WorldWideScience

Sample records for cho cells

  1. Towards dynamic metabolic flux analysis in CHO cell cultures.

    Science.gov (United States)

    Ahn, Woo Suk; Antoniewicz, Maciek R

    2012-01-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance. PMID:22102428

  2. Methods for modeling chinese hamster ovary (cho) cell metabolism

    DEFF Research Database (Denmark)

    2015-01-01

    Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods for...

  3. Identification of a novel temperature sensitive promoter in cho cells

    Directory of Open Access Journals (Sweden)

    Hesse Friedemann

    2011-05-01

    Full Text Available Abstract Background The Chinese hamster ovary (CHO expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. Results Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. Conclusions This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

  4. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan; Schulz, Morten Alder; Frodin, Morten; Rahman, Shamim H.; Vester-Christensen, Malene Bech; Behrens, Carsten; Kristensen, Claus; Vakhrushev, Sergey Y.; Bennett, Eric Paul; Wandall, Hans H.; Clausen, Henrik

    2015-01-01

    genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like alpha 2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic...

  5. Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness.

    Science.gov (United States)

    Sellick, Christopher A; Croxford, Alexandra S; Maqsood, Arfa R; Stephens, Gill M; Westerhoff, Hans V; Goodacre, Royston; Dickson, Alan J

    2015-09-01

    Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing. PMID:26198903

  6. Radiosensitization and radiation chemistry studies using CHO cells

    International Nuclear Information System (INIS)

    The cytotoxicity, radiosensitization and radiation chemistry studies on several recently synthesized isoindole-4,7-diones using chinese hamster ovary cells (CHO) have been carried out. The cytotoxicity studies have shown that these quinones have cytotoxic activity under oxic and hypoxic conditions. Radiosensitization studies using a Cs-137 irradiator at different radiation doses demonstrate that these isoindole-diones have radiosensitization characteristics under hypoxic conditions. The results are compared with the well known radiosensitizer, misoidazole, under the same conditions. The electron redox potential of these quinones are in the vicinity of -440mv to -360mv, which demonstrates that they have the appropriate electron affinity to transfer electrons quite readily. The interaction of glutathione, a well known radioprotector, with these quinones was also studied. The concentration of glutathione in CHO cells decreases very little in the presence of the isoindole-diones. The results of these experiments show that the radiosensitization mechanism of these isoindole-diones is mainly due to electron transfer reactions and not to interaction with chemical radioprotectors such as glutathione in the CHO cells

  7. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  8. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU106 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU106 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  9. Detachment factors for enhanced carrier to carrier transfer of CHO cell lines on macroporous microcarriers

    OpenAIRE

    Landauer, K.; Dürrschmid, M.; Klug, H.; Wiederkum, S.; Blüml, G.; Doblhoff-Dier, O.

    2002-01-01

    In this publication different detachment factors were tested for enhancing carrier to carrier transfer for scale-up of macroporous microcarrier based bioprocesses. Two Chinese hamster ovary cell lines, CHO-K1 and a genetically engineered CHO-K1 derived cell line (CHO-MPS), producing recombinant human Arylsulfatase B, were examined. The cells were grown on Cytoline 1microcarriers (Amersham Biosciences, Uppsala, Sweden) in protein-free and chemically defined medium respectively. Fully colonised...

  10. Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

    Science.gov (United States)

    Romand, Sandrine; Jostock, Thomas; Fornaro, Mara; Schmidt, Joerg; Ritter, Anett; Wilms, Burkhard; Laux, Holger

    2016-05-01

    Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells. Biotechnol. Bioeng. 2016;113: 1094-1101. © 2015 Wiley Periodicals, Inc. PMID:26523469

  11. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin; Chen, Wenbin; Xie, Min; Wang, Wenliang; Nagarajan, Harish; Lewis, Nathan; Famili, Iman; Palsson, Bernhard O.; Cai, Zhiming; Gui, Yaoting; Hammond, Stephanie; Lee, Kelvin H.; Andersen, Mikael Rørdam; Neff, Norma; Passarelli, Benedetto; Koh, Winston; Fan, H. Christina; Wang, Jianbin; Quake, Stephen R.; Betenbaugh, Michael; Palsson, Bernhard; Wang, Jun

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  12. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup; Kristensen, Claus; Andersen, Mikael Rørdam

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies of...

