction in serine hydroxymethyltransferase (SHMT) activity is localized in the mitochondria. Intact mitochondria from GLYB and AUXB2 convert labeled serine to glycine at 1-4% the rate and with only 1-3% of the total capacity of parental CHO mitochondria. Yet, GLYB and AUXB2 contain parental cell amounts of cytosolic and mitochondrial SHMT, the latter displaying normal substrate K/sub m/ values. The whole cell and mitochondrial impairments in glycine formation are corrected in GLYB (but not AUXB2) by a prior growth with 100 ..mu..M dl-folinate. They are also partially restored in spontaneous or chemically induced Gly/sup +/ revertants of GLYB and AUXB2. Subcellular fractionation experiments suggest that a low content (one-fifth parental) of mitochondrial folylpolyglutamates contributes to the auxotrophy of GLYB. These studies demonstrate that mitochondrial SHMT is potentially functional in the Gly/sup -/ mutant classes B (GLYB) and C (AUXB2). The impaired SHMT activity in vivo and in isolated mitochondria may result from a deficiency in mitochondrial recycling of 5,10-methylenetetrahydrofolate back to tetrahydrofolate.

  14. Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells

    SciTech Connect

    Kenne, K.; Ljungquist, S.; Ringertz, N.R.

    1986-04-01

    The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo(a)pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 ..mu..m), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possible be used as an index of the degree of mitotic disturbances induced by mineral fibers.

  15. Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant.

    PubMed

    Zhang, Peiqing; Haryadi, Ryan; Chan, Kah Fai; Teo, Gavin; Goh, John; Pereira, Natasha Ann; Feng, Huatao; Song, Zhiwei

    2012-07-01

    The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect. PMID:22492235

  16. Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells.

    PubMed

    Dufour, Eric K; Kumaravel, Tirukalikundram; Nohynek, Gerhard J; Kirkland, David; Toutain, Hervé

    2006-09-01

    Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in

  17. Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

    PubMed

    Liu, B; Nakashima, S; Adachi, T; Ito, Y; Takano, T; Shimizu, T; Nozawa, Y

    1997-10-01

    The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that

  18. Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

    PubMed

    Renzetti, A R; Catalioto, R M; Carloni, C; Criscuoli, M; Cucchi, P; Giolitti, A; Zappitelli, S; Rotondaro, L; Maggi, C A

    1999-04-15

    A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists. PMID:10086323

  19. Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    PubMed Central

    Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

    2015-01-01

    Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites

  20. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells

    PubMed Central

    2014-01-01

    Background Establishing highly productive clonal cell lines with constant productivity over 2–3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure. Results We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative. Conclusions The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX

  1. Cytotoxic and mutagenic properties of shale oil byproducts. II. Comparison of mutagenic effects at five genetic markers induced by retort process water plus near ultraviolet light in Chinese hamster ovary cells

    SciTech Connect

    Chen, D.J.C.; Strniste, G.F.

    1982-01-01

    A Chinese hamster ovary (CHO) cell line heterozygous at the adenine phosphoribosyl transferase (APRT) locus was used for selection of induced mutants resistant to 8-azaadenine (8AA), 6-thioguanine (6TG), ouabain (OUA), emetine (EMT) and diphtheria toxin (DIP). The expression times necessary for optimizing the number of mutants recovered at the different loci have been determined using the known direct acting mutagen, far ultraviolet light (FUV), and a complex aqueous organic mixture (shale oil process water) activated with near ultraviolet light (NUV). The results indicate that optimal expression times following treatment with either mutagen was between 2 and 8 days. For CHO cells treated with shale oil process water and subsequently exposed to NUV a linear dose response for mutant induction was observed for all five genetic loci. At 10% surviving fraction of cells, between 35- and 130-fold increases above backgound mutation frequencies were observed for the various markers examined.

  2. The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells.

    PubMed Central

    Lidell, Martin E; Johansson, Malin E V; Mörgelin, Matthias; Asker, Noomi; Gum, James R; Kim, Young S; Hansson, Gunnar C

    2003-01-01

    The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2. PMID:12597771

  3. Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent mechanism in Chinese hamster ovary cells: regulation by Nrf2 and MafG transcription factors.

    PubMed

    Gong, P; Hu, B; Stewart, D; Ellerbe, M; Figueroa, Y G; Blank, V; Beckman, B S; Alam, J

    2001-07-20

    We have shown previously that activation of the heme oxygenase-1 (ho-1) gene by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking HIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia-mimetic CoCl(2) was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIF-independent mechanisms for ho-1 gene activation by hypoxia and CoCl(2). In Ka13 cells, CoCl(2) stimulated expression of a luciferase reporter gene under the control of a 15-kilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified the cobalt-responsive sequences as the stress-response elements (StREs). In electrophoretic mobility shift assays, two specific StRE-protein complexes were observed using extracts from Ka13 cells. In response to cobalt, the level of the slower migrating complex X increased, whereas that of complex Y decreased, in a time-dependent manner. Members of the AP-1 superfamily of basic-leucine zipper factors bind to the StRE. Antibody supershift electrophoretic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of complex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but not of other bZIP factors, attenuated cobalt-mediated gene activation. Additional experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 but is dependent on cellular oxidative stress. Unlike cobalt, hypoxia did not stimulate pHO15luc expression and did not increase StRE binding activity, indicating distinct mechanisms for ho-1 gene activation by cobalt and hypoxia in Chinese hamster ovary cells. PMID:11356853

  4. Latent transforming growth factor-beta complex in Chinese hamster ovary cells contains the multifunctional cysteine-rich fibroblast growth factor receptor, also termed E-selectin-ligand or MG-160.

    PubMed Central

    Olofsson, A; Hellman, U; Ten Dijke, P; Grimsby, S; Ichijo, H; Morén, A; Miyazono, K; Heldin, C H

    1997-01-01

    Transforming growth factor-beta (TGF-beta) is secreted as latent high molecular mass complexes from producer cells. The N-terminal precursor remnant, also called latency-associated peptide (LAP), forms a non-covalently linked complex with TGF-beta and confers the latency to TGF-beta. In human platelets and certain other cell types, latent TGF-beta binding protein-1 (LTBP-1) is disulphide-linked to LAP, and forms complexes of more than 230 kDa. In addition, LTBP-2 and -3, which are structurally similar to LTBP-1, can be part of latent TGF-beta complexes. In Chinese hamster ovary (CHO) cells transfected with the TGF-beta1 cDNA, a major part of the latent TGF-beta secreted into the medium is a 100-kDa small latent complex containing TGF-beta and LAP. In addition, we found two other forms of latent TGF-beta complexes, i.e. a 220-kDa complex containing LTBP-1, and a 220-kDa complex containing a 140-kDa protein. Purification of the 140-kDa component, termed latent TGF-beta complexed protein-1 (LTCP-1), followed by amino acid sequencing and cDNA cloning from a CHO cell cDNA library, revealed that it is a hamster counterpart of a previously identified, multifunctional protein known as chicken cysteine-rich fibroblast growth factor (FGF) receptor, mouse E-selectin-ligand and rat MG-160 (a 160-kDa membrane sialoglycoprotein of the Golgi apparatus). Immunoprecipitation of LTCP-1 and TGF-beta1 from CHO cells stably transfected with TGF-beta1 precursor cDNA revealed that the expressed protein forms a complex with LAP, and that a major part of the complex is secreted. Northern blot analysis showed that mRNA for LTCP-1 was expressed in large amounts in testis, ovary and placenta, but less abundantly in other tissues. These results suggest that TGF-beta, produced in certain cell types, may form a complex with LTCP-1, which may have different properties compared with other latent TGF-beta complexes. It remains to be investigated whether the complex formation between LTCP-1 and TGF

  5. Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1).

    PubMed

    Sealover, Natalie R; Davis, Angela M; Brooks, Jeanne K; George, Henry J; Kayser, Kevin J; Lin, Nan

    2013-08-10

    While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production. PMID:23777858

  6. Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: Lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein

    SciTech Connect

    Freeman, M. Massachusetts General Hospital, Boston ); Ekkel, Y.; Rohrer, L.; Penman, M.; Freedman, N.J.; Krieger, M. ); Chisolm, G.M. )

    1991-06-01

    Type I and type II scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-associated functions, differ only by the presence in the type I receptor of an extracellular cysteine-rich C-terminal domain. Stable Chinese hamster ovary (CHO) cell transfectants expressing high levels of either the type I or type II bovine scavenger receptors have been generated. Type I and type II receptors in these cells mediated high-affinity saturable endocytosis of both {sup 125}I-labeled acetylated low density lipoprotein (LDL) and {sup 125}I-labeled oxidized LDL with the distinctive broad ligand specificity characteristic of scavenger receptors. After incubation for 2 days with acetylated LDL, the transfected cells accumulated oil red O-staining lipid droplets reminiscent of those in macrophage foam cells, whereas untransfected CHO cells did not. Thus, macrophage-specific gene products other than the scavenger receptor are not required for modified-LDL-induced intracellular lipid accumulation. In transfected cells, acetylated LDL efficiently competed for both its own endocytosis and that of oxidized LDL. This nonreciprocal cross competition suggests that these ligands may bind to nonidentical but interacting sites on a single receptor. Results were similar for transfectants expressing either type I or type II scavenger receptors. The nonreciprocal cross competition seen in the transfected CHO cells differs from that previously observed with cultured macrophages.

  7. The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells.

    PubMed

    Owen, D G; Hall, A; Stephens, G; Stow, J; Robertson, B

    1997-03-01

    1. The mKv1.1 voltage-gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole-cell currents recorded by the patch-clamp method. 2. A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration-inhibition relationships. 3. The rank order of potency, thus determined was: Toxin K > 7-dendrotoxin(7-Dtx) > delta-Dtx > Toxin I = alpha-Dtx > beta-Dtx. 4. Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels. 5. A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pM compared with 1.8 nM for delta-Dtx in spite of 95% sequence identity. PMID:9134213

  8. The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells

    PubMed Central

    Owen, David G; Hall, Adam; Stephens, Gary; Stow, John; Robertson, Brian

    1997-01-01

    The mKv1.1 voltage-gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole-cell currents recorded by the patch-clamp method.A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration-inhibition relationships.The rank order of potency, thus determined was: Toxin K>γ-dendrotoxin(γ-Dtx)>δ-Dtx>Toxin I=α-Dtx>β-Dtx.Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels.A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pM compared with 1.8 nM for δ-Dtx in spite of 95% sequence identity. PMID:9134213

  9. Effects of chronic pH 6.6 on growth, intracellular pH, and response to 42.0 degrees C hyperthermia of Chinese hamster ovary cells.

    PubMed

    Cook, J A; Fox, M H

    1988-05-01

    Culturing Chinese hamster ovary cells in low pH (6.6) medium for several months altered the reproductive survival of these cells to combined low pH treatments and 42.0 degrees C heating. We isolated new pH-resistant cells (identified as pHV-2) with enhanced ability to grow and divide under a low pH (6.6) environment. Their growth characteristics include (a) a plating efficiency of 70%, (b) a doubling time of 16 to 17 h, and (c) a steady state intracellular pH 0.12 pH units higher than for cells grown at a normal pH of 7.3. The pHV-2 cells had 100- to 200-fold increases in survival after 5 h of heating compared to cells incubated at low pH (6.6) for 4 h prior to and during the heat treatments. In addition, they developed a significant degree of thermotolerance. We measured a progressive decline in the intracellular pH as a function of time at 42.0 degrees C. However, the decrease in the intracellular pH did not seem to be correlated with the increased heat sensitivity. The ability to select for low pH variants may have important implications in the extrapolation of in vitro hyperthermic data to the in vivo situation. PMID:3356006

  10. High affinity binding of [3H]propionyl-[Met(O2)11]substance P(7-11), a tritiated septide-like peptide, in Chinese hamster ovary cells expressing human neurokinin-1 receptors and in rat submandibular glands.

    PubMed

    Sagan, S; Beaujouan, J C; Torrens, Y; Saffroy, M; Chassaing, G; Glowinski, J; Lavielle, S

    1997-07-01

    Propionyl-[Met(O2)11]substance P(7-11) [ALIE-124 or propionyl-[Met(O2)11]SP(7-11)] has been designed as a septide-like ligand adequate for tritiation and, therefore, adequate for binding studies. In Chinese hamster ovary (CHO) cells expressing human tachykinin neurokinin (NK)-1 receptors, ALIE-124 displaced [3H][Pro9]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [3H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (Kd = 6.6 +/- 1.0 nM), with a low maximal binding capacity. [3H]ALIE-124 specific maximal binding represented only 15-20% of that observed with [3H][Pro9]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [3H]ALIE-124 specific binding site. Interestingly, SP and [Pro9]SP were also potent competitors, with 10-fold greater potency for sites labeled with [3H]ALIE-124 than for sites labeled with [3H][Pro9]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [3H]ALIE-124 than for [3H][Pro9]SP-specific binding sites. NKB and [Lys5,methyl-Leu9,Nle10]NKA(4-10) displaced [3H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [3H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [3H]ALIE-124 specific maximal binding was higher, reaching 40-50% of that achieved with [3H][Pro9]SP. PMID:9224821

  11. Assay of SF/sub 6/ and spark-decomposed SF/sub 6/ for mutagenic activity in the CHO/HGPRT (Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase) mammalian cell system

    SciTech Connect

    Kurka, K.; Griffin, G.D.

    1987-01-01

    The potential mutagenic (and cytotoxic) activity of SF/sub 6/ and spark-decomposed SF/sub 6/ was investigated in an in vitro mammalian cell culture system using Chinese Hamster Ovary (CHO) cells. The CHO cells were exposed to the gases in vacutainer tubes which were constantly rotated. After a 4 h exposure the mutagenic and cytotoxic activity was assayed with the CHO/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system. Results indicated that SF/sub 6/ was neither cytotoxic nor mutagenic to CHO cells. Spark-decomposed SF/sub 6/ was found to be strongly cytotoxic (-80% cell death following 4 h exposure to 2 kJ spark discharge in 60 cm/sup 3/ at 1000 torr of SF/sub 6/) but not mutagenic. Increasing spark energy increased cytotoxicity but the spark samples remained nonmutagenic. The CHO/HGPRT system was coupled with a metabolic activation (S9 fraction) system used for detecting promutagens. When exposures were carried out in the presence of S9 fraction, SF/sub 6/ was still neither cytotoxic nor mutagenic; spark-decomposed SF/sub 6/ was again strongly cytotoxic but not mutagenic. It appears that SF/sub 6/ and sparked SF/sub 6/ are neither promutagens nor direct acting mutagens in the CHO/HGPRT system. Studies have begun using a more mutagenically sensitive subclone of the CHO cells known as CHO-AS/sub 52/. The results of initial experiments using the CHO-AS/sub 52/ cells remain unchanged. 9 refs., 1 tab.