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

    Science.gov (United States)

    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

  14. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  15. N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

    DEFF Research Database (Denmark)

    Fan, Yuzhou

    cell culture technology, upstream process engineering, metabolic engineering, and glycobiology into a systematic framework allow us to improve the production of recombinant therapeutic protein towards an optimal balance between quantity and quality. In the presented work, recent know-how on impact......Thanks to the recent advances in Chinese hamster ovary (CHO) “omic” revolution, the development of recombinant therapeutic protein bioprocessing using CHO cell factory started to merge with the new biological mindset called systems biology. In order to produce a CHO-derived recombinant therapeutic......, analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...

  16. Benchmarking of commercially available CHO cell culture media for antibody production

    OpenAIRE

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-01-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but hi...

  17. Cell-free protein expression based on extracts from CHO cells.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the

  18. The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

    DEFF Research Database (Denmark)

    Zhang, Yang; Halim, Adnan; Narimatsu, Yoshiki;

    2014-01-01

    The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors...

  19. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.;

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  20. Strategies for adaptation of mAb-producing CHO cells to serum-free medium

    OpenAIRE

    Costa A; Rodrigues M.; Henriques Mariana; Oliveira Rosário; Azeredo Joana

    2011-01-01

    Large-scale production of biopharmaceuticals commonly requires the use of serum-free medium, for safety and cost reasons. However, serum is essential to most mammalian cells growth, and its removal implies a very time-consuming process for cell adaptation. Thus, the aim of the study was to evaluate different strategies for cell adaptation to serum-free medium. Three cell types were used to assess the impact of transfection on adaptation: one common CHO-K1 cell line and two CHO-K1 cells tr...

  1. CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

    OpenAIRE

    Pawłowska, Monika; Augustin, Ewa; Mazerska, Zofia

    2013-01-01

    Aim: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. Methods: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 act...

  2. Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

    Directory of Open Access Journals (Sweden)

    Herbert Rodrigues Goulart

    2010-01-01

    Full Text Available Sodium butyrate (NaBu has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM. Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2 and mouse pro-B lymphoma (Ba/F3-LLP cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714. Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

  3. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    Science.gov (United States)

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  4. [Metabolic characteristics and kinetic model of recombinant CHO cells in serum-free suspension batch culture].

    Science.gov (United States)

    Liu, Xingmao; Liu, Hong; Ye, Lingling; Li, Shichong; Wu, Benchuan; Wang, Haitao; Xie, Jing; Chen, Zhaolie

    2010-01-01

    By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established. PMID:20353097

  5. Mannose metabolism in recombinant CHO cells and its effect on IgG glycosylation.

    Science.gov (United States)

    Slade, Peter G; Caspary, R Guy; Nargund, Shilpa; Huang, Chung-Jr

    2016-07-01

    Understanding the causes of high-mannose (HM) glycosylation of recombinant IgG in CHO cells would facilitate the production of therapeutics. CHO cells grown with mannose as the major carbon source demonstrated a dramatic increase in total HM glycosylation in recombinant IgG, with no effect on cell growth, viability, or titer. Quantitative metabolomics and (13) C flux analysis were used to explore the mechanism for increased HM glycosylation and understand the metabolism of mannose in CHO cells. It was demonstrated that mannose was a good carbon source for CHO cell growth and IgG production, readily entering both glycolysis and the TCA Cycle. Previous mechanisms for increased HM glycosylation during antibody production have been attributed to changes in pH, osmolality, increased specific productivity, and nutrient limitation. The results from this study propose a novel mechanism where an increased carbon flux in the GDP-mannose synthetic pathway increased the intracellular concentration of mannose-containing metabolites. The abnormally high concentration of mannose and mannose-metabolites were shown to inhibit α-mannosidase activity and it was proposed that this inhibition in the ER and Golgi caused the production of IgG with increased high-mannose glycosylation. Biotechnol. Bioeng. 2016;113: 1468-1480. © 2016 Wiley Periodicals, Inc. PMID:26724786