  12. Poly(N-isopropylacrylamide)-coated thermo-responsive nanoparticles for controlled delivery of sulfonated Zn-phthalocyanine in Chinese hamster ovary cells in vitro and zebra fish in vivo

    NASA Astrophysics Data System (ADS)

    He, Jia; Chen, Ji-Yao; Wang, Pu; Wang, Pei-Nan; Guo, Jia; Yang, Wu-Li; Wang, Chang-Chun; Peng, Qian

    2007-10-01

    Poly(N-isopropylacrylamide) (PNIPAM)-coated Fe3O4@SiO2@CdTe multifunctional nanoparticles with photoluminescent (PL), thermosensitive and magnetic properties, were investigated as carriers to deliver water-soluble, fluorescent sulfonated Zn-phthalocyanine (ZnPcS), a photosensitizing drug for photodynamic therapy of cancer, in Chinese hamster ovary (CHO) cells in vitro and zebra fish in vivo. PNIPAM is a well-known thermo-responsive polymer with a volume phase transition temperature. This property allows it to be swollen in water at temperatures lower than 32-34 °C to take up ZnPcS and shrunken to expel the drug at higher temperatures. Since the PL band of CdTe quantum dots (QDs) as indicators for the nanoparticles is at 585 nm and the emission band of ZnPcS is at 680 nm, it is possible to study the temperature-dependent release of ZnPcS from the nanoparticles by fluorescence measurements. ZnPcS was embedded in the PNIPAM of the nanoparticles at 25 °C in phosphate buffered saline (PBS) solution and released at 37 °C, measured with a spectrophotometer. When CHO cells had been incubated with the ZnPcS-loaded nanoparticles at 27 °C, a similar intracellular localization pattern of CdTe QDs and ZnPcS was seen by multichannel measurements in confocal laser scanning microscopy (CLSM), but a diffuse pattern of only ZnPcS fluorescence was detected in the cytoplasm of the cells at 37 °C, indicating a release of ZnPcS from the nanoparticles. Similar results were also found in the intestinal tract of zebra fish in vivo after intake of the nanoparticles. Since the nanoparticles contain magnetic (Fe3O4) material, the nanoparticles could also be manipulated to change their location in the intestinal tract of the zebra fish with an external magnetic field gradient of 300 G mm-1. The results presented suggest that such multifunctional nanoparticles may have combined potential for temperature-dependent drug delivery, QD photodetection and magnetic manipulation in diagnosis and

  13. 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.

    PubMed

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R; Metry, Kristin J; Doll, Mark A; States, J Christopher; Pierce, William M; Hein, David W

    2007-07-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism

  14. Lack of carcinogenicity of cadmium chloride in female Syrian hamsters.

    PubMed

    Waalkes, M P; Rehm, S

    1998-04-01

    Cadmium is very effective at inducing necrosis within the ovaries of rodents, and the Syrian hamster appears particularly sensitive. The extent of cadmium-induced necrosis depends on the stage of the estrous cycle and is most pronounced when injected on the day prior to ovulation (proestrous). In male rodents cadmium induces a similar necrosis within the testes, which given sufficient time can lead to the development of testicular tumors. In this study we tested the hypothesis that cadmium-induced ovarian necrosis could eventually lead to tumor formation. In sexually mature groups of female Syrian hamsters (> 8 weeks old; n = 50-59), the estrous cycle was determined by visual inspection of vaginal discharge for four consecutive cycles. The animals were then given cadmium (0, 30, 40 and 50 micromol/kg) subcutaneously as a single injection in the dorsal thoracic midline on cycle day 4 (proestrous). Based on prior work, these doses are sufficient to induce extensive acute ovarian damage. Animals were then observed over the next 78 weeks. Although survival and body weight were reduced by cadmium, treatment with the metal did not result in an enhanced incidence of tumors at any site including the ovaries. Non-neoplastic lesions such as amyloidosis and pancreatic hepatocytes were linked to cadmium exposure. These results indicate that the association of cadmium-induced testicular necrosis with tumor development seen in males does not occur in the Syrian hamster ovaries. PMID:9674965

  15. Polycystic Ovary Syndrome FAQ

    MedlinePlus

    f AQ FREQUENTLY ASKED QUESTIONS FAQ121 GYNECOLOGIC PROBLEMS Polycystic Ovary Syndrome (PCOS) • What are common signs and symptoms of polycystic ovary syndrome (PCOS)? • What causes PCOS? • What is insulin resistance? • ...

  16. Polycystic Ovary Syndrome

    MedlinePlus

    Polycystic ovary syndrome (PCOS) happens when a woman's ovaries or adrenal glands produce more male hormones than normal. One result is that cysts ( ... who are obese are more likely to have polycystic ovary syndrome. Symptoms of PCOS include: Infertility Pelvic pain Excess ...

  17. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  18. Increasing effect of tri-n-butyltins and triphenyltins on the frequency of chemically induced chromosome aberrations in cultured Chinese hamster cells.

    PubMed

    Sasaki, Y F; Yamada, H; Sugiyama, C; Kinae, N

    1993-06-01

    Organotins have been widely used as anti-fouling coatings for fishing nets and ship bottoms, and marine pollution by them has become a serious environmental problem. In this communication, the potentiating effects of three kinds of tri-n-butyltins and three thiphenyltins on chromosome aberrations were studied in Chinese hamster CHO K1 cells. None of the organotins studied showed any clastogenic activity under the experimental conditions without rat liver S9. Post-treatment with organotins, however, increased the number of breakage-type (but not exchange-type) chromatid aberrations induced by five kinds of S-phase-dependent clastogens: MMC, cisPt, 4NQO, MMS, and AMD). Enhancement of the induction of chromosome aberrations by MMC was observed when cells were treated with organotins during the G2 phase. These results suggest that organotin G2 effect causes potentiating effects. Organotins also enhanced the induction of breakage-type chromatid aberrations by clastogenic pollutants in chlorinated tap water, indicating their potential for a more realistic health risk. PMID:7683769

  19. Polycystic ovary disease

    MedlinePlus

    ... hormones. This may lead to changes in the menstrual cycle, cysts in the ovaries , trouble getting pregnant, and ... Symptoms Symptoms of PCOS include changes in the menstrual cycle, such as: Not getting a period after you ...

  20. A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

    SciTech Connect

    O'Donnell, Christopher D.; Tiwari, Vaibhav; Oh, Myung-Jin; Shukla, Deepak . E-mail: dshukla@uic.edu

    2006-03-15

    Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.

  1. A role for 3-O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread

    SciTech Connect

    Tiwari, Vaibhav; O'Donnell, Christopher D.; Oh, Myung-Jin; Valyi-Nagy, Tibor; Shukla, Deepak . E-mail: dshukla@uic.edu

    2005-12-16

    Many heparan sulfate (HS) 3-O-sulfotransferase (3-OST) isoforms generate cellular receptors for herpes simplex virus type-1 (HSV-1) glycoprotein D (gD). Interestingly, the ability of 3-OST-4 to mediate HSV-1 entry and cell-to-cell fusion has not been determined, although it is predominantly expressed in the brain, a primary target of HSV-1 infections. We report that expression of 3-OST-4 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and a mutant (Rid1) strain of HSV-1. Evidence for generation of gD receptors by 3-OST-4 was suggested by gD-mediated interference assay and the ability of 3-OST-4 expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases-II/III). In addition, 3-OST-4 expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together our results suggest a role of 3-OST-4 in HSV-1 pathogenesis.

  2. Chinese hamster pleiotropic multidrug-resistant cells are not radioresistant

    SciTech Connect

    Mitchell, J.B.; Gamson, J.; Russo, A.; Friedman, N.; DeGraff, W.; Carmichael, J.; Glatstein, E.

    1988-01-01

    The inherent cellular radiosensitivity of a Chinese hamster ovary pleiotropic cell line that is multidrug resistant (CHRC5) was compared to that of its parental cell line (AuxB1). Radiation survival curve parameters n and D0 were 4.5 and 1.1 Gy, respectively, for the CHRC5 line and 5.0 and 1.2 Gy, respectively, for the parental line. Thus, the inherent radiosensitivity of the two lines was similar even though key intracellular free radical scavenging and detoxifying systems employing glutathione, glutathione transferase, and catalase produced enzyme levels that were 2.0-, 1.9-, and 1.9-fold higher, respectively, in the drug-resistant cell line. Glutathione depletion by buthionine sulfoximine resulted in the same extent of aerobic radiosensitization in both lines (approximately 10%). Incorporation of iododeoxyuridine into cellular DNA sensitized both cell lines to radiation. These studies indicate that pleiotropic drug resistance does not necessarily confer radiation resistance.

  3. Role of luteinizing hormone in luteotropic complex of pregnant hamster

    SciTech Connect

    Tamura, H.; Greenwald, G.S.

    1987-04-01

    Hamsters were hypophysectomized on day 4 of pregnancy and injected subcutaneously on days 4-7 with various combinations of 200 ..mu..g prolactin (Prl), 10 ..mu..g follicle-stimulating hormone (FSH), and 20 ..mu..g luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) to decrease its rate of absorption or in saline. End points for luteal function on day 8 were maintenance of pregnancy, serum progesterone (P/sub 4/), luteal weight, and luteal binding for human chorionic gonadotropin, FSH, and Prl. After hypophysectomy, a drastic decline occurred in all parameters including an 89% decrease in luteal weight. Injection of Prl did not maintain pregnancy nor serum P/sub 4/ but partially maintained luteal weight and human chorionic gonadotropin binding sites per corpus luteum. The minimal luteotropic complex of Prl and FSH was effective in maintaining pregnancy and significantly increased serum P/sub 4/ and Prl and FSH receptors but not to control levels. Thus, the luteotropic activity of LH was only demonstrable when it was injected in a long-acting form; when delivered as a bolus, LH (saline) was luteolytic. P/sub 4/ and estradiol were measured by radioimmunoassay. Radioiodinated gonadotropins were prepared. The percentage of tracer reacting with an excess of receptor were 51% of /sup 125/I-FSH and 45.9% of /sup 125/I-hCG using whole homogenates of hamster ovaries.

  4. Analysis of the DNA replication competence of the xrs-5 mutant cells defective in Ku86.

    PubMed

    Matheos, Diamanto; Novac, Olivia; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2003-01-01

    The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein, previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2, PCNA, DNA polymerase epsilon and delta, Primase and Topoisomerase IIalpha) were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oribeta) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oribeta origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells. PMID:12456721

  5. Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

    PubMed

    Vishwanathan, Nandita; Bandyopadhyay, Arpan A; Fu, Hsu-Yuan; Sharma, Mohit; Johnson, Kathryn C; Mudge, Joann; Ramaraj, Thiruvarangan; Onsongo, Getiria; Silverstein, Kevin A T; Jacob, Nitya M; Le, Huong; Karypis, George; Hu, Wei-Shou

    2016-09-01

    Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines. PMID:27374913

  6. Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Albanetti, Thomas; Linkous, Travis; Larkin, Christopher; Schoner, Ronald; McGivney, James B; Dovichi, Norman J

    2016-10-01

    We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed. For each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. Over 70% of the supernatant proteins in the CHO K1SV host cell line are present in the CHO CAT-S cell line. For the CHO K1SV cell line, the overlap in supernatant protein identifications between the host cell line and the transfected clones is >59%. For the CHO CAT-S cell line, the overlap between supernatant protein identifications for the transfected clone and host cell is >45%. These differences in the supernatant protein identifications between transfected clones in each cell line and between the two host cell lines are not significant. We used cluster analysis to characterize the change in supernatant protein expression as a function of cell culture time. Roughly <60% of the supernatant proteins show significant change across the three time points (ratio >1.3 or <0.7). We also used cluster analysis to compare changes in supernatant protein expression between the host and three transfected clones at each time point. Greater than 65% of the common proteins in the CHO K1SV cell line supernatant and over 54% in the CHO CAT-S cell line supernatant show no significant expression difference between host and the three transfected clones. Data are available via ProteomeXchange with identifier PXD003462. Biotechnol. Bioeng. 2016;113: 2140-2148. © 2016 Wiley Periodicals, Inc. PMID:27070921

  7. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    PubMed

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. PMID:25792187

  8. Polycystic ovary syndrome.

    PubMed

    Azziz, Ricardo; Carmina, Enrico; Chen, ZiJiang; Dunaif, Andrea; Laven, Joop S E; Legro, Richard S; Lizneva, Daria; Natterson-Horowtiz, Barbara; Teede, Helena J; Yildiz, Bulent O

    2016-01-01

    Polycystic ovary syndrome (PCOS) affects 5-20% of women of reproductive age worldwide. The condition is characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology (PCOM) - with excessive androgen production by the ovaries being a key feature of PCOS. Metabolic dysfunction characterized by insulin resistance and compensatory hyperinsulinaemia is evident in the vast majority of affected individuals. PCOS increases the risk for type 2 diabetes mellitus, gestational diabetes and other pregnancy-related complications, venous thromboembolism, cerebrovascular and cardiovascular events and endometrial cancer. PCOS is a diagnosis of exclusion, based primarily on the presence of hyperandrogenism, ovulatory dysfunction and PCOM. Treatment should be tailored to the complaints and needs of the patient and involves targeting metabolic abnormalities through lifestyle changes, medication and potentially surgery for the prevention and management of excess weight, androgen suppression and/or blockade, endometrial protection, reproductive therapy and the detection and treatment of psychological features. This Primer summarizes the current state of knowledge regarding the epidemiology, mechanisms and pathophysiology, diagnosis, screening and prevention, management and future investigational directions of the disorder. PMID:27510637

  9. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    SciTech Connect

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light.

  10. Carcinoma of the Ovary

    PubMed Central

    Weekes, Leroy R.; Watkins, Sue A.