  6. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup;

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  7. BEHAVIOR OF CHO CELLS ON MODIFIED POLYPROPYLENE BY LOW TEMPERATURE AMMONIA PLASMA

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong; YU Yaoting; PAN Jilun; XU Yuanping; ZHU Hesun

    2001-01-01

    The surface of polypropylene (PP) membrane was modified by low temperature plasma with ammonia. The effect of exposure time was investigated by means of contact angle measurement. The results show that low temperature ammonia plcsma treatment can enhance its hydrophilicity. Chinese hamster ovary (CHO) cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.

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

    Science.gov (United States)

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

    Science.gov (United States)

    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 1.3 or 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

  10. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

    OpenAIRE

    Carrillo-Cocom, L. M.; Genel-Rey, T.; Araíz-Hernández, D.; López-Pacheco, F.; López-Meza, J.; Rocha-Pizaña, M. R.; Ramírez-Medrano, A.; Alvarez, M. M.

    2014-01-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration prof...

  11. Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification

    OpenAIRE

    Orlova, N.; Kovnir, S.; Vorobiev, I.; Yuriev, A.; Gabibov, A.; Vorobiev, A.

    2012-01-01

    Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methot...

  12. Optimization of Seeding Density in Microencapsulated Recombinant CHO Cell Culture

    OpenAIRE

    Zhang, Ying; Zhou, Jing; Zhang, Xulang; Yu, Weiting; Guo, Xin; Wang, Wei; Ma, Xiaojun

    2008-01-01

    Microencapsulation technology is an alternative large-scale mammalian cell culture method. The semi-permeable membrane of the microcapsule allows free diffusion of nutrients, oxygen and toxic metabolites to support cell growth, and the microcapsule membrane can protect the cells from the mechanical damage of shear forces associated with agitation and aeration. Many polymers have been used to make microcapsules, such as chitosan, polyacrylates, alginate, polyamino acids, and polyamides. One of...

  13. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  14. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    International Nuclear Information System (INIS)

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV–Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408–410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 ± 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC50) for CHO cells is 68.0 ± 2.65 μg/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 μg/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity

  15. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Science.gov (United States)

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. PMID:26721629

  16. Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions

    Energy Technology Data Exchange (ETDEWEB)

    Czub, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Banas, D. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Blaszczyk, A. [Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun (Poland); Braziewicz, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Buraczewska, I. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Choinski, J. [Heavy Ion Laboratory, Warsaw University, ul. Pasteura 5A, 02-093 Warsaw (Poland); Gorak, U. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Jaskola, M.; Korman, A. [Andrzej Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland); Lankoff, A.; Lisowska, H. [Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Lukaszek, A. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Main School of Fire Service, ul. Slowackiego 52/54, 01-629 Warsaw (Poland); Szeflinski, Z. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland)], E-mail: szef@fuw.edu.pl; Wojcik, A. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland)

    2009-03-15

    Chinese hamster ovary CHO-K1 cells were exposed to high LET {sup 12}C-beam (LET: 830 keV/{mu}m) in the dose range of 0-6 Gy and to {sup 60}Co irradiation and the RBE value was obtained. Effects of {sup 12}C-beam exposure on cell survival and chromosomal aberrations were calculated. The chromosomal aberration data were fitted with linear equation. The distribution of aberration in cells was examined with a standard u-test and used to evaluate the data according to Poisson probabilities. The variance to the mean ratio {sigma}{sup 2}/Y and the dispersion index (u) were determined. Overdispersion was significant (p<0.05) when the value of u exceeded 1.96.