    1981-01-01

    The data for this paper are based on 50 patients discharged from the Queen of Angels Hospital with a diagnosis of carcinoma of the ovary from 1972 to 1978. Currently, ovarian cancer is the leading cause of death of all pelvic malignancies. Peak incidence of ovarian cancer is found in women between 40 to 65 years of age. Symptomatology includes often vague abdominal discomfort, dyspepsia, and other digestive disorders which may be present for several months prior to diagnosis. The workup for suspected ovarian cancer should include a careful history, physical examination, pelvic, and rectal examinations, Pap smear, CBC, urinalysis, SMA 12 (blood chemistries), chest x-ray, and intravenous pyelography as indicated. Sonography, lymphangiography are optional. Traditionally, operative treatment has been the keystone of management for ovarian carcinoma. In view of the unsatisfactory results with operation and radiotherapy in disseminated disease, chemotherapy has been used widely. Hope for the future lies in further development of immunodiagnosis and immunotherapy. PMID:7310921

  11. Directed Student Inquiry: Modeling in Roborovsky Hamsters

    ERIC Educational Resources Information Center

    Elwess, Nancy L.; Bouchard, Adam

    2007-01-01

    In this inquiry-based activity, Roborovsky hamsters are used to provide students with an opportunity to develop their skills of analysis, inquiry, and design. These hamsters are easy to maintain, yet offer students a means to use conventional techniques and those of their own design to make further observations through measuring, assessing, and…

  12. FATE OF INHALED FLY ASH IN HAMSTERS

    EPA Science Inventory

    To determine pulmonary deposition, translocation, and clearance of inhaled fly ash, hamsters received a single 95-min nose-only exposure to neutron-activated fly ash. Over a period of 99 days postexposure, the hamsters were sacrificed in groups of six animals. Lungs, liver, kidne...

  13. Tumours of the ovary

    PubMed Central

    Nielsen, Svend W.; Misdorp, W.; McEntee, Kenneth

    1976-01-01

    Ovarian tumours are common in animals, the majority occurring in bitches and cows. The two most important germ cell tumours were dysgerminoma and teratoma; these morphologically resemble their counterparts in women, with the exception that teratomas in animals tend less to malignancy. The granulosa cell tumour is the most frequent sex cord-stromal tumour in all six species and it may contain luteinized areas or show differentiation towards a Sertoli cell pattern. The canine papillary adenoma and papillary adenocarcinoma, which are as common as granulosa tumours, have several features in common with their counterparts in women: they are of similar histological appearance, are frequently bilateral, and the adenocarcinomas have a great propensity for peritoneal implantation metastasis. Ovarian cysts are frequent in the bitch, sow, and cow and may originate from five different anatomical structures in the ovary. ImagesFig. 1Fig. 2 and 3Fig. 20-22Fig. 8-10Fig. 15 and 16Fig. 23Fig. 24Fig. 25Fig. 26Fig. 17-19Fig. 4 and 5Fig. 6 and 7Fig. 11Fig. 12Fig. 13 and 14 PMID:1086151

  14. [Polycystic ovary syndrome].

    PubMed

    Vrbíková, Jana

    2015-10-01

    For diagnosing of polycystic ovary syndrome (PCOS) it is currently recommended to follow the ESHRE criteria. For diagnosis according to them two of the following three symptoms are sufficient: 1. morphology of polycystic ovaria, 2. clinical manifestations of hyperandrogenism or laboratory proof of hyperandrogenemia, and 3. oligo-anovulation. PCOS is a complex disorder in whose pathogenesis genetic and environmental effects interact. It is not a gynecological disorder alone, the syndrome is accompanied by insulin resistance which leads to increased incidence of type 2 diabetes mellitus and impaired glucose tolerance (4 times and twice, independently of BMI). Also gestational DM occurs more frequently. Dyslipidemia, arterial hypertension, elevated CRP and homocysteine levels, endothelial dysfunction and greater intima-media thickness are also more frequent. It is not quite clear, however, whether women with PCOS suffer cardiovascular events more frequently as well. More often than is accidental PCOS is associated with depression, anxiety and eating disorders, further with nonalcoholic steatohepatitis and with the sleep apnoea syndrome - especially in obese women. Therapeutic measures include non-pharmacological methods - lifestyle adjustments focused on weight reduction in obese individuals, cosmetic measures for dermatologic manifestation of hyperandrogenism, in particular laser and pharmacotherapy (combined hormonal contraceptives and antiandrogens). Menstrual irregularities can be treated with contraceptives or cyclical administration of gestagens, also metformin can be used. PMID:26486483

  15. The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

    PubMed Central

    Altman, Amy L.; Fanning, Ellen

    2001-01-01

    To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-β) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-β in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-β region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-β deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-β region is sufficient to direct initiation and that specific DNA sequences in the ori-β region are required for efficient initiation activity. PMID:11158297

  16. A three-step purification strategy for isolation of hamster TIG2 from CHO cells: characterization of two processed endogenous forms.

    PubMed

    Busmann, Annette; Walden, Michael; Wendland, Martin; Kutzleb, Christian; Forssmann, Wolf-Georg; John, Harald

    2004-11-25

    We have recently isolated a bioactive, circulating protein of human tazarotene-induced gene-2 (TIG2) as the natural ligand of the orphan receptor ChemR23. Here we describe a simplified method for the isolation of hamster TIG2 protein from Chinese hamster ovary (CHO) cell supernatant. Using a heparin-affinity column followed by two reversed phase chromatography steps resulted in the isolation of pure biologically active material. Two processed bioactive forms of Chinese hamster TIG2 were identified by Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) mass fingerprint analysis, representing the amino acid residues T20 to F156, and T20 to A155 of the 163 amino acid propeptide. Comparison with the predicted aa-sequence indicates a mutation or modification within the C-terminal end of the peptide. PMID:15522723

  17. Cryopreservation of ovaries from neonatal marmoset monkeys

    PubMed Central

    Motohashi, Hideyuki H.; Ishibashi, Hidetoshi

    2016-01-01

    The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries. PMID:26876597

  18. Cryopreservation of ovaries from neonatal marmoset monkeys.

    PubMed

    Motohashi, Hideyuki H; Ishibashi, Hidetoshi

    2016-07-29

    The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries. PMID:26876597

  19. [Laparoscopic fenestration of the ovaries].

    PubMed

    Nalbanski, B; Pŭnevska, M; Veltova, L

    1997-01-01

    The authors have performed an ovary fenestration in 220 women with primary or secondary amenorrhea. This was made with the help of instrumentation for laparoscopy and a monopolar electrodiathermic coagulator with differently shaped endings. The power supply was provided from Martin Elektrotom 2000. A spontaneous recovers of the menstruation was achieved in 36.84% of the cases as a result of the endoscopic intervention. A follows up pregnancy was achieved in 12.4% of the women. Considering the obtained results, the authors recommend the use of a laparoscopic ovary fenestration when a women with ovarian cysts is treated. PMID:9471896

  20. Induction of lyme arthritis in LSH hamsters

    SciTech Connect

    Schmitz, J.L.; Schell, R.F.; Hejka, A.; England, D.M.; Konick, L.

    1988-09-01

    In studies of experimental Lyme disease, a major obstacle has been the unavailability of a suitable animal model. We found that irradiated LSH/Ss Lak hamsters developed arthritis after injection of Borrelia burgdorferi in the hind paws. When nonirradiated hamsters were injected in the hind paws with B. burgdorferi, acute transient synovitis was present. A diffuse neutrophilic infiltrate involved the synovia and periarticular structures. The inflammation was associated with edema, hyperemia, and granulation tissue. Numerous spirochetes were seen in the synovial and subsynovial tissues. The histopathologic changes were enhanced in irradiated hamsters. The onset and duration of the induced swelling were dependent on the dose of radiation and the inoculum of spirochetes. Inoculation of irradiated hamsters with Formalin-killed spirochetes or medium in which B. burgdorferi had grown for 7 days failed to induce swelling. This animal model should prove useful for studies of the immune response to B. burgdorferi and the pathogenesis of Lyme arthritis.

  1. Spontaneous endomyometrial neoplasms in aging Chinese hamsters

    SciTech Connect

    Brownstein, D.G.; Brooks, A.L.

    1980-05-01

    Twenty-one endomyometrial neoplasms among 93 nulliparous noninbred Chinese hamsters were evaluated. The median survival time of the 93 females was 1040 days. The median age of hamsters with endomyometrial neoplasms was 1200 days. Neoplasms were classified as carcinomas or malignant mixed muellerian tumors of the endometrium and benign or malignant myometrial neoplasms. There were 13 endometrial adenocarcinomas. Three tumors were mixed adenosquamous carcinomas, which occurred in significantly older Chinese hamsters than did adenocarcinomas. Three malignant mixed muellerian tumors consisted of 2 carcinosarcomas and 1 mixed mesodermal tumor. The 2 myometrial neoplasms were a lelomyoma and a lelomyosarcoma. The classification and relative frequency of these neoplasms were similar to endomyometrial neoplasms of women, which makes Chinese hamsters useful subjects for studies of spontaneous endomyometrial cancers.

  2. 4-Vinylcyclohexene diepoxide reduces fertility in female Siberian hamsters when treated during their reproductively active and quiescent states.

    PubMed

    Roosa, Kristen A; Mukai, Motoko; Place, Ned J

    2015-01-01

    The industrial compound 4-vinylcyclohexene diepoxide (VCD) destroys ovarian follicles and reduces fertility in rodents, but to date VCD has not been tested in species that experience seasonal anestrus. To determine if VCD destroys follicles when administered during reproductive quiescence, Siberian hamsters were treated with VCD (240mg/kg i.p. daily for 10 days) during short days, and outcomes were compared with reproductively active females that were maintained and treated in long days. Primordial follicle numbers were significantly reduced by VCD under both day lengths, and reproductive quiescence in short days did not appear to render the ovaries less susceptible to VCD-induced follicle depletion. Independent of day length and reproductive state, VCD-treated hamsters weaned substantially fewer offspring than controls. These results suggest that time of year may not be an important consideration for optimizing use of VCD in the field when the target pest species is a seasonally breeding rodent. PMID:25511107

  3. SV40 lymphomagenesis in Syrian golden hamsters

    PubMed Central

    McNees, Adrienne L.; Vilchez, Regis A.; Heard, Tiffany C.; Sroller, Vojtech; Wong, Connie; Herron, Alan J.; Hamilton, Mary J.; Davis, William C.; Butel, Janet S.

    2013-01-01

    Simian virus 40 (SV40) isolates differ in oncogenic potential in Syrian golden hamsters following intraperitoneal inoculation. Here we describe the effect of intravenous exposure on tumor induction by SV40. Strains SVCPC (simple regulatory region) and VA45-54(2E) (complex regulatory region) were highly oncogenic following intravenous inoculation, producing a spectrum of tumor types. Three lymphoma cell lines were established; all expressed SV40 T-antigen, were immortalized for growth in culture, and were tumorigenic following transplantation in vivo. New monoclonal antibodies directed against hamster lymphocyte surface antigens are described. The cell lines expressed MHC class II and macrophage markers and were highly phagocytic, indicating a histiocytic origin. Many hamsters that remained tumor-free developed SV40 T-antigen antibodies, suggesting that viral replication occurred. This study shows that route of exposure influences the pathogenesis of SV40-mediated carcinogenesis, that SV40 strain VA45-54(2E) is lymphomagenic in hamsters, that hamster lymphoid cells of histiocytic origin can be transformed in vivo and established in culture, and that reagents to hamster leukocyte differentiation molecules are now available. PMID:19038412

  4. APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Apoptosis in Whole Mouse Ovaries
    Robert M. Zucker Susan C. Jeffay and Sally D. Perreault
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  5. Effect of the drug Lonidamine on Chinese hamster ovary cells in vitro and on experimental tumors

    SciTech Connect

    Rotin, D.; Tannock, I.F.

    1984-09-01

    Hypoxic cells in solid tumors are known to be resistant to radiation, and may also be resistant to some anti-cancer drugs. Biochemical properties of hypoxic cells, such as their dependence on anaerobic glycolysis leading to production of lactate and low pH might have potential for inhibition by drugs with selective activity against hypoxic cells. Such drugs might improve the Therapeutic Index when used with radiation or some conventional anti-cancer drugs. Lonidamine, an inhibitor of mitochondrially-bound hexokinase and lactate transport, was cytotoxic to CHO cells at low pH, but had no effect at physiological pH under aerobic or hypoxic conditions. Lonidamine has also been tested for in vivo effects against three murine tumors: the KHT fibrosarcoma, 16/C mammary carcinoma and the Lewis Lung Tumor. The drug was tested either alone, or with radiation or Adriamycin to kill aerobic cells, and/or with glucose and insulin to lower intra-tumor pH. No major therapeutic effects have been demonstrated.

  6. Size and number of DNA molecules from Chinese hamster ovary cells determined by molecular autoradiography

    SciTech Connect

    Todd, M.B.

    1980-06-01

    A new method for visualization of separable subunits of DNA is described. Autoradiography of tritium-labeled DNA from one or a few nuclei, lysed with detergent, moderate salt, and proteases, and gently deposited on a filter, allows determination of subunit molecular weight, size distribution, number per nucleus, and organization. The shape of the size distribution of CHO subunit images is similar to that of CHO mitotic chromosomes, and the numbers of subunits per nucleus supports a model of eight subunits per chromosome.

  7. Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

    PubMed

    Lee, Suk Kyoo; Lee, Gyun Min

    2003-06-30

    Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. PMID:12701155

  8. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

    PubMed Central

    Looney, J E; Hamlin, J L

    1987-01-01

    We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line. Images PMID:3821723

  9. A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

    PubMed Central

    del Val, Ioscani Jimenez; Polizzi, Karen M.; Kontoravdi, Cleo

    2016-01-01

    Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation. PMID:27345611

  10. A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation.

    PubMed

    Del Val, Ioscani Jimenez; Polizzi, Karen M; Kontoravdi, Cleo

    2016-01-01

    Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation. PMID:27345611

  11. Isolation of a human DNA repair gene by selection in Chinese hamster ovary cells

    SciTech Connect

    Ding, R.C.; Eastman, A.; Bresnick, E.

    1987-05-01

    Alkylation of DNA at the O/sup 6/-position of guanine represents a potent mutagenic and carcinogenic lesion. O/sup 6/-Methylguanine DNA methyltransferase is the repair system responsible for catalyzing the transfer of the methyl group to a cysteine of the protein in a suicide reaction. The gene controlling its expression in mammalian systems is designated mex. Resistance to chloroethylnitrosourea (CNU) is also mediated by this protein; this was used to select cells into which the max gene has been introduced. DNA purified from human liver has been transfected into mex/sup -/ CHO cells by the CaPO/sub 4/ method. pSV2gpt, containing a marker gene, gpt, was cotransfected. The transformed cells were initially selected for the expression of gpt (mycophenolic acid resistance) and reselected in CNU for mex/sup +/. Several clones were resistant to both demonstrating the linkage of these genes. A cosmid library was made from a mex/sup +/gpt/sup +/ clone and grown in a gpt/sup -/ strain of E. coli. gpt/sup +/ colonies were selected and the cosmid DNA rescued. One of the tested cosmid DNA's produced CNU resistance upon introduction into CHO cells. This cosmid was subcloned, restriction endonuclease-treated and a 5.3 kb fragment showed mex activity. This fragment is being further characterized and the DNA sequenced.