  17. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Directory of Open Access Journals (Sweden)

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  18. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    OpenAIRE

    Pilbrough, Warren; Munro, Trent P.; Gray, Peter

    2009-01-01

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are...

  19. Intracellular boron accumulation in CHO-K1 cells using amino acid transport control

    International Nuclear Information System (INIS)

    BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0 mM BPA, with 1.0 mM L-Tyr-O-Et and incubating for 60 min. This increase is caused by activation of a system L amino acid exchanger between BPA and L-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation. - Highlights: • We examined optimal L-p-boronophenylalanine (BPA) loading in CHO-K1 cells. • Optimal BPA loading parameters were 1.0 mM and incubation for 60 min. • Intracellular boron accumulation increased upon co-loading BPA with L-Tyr-O-Et. • Optimal L-Tyr-O-Et loading parameters were 1.0 mM and incubation for 60 min. • Co-loading BPA with L-Tyr-O-Et can increase intracellular boron accumulation

  20. Influence of catechins on bystander responses in CHO cells induced by alpha-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Law, Y.L.; Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Yu, K.N. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)], E-mail: peter.yu@cityu.edu.hk

    2010-04-15

    In this work, we studied alpha-particle induced and medium-mediated bystander effects in Chinese hamster ovary (CHO) cells through micronucleus (MN) assay. We showed that signal transduction from irradiated cells to bystander cells occur within a short time after irradiation. We then studied the effects of ROS (reactive oxygen species)-scavenging catechins in the medium before irradiation. We observed decreases in the percentage of bystander cells with MN formation and thus proved the protection effect of catechins on bystander cells from radiation.

  1. BEHAVIOR OF CHO CELLS ON MODIFIED POLYPROPYLENE BY LOW TEMPERATURE AMMONIA PLASMA

    Institute of Scientific and Technical Information of China (English)

    ZHANGHong; ZHUHesun; 等

    2001-01-01

    The surface of polypropylene(PP) membrane was modified by low temperature plasma with ammonia.The effect of exposure time was investigated by means of contact angle measurement.The results show that low temperature ammonia plasma treatment can enhance its hydrophilicity.Chinese hamster ovary(CHO)cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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 μM 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

  4. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

    DEFF Research Database (Denmark)

    Grav, Lise Marie; Lee, Jae Seong; Thomsen, Signe Gerling;

    2015-01-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a....... Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further....

  5. Enhancement of monoclonal antibody production in CHO cells by exposure to He–Ne laser radiation

    OpenAIRE

    Ghaleb, Rana; Naciri, Mariam; Al-Majmaie, Rasoul; Maki, Amel; Al-Rubeai, Mohamed

    2013-01-01

    This study tested the effectiveness of laser biostimulation in small-scale cultures in vitro. We investigated the response of recombinant CHO cells, which are used for the production of monoclonal antibody, to low level laser radiation. The cells were irradiated using a 632.8 nm He–Ne laser in a continuous wave mode at different energy doses. We incubated the irradiated cells in small batch cultures and assessed their proliferation and productivity at various time intervals. Compared to untre...

  6. Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

    Directory of Open Access Journals (Sweden)

    W Warchoł

    2004-07-01

    Full Text Available 5-aminolevulinic acid (ALA is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX, which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.

  7. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    International Nuclear Information System (INIS)

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S9-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis

  8. Genetically modified CHO cells for studying the genotoxicity of heterocyclic amines from cooked foods

    International Nuclear Information System (INIS)

    We have developed metabolically competent CHO cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with IQ but not with PhIP. This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines

  9. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

    OpenAIRE

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven CL; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC ...