  12. Mathematical Models Describing Chinese Hamster Ovary Cell Death Due to Electroporation In Vitro.

    PubMed

    Dermol, Janja; Miklavčič, Damijan

    2015-10-01

    Electroporation is a phenomenon used in the treatment of tumors by electrochemotherapy, non-thermal ablation with irreversible electroporation, and gene therapy. When treating patients, either predefined or variable electrode geometry is used. Optimal pulse parameters are predetermined for predefined electrode geometry, while they must be calculated for each specific case for variable electrode geometry. The position and number of electrodes are also determined for each patient. It is currently assumed that above a certain experimentally determined value of electric field, all cells are permeabilized/destroyed and under it they are unaffected. In this paper, mathematical models of survival in which the probability of cell death is continuously distributed from 0 to 100 % are proposed and evaluated. Experiments were performed on cell suspensions using electrical parameters similar to standard electrochemotherapy and irreversible electroporation parameters. The proportion of surviving cells was determined using clonogenic assay for assessing the ability of a cell to grow into a colony. Various mathematical models (first-order kinetics, Hülsheger, Peleg-Fermi, Weibull, logistic, adapted Gompertz, Geeraerd) were fitted to experimental data using a non-linear least-squares method. The fit was evaluated by calculating goodness of fit and by observing the trend of values of models' parameters. The most appropriate models of cell survival as a function of treatment time were the adapted Gompertz and the Geeraerd models and, as a function of the electric field, the logistic, adapted Gompertz and Peleg-Fermi models. The next steps to be performed are validation of the most appropriate models on tissues and determination of the models' predictive power. PMID:26223863

  13. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    PubMed Central

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  14. [Diagnostics of polycystic ovary syndrome].

    PubMed

    Lazúrová, Ivica; Figurová, Jana; Lazúrová, Zora

    2015-12-01

    Polycystic ovary syndrome (PCOS) is the most frequent endocrinopathy among women of reproductive age and the most frequent cause of menstruation cycle disorders. It is marked by a hyperandrogenic state (clinical and/or biochemical) and ovulatory dysfunction (anovulation and/or ultrasonographic finding of polycystic ovaries), which are also criteria for its diagnosis according to Androgen Excess and PCOS Society. The syndrome has multiple phenotypic expressions, among them besides the above characteristics also a metabolic syndrome, primarily obesity and insulin resistance. Diagnosing of PCOS may be rather exacting in clinical practice and it remains to be a diagnosis per exclusionem, following elimination of other causes of hyperandrogenic state and chronic oligo-anovulation. It requires a close cooperation between a gynecologist and endocrinologist and with regard to frequent metabolic complications also with an internist, diabetologist and possibly cardiologist. PMID:27124971

  15. Metabolic Syndrome: Polycystic Ovary Syndrome.

    PubMed

    Mortada, Rami; Williams, Tracy

    2015-08-01

    Polycystic ovary syndrome (PCOS) is a heterogeneous condition characterized by androgen excess, ovulatory dysfunction, and polycystic ovaries. It is the most common endocrinopathy among women of reproductive age, affecting between 6.5% and 8% of women, and is the most common cause of infertility. Insulin resistance is almost always present in women with PCOS, regardless of weight, and they often develop diabetes and metabolic syndrome. The Rotterdam criteria are widely used for diagnosis. These criteria require that patients have at least two of the following conditions: hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. The diagnosis of PCOS also requires exclusion of other potential etiologies of hyperandrogenism and ovulatory dysfunction. The approach to PCOS management differs according to the presenting symptoms and treatment goals, particularly the patient's desire for pregnancy. Weight loss through dietary modifications and exercise is recommended for patients with PCOS who are overweight. Oral contraceptives are the first-line treatment for regulating menstrual cycles and reducing manifestations of hyperandrogenism, such as acne and hirsutism. Clomiphene is the first-line drug for management of anovulatory infertility. Metformin is recommended for metabolic abnormalities such as prediabetes, and a statin should be prescribed for cardioprotection if the patient meets standard criteria for statin therapy. PMID:26280343

  16. Human follicular fluid adverses hamster spermatozoa motility.

    PubMed

    Wetzels, A; Goverde, H J; Bastiaans, L A; Rolland, R

    1989-01-01

    To determine the optimal conditions for in vitro spermatozoa vitality, human and hamster spermatozoa were incubated at 37 degrees C in T6 medium supplemented with different biologic fluids (10% v/v). The fluids tested were human serum (HUS), hamster serum (HAS), and human follicular fluid (HUF). After incubation the spermatozoa were investigated for their qualitative and quantitative motility. Human spermatozoa maintained a good vitality in all fluids tested (approximately 25% motility after 18-h incubation). The hamster spermatozoa had after an incubation of 4 h a motility of 28.4% in HUS, 14.2% in HAS, and 2.2% in HUF. The quality of the motility was also extremely low in HUF, whereas it was adequate in HUS and in HAS. The presence of species-specific substances in mammalian follicular fluid is discussed. PMID:2589906

  17. Histopathology of Lyme arthritis in LSH hamsters

    SciTech Connect

    Hejka, A.; Schmitz, J.L.; England, D.M.; Callister, S.M.; Schell, R.F.

    1989-05-01

    The authors studied the histopathologic evolution of arthritis in nonirradiated and irradiated hamsters infected with Borrelia burgdorferi. Nonirradiated hamsters injected in the hind paws with B. burgdorferi developed an acute inflammatory reaction involving the synovium, periarticular soft tissues, and dermis. This acute inflammatory reaction was short-lived and was replaced by a mild chronic synovitis as the number of detectable spirochetes in the synovium, periarticular soft tissues, and perineurovascular areas diminished. Exposing hamsters to radiation before inoculation with B. burgdorferi exacerbated and prolonged the acute inflammatory phase. Spirochetes also persisted longer in the periarticular soft tissues. A major histopathologic finding was destructive and erosive bone changes of the hind paws, which resulted in deformation of the joints. These studies should be helpful in defining the immune mechanism participating in the onset, progression, and resolution of Lyme arthritis.