  10. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

    2010-04-23

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  11. Recovery of CHO cells from hyperthermic potentiation to x rays: repair of DNA and chromatin

    International Nuclear Information System (INIS)

    Above the critical temperature, ca. 42.50C, hyperthermic potentiation of Chinese hamster ovary (CHO) cells to x irradiation was accompanied by increased binding of nonhistone proteins to DNA and by reduced rates of rejoining of DNA strand breaks. These biochemical changes were reversed as the cells recovered from the hyperthermic exposures at 370C. If the hyperthermically treated cells were incubated at 370C before x irradiation, the ratio of nonhistone protein to DNA returned to normal in 12 h but the depressed rate of rejoining of DNA strand breaks and increased cell radiosensitivity remained unaltered. Cell radiosensitivity began to decrease after 12 h and recovery from hyperthermia-potentiated radiosensitivity was complete by 48 h. In the same interval, the rate of rejoining of DNA strand breaks also returned to normal. From this behavior, we conclude that the reduction in the rate of rejoining of DNA strand breaks involved changes in DNA structure which were restored only after the thermal enhancement of protein binding was reversed. These experiments provide support for the viewpoint that critical hyperthermic potentiation (i.e., above 42.50C for CHO cells) may have logistical advantages over subcritical hyperthermic potentiation (i.e., below 42.50C) in clinical situations

  12. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    Science.gov (United States)

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  13. Benchmarking of commercially available CHO cell culture media for antibody production.

    Science.gov (United States)

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  14. Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time.

    Science.gov (United States)

    Feichtinger, Julia; Hernández, Inmaculada; Fischer, Christoph; Hanscho, Michael; Auer, Norbert; Hackl, Matthias; Jadhav, Vaibhav; Baumann, Martina; Krempl, Peter M; Schmidl, Christian; Farlik, Matthias; Schuster, Michael; Merkel, Angelika; Sommer, Andreas; Heath, Simon; Rico, Daniel; Bock, Christoph; Thallinger, Gerhard G; Borth, Nicole

    2016-10-01

    The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA-methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA-methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241-2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:27072894

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  17. Measurement of DNA strand break repair and survival rate after X-irradiation of synchronized CHO cells

    International Nuclear Information System (INIS)

    The author investigated the induction and repair of DNA strand breaks and the survival rate of CHO cells after X-radiation at different stages of the cell cycle. His particular concern was the interdependence between cell inactivation and double strand break repair. (orig./AJ)

  18. Induction of sister-chromatid exchanges and chromosome aberrations by γ-irradiated nucleic acid constituents in CHO cells

    International Nuclear Information System (INIS)

    Chinese hamster ovary (CHO) cells were exposed to different concentrations of hydrogen peroxide and to γ-irradiated, oxygenated solutions of thymine, thymidine and 2-deoxy-D-ribose. By using a modified Brd Zrd-labelling method sister-chromated exchanges (SCEs) and chromosomal aberrations were scored in the same cell population, one cycle after treatment. Irradiated, oxygenated solutions of 2-deoxy-D-ribose clearly induced SCEs and chromosomal aberrations. In comparison, hydrogen peroxide and irradiated solutions of thymine and thymidine were less effective in CHO cells. (orig.)

  19. Fibrinogen interaction of CHO cells expressing chimeric αIIb/αvβ3 integrin

    Institute of Scientific and Technical Information of China (English)

    Juan-juan CHEN; Xiao-yu SU; Xiao-dong XI; Li-ping LIN; Jian DING; He LU

    2008-01-01

    Aim: The molecular mechanisms of the affinity regulation of αvβ3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of αvβ3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-func-tion within these domains remains unclear. Methods: The extracellular and trans-membrane domain of αⅡb was fused to the αv integrin cytoplasmic domain, and the chimeric α subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type β3 subunit or with 3 mutant 133 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. Results: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric α subunit with the wild-type β3 subunit, but not those with truncated β3, could adhere to and spread on immobilized fibrinogen. Conclusion: The substitution αⅡb at the cytoplasmic domain with the ctv cyto-plasmic sequence rendered the extracellular αⅡbβ3 a constitutively activated con-formation for ligands without the need of "inside-out" signals. Our results also indicated that the COOH-terminal sequence of β3 might play a key role in integrin αⅡb/αvβ3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing αⅡb/αvβ3 have enormous potential for facilitating drug screen-ing for antagonists either to αvβ3 intracellular interactions or to αⅡbβ3 receptor functions.

  20. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc. PMID:26914152

  1. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian;

    2015-01-01

    Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream proces...

  2. Developement of serum-free media in CHO-DG44 cells using a central composite statistical design

    OpenAIRE

    Parampalli, Ananth; Eskridge, Kent; Smith, Leonard; Meagher, Michael M.; Mowry, Mark C.; Subramanian, Anuradha

    2007-01-01

    A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S2...

  3. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

    Directory of Open Access Journals (Sweden)

    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  4. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram;

    2014-01-01

    of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was...... achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genomeediting methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in...

  5. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

    Science.gov (United States)

    McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

    2016-09-01

    The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. PMID:26888596

  6. Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells.

    Science.gov (United States)

    Sun, Qiu-Li; Zhao, Chun-Peng; Chen, Shao-Nan; Wang, Li; Wang, Tian-Yun

    2016-05-15

    Chinese hamster ovary (CHO) cells offer many advantages for recombinant gene expression, including proper folding and post-translational modification of the recombinant protein. However, due to positional effects resulting from the neighboring chromatin, transgenes are often expressed at low levels in these cells. While previous studies demonstrated that matrix attachment regions (MARs) can be utilized to increase transgene expression by buffering transgene silencing, the mechanism by which this occurs is poorly understood. We therefore performed a deletion analysis of the human β-globin MAR sequence to characterize the regions that are necessary to enhance transgene expression in CHO cells. Our results indicate that of the six β-globin MAR fragments tested (MAR-1-6; nucleotides 1-540, 420-1020, 900-1500, 1380-1980, 1860-2460, and 2340-2999, respectively), MAR-2, followed by MAR-3, was the most effective region for promoting stable and elevated transgene expression. Meanwhile, bioinformatic analyses demonstrated that these fragments encode a MAR-like motif and several transcription factor binding sites, including special AT-rich binding protein 1 (SATB1), CCAAT-enhancer-binding proteins (C/EBP), CCCTC-binding factor (CTCF), and Glutathione (GSH) binding motifs, indicating that these elements may contribute to the MAR-mediated enhancement of transgene expression. In addition, we found that truncated MAR derivatives yield more stable transgene expression levels than transgenes lacking the MAR. We concluded that the MAR-mediated transcriptional activation of transgenes requires a specific AT-rich sequence, as well as specific transcription factor-binding motifs. PMID:26869318

  7. Reversal of acquired resistance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with alpha 1 acid glycoprotein.

    OpenAIRE

    Chatterjee, M.; Harris, A. L.

    1990-01-01

    Tamoxifen and 4-OH tamoxifen were used to reverse multidrug resistance (MDR) in CHO cells with acquired resistance to adriamycin (CHO-Adrr). Because alpha 1 acid glycoprotein (AAG) can bind a range of calcium channel blockers that also reverse MDR and rises in malignancy, its interactions with tamoxifen and 4-OH tamoxifen were also studied. Tamoxifen decreased the IC50 of 10 microM adriamycin 4.8-fold in the parent CHO-K1 cell line and 16-fold in CHO-Adrr. Similarly 4-OH tamoxifen decreased t...

  8. Performance evaluation of CHO-K1 cell in culture medium supplemented with hemolymph

    Directory of Open Access Journals (Sweden)

    Tássia Raffoul

    2005-06-01

    Full Text Available