  18. Decreased adult neurogenesis in hibernating Syrian hamster.

    PubMed

    León-Espinosa, Gonzalo; García, Esther; Gómez-Pinedo, Ulises; Hernández, Félix; DeFelipe, Javier; Ávila, Jesús

    2016-10-01

    Generation of new neurons from adult neural stem cells occurs in the dentate gyrus (DG) of the hippocampus and the lateral walls of the lateral ventricles. In this article, we study the neurogenesis that takes place during the hibernation of the Syrian hamster (Mesocricetus auratus). Using a variety of standard neurogenesis markers and 5-bromo-2-deoxyuridine (BrdU) incorporation, we describe a preferential decrease in the proliferation of newborn neurons in the subventricular zone (SVZ) of the hibernating hamsters (torpor) rather than in the hippocampus. Furthermore, we demonstrate that the proliferative capacity is recovered after 3-4days of torpor when arousal is triggered under natural conditions (i.e., not artificially provoked). In addition, we show that tau3R, a tau isoform with three microtubule-binding domains, is a suitable marker to study neurogenesis both in the SVZ and subgranular zone (SGZ) of the Syrian hamster brain. PMID:27436535

  19. Identification of the Syrian hamster cardiomyopathy gene.

    PubMed

    Nigro, V; Okazaki, Y; Belsito, A; Piluso, G; Matsuda, Y; Politano, L; Nigro, G; Ventura, C; Abbondanza, C; Molinari, A M; Acampora, D; Nishimura, M; Hayashizaki, Y; Puca, G A

    1997-04-01

    The BIO14.6 hamster is a widely used model for autosomal recessive cardiomyopathy. These animals die prematurely from progressive myocardial necrosis and heart failure. The primary genetic defect leading to the cardiomyopathy is still unknown. Recently, a genetic linkage map localized the cardiomyopathy locus on hamster chromosome 9qa2.1-b1, excluding several candidate genes. We now demonstrate that the cardiomyopathy results from a mutation in the delta-sarcoglycan gene that maps to the disease locus. This mutation was completely coincident with the disease in backcross and F2 pedigrees. This constitutes the first animal model identified for human sarcoglycan disorders. PMID:9097966

  20. Southern analysis of genomic alterations in gamma-ray-induced aprt- hamster cell mutants

    SciTech Connect

    Grosovsky, A.J.; Drobetsky, E.A.; deJong, P.J.; Glickman, B.W.

    1986-06-01

    The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.

  1. Congenital Transmission of Experimental Leishmaniasis in a Hamster Model

    PubMed Central

    Osorio, Yaneth; Rodriguez, Luz D.; Bonilla, Diana L.; Peniche, Alex G.; Henao, Hector; Saldarriaga, Omar; Travi, Bruno L.

    2012-01-01

    Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection. PMID:22556079

  2. Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters

    PubMed Central

    delBarco-Trillo, Javier; Johnston, Robert E.

    2012-01-01

    If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8 days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters. PMID:22658324

  3. Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters.

    PubMed

    delBarco-Trillo, Javier; Johnston, Robert E

    2012-08-01

    If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters. PMID:22658324

  4. Cloning, Identification and Functional Characterization of Bovine Free Fatty Acid Receptor-1 (FFAR1/GPR40) in Neutrophils

    PubMed Central

    Manosalva, Carolina; Mena, Jaqueline; Velasquez, Zahady; Colenso, Charlotte K.; Brauchi, Sebastian; Burgos, Rafael A.; Hidalgo, Maria A.

    2015-01-01

    Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid. PMID:25790461

  5. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    PubMed Central

    Lo, Shih-Hsiang; Cheng, Kai-Chung; Li, Ying-Xiao; Chang, Chin-Hong; Cheng, Juei-Tang; Lee, Kung-Shing

    2016-01-01

    Background G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time. Conclusion Similar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future. PMID:27578964

  6. Enhanced sialylation of a human chimeric IgG1 variant produced in human and rodent cell lines.

    PubMed

    Mimura, Yusuke; Kelly, Ronan M; Unwin, Louise; Albrecht, Simone; Jefferis, Roy; Goodall, Margaret; Mizukami, Yoichi; Mimura-Kimura, Yuka; Matsumoto, Tsuneo; Ueoka, Hiroshi; Rudd, Pauline M

    2016-01-01

    Glycosylation of the IgG-Fc is essential for optimal binding and activation of Fcγ receptors and the C1q component of complement. However, it has been reported that the effector functions are down-regulated when the Fc glycans terminate in sialic acid residues and that sialylated IgG mediates anti-inflammatory effects of intravenous immunoglobulin (IVIG). Although recombinant IgG is hypo-sialylated, Fc sialylation is shown to be markedly increased when a mouse/human chimeric IgG3 Phe243Ala (F243A) variant is expressed in Chinese hamster ovary (CHO)-K1 cells. Here we investigate whether sialylation is increased in IgG1 F243A when expressed in CHO-K1, mouse myeloma J558L and human embryonic kidney (HEK) 293. Although the sialylation level was 2-5% for IgG1 wild type (WT), it was increased to 31%, 10% and 33% for the variant from CHO-K1, J558L and HEK293 cells, respectively. Interestingly, an increased addition of bisecting GlcNAc and α(1-3)-galactose residues to the Fc glycan was observed for HEK293-derived and J558L-derived IgG1 F243A, respectively. Fucosylation of HEK293-derived IgG1 F243A was maintained despite increased bisecting GlcNAc content. Although sialic acid and bisecting GlcNAc residues are reported to have an opposing effect on antibody-dependent cellular cytotoxicity (ADCC), IgG1 F243A showed 7 times lower ADCC activities than IgG1 WT, irrespective of bisecting GlcNAc residue. Thus, highly sialylated, human cell-derived IgG1 F243A with lowered ADCC activity may be of interest for the development of therapeutic antibodies with anti-inflammatory properties as an alternative to IVIG. PMID:26627984

  7. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

    2014-03-01

    Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

  8. Polycystic Ovary Syndrome in Adolescents.

    PubMed

    Witchel, Selma Feldman; Roumimper, Hailey; Oberfield, Sharon

    2016-06-01

    Polycystic ovary syndrome (PCOS) is a familial heterogeneous disorder affecting 6% to 10% of reproductive-age women. The use of criteria developed for adult women is problematic for the adolescent girl because the clinical features associated with PCOS are normal pubertal events. The recent consensus statement on PCOS in adolescents stated that hyperandrogenism and oligomenorrhea need to persist for at least 2 years to consider the diagnosis of PCOS. Although insulin resistance, hyperinsulinism, and obesity are often associated with PCOS, these features are not considered valid diagnostic criteria. Recent genomewide association studies implicate genetic loci involved in the hypothalamic-pituitary-ovarian axis. PMID:27241968

  9. IN VITRO CULTURE OF POSTIMPLANTATION HAMSTER EMBRYOS

    EPA Science Inventory

    In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium sa...

  10. Preference for bedding material in Syrian hamsters.

    PubMed

    Lanteigne, M; Reebs, S G

    2006-10-01

    This study aimed to determine whether Syrian (golden) hamsters, Mesocricetus auratus, prefer certain bedding materials and whether bedding material can affect paw condition, body weight gain and wheel-running activity. In a first experiment, 26 male hamsters had access to two connected cages, each cage containing a different bedding material (either pine shavings, aspen shavings, corn cob or wood pellets). In a second experiment, 14 male hamsters had access to four connected cages that contained the different bedding materials and also a piece of paper towel to serve as nest material. In a third experiment, 30 male hamsters were each placed in a single cage, 10 of them with pine shavings, 10 with aspen shavings and 10 with corn cob, and they were monitored for 50 days. Significant preferences in the first experiment were: pine shavings over aspen shavings, corn cob over wood pellets, pine shavings over corn cob and aspen shavings over wood pellets (aspen shavings versus corn cob was not tested). However, there was no significant preference expressed in the second experiment, suggesting that the general preference for shavings in the first experiment was based on bedding material suitability as a nesting material. No significant effect of bedding material on paw condition, body weight gain and wheel-running activity was detected. None of the four bedding materials tested in this study can be judged to be inappropriate in the short term if nesting material is added to the cage and if the litter is changed regularly. PMID:17018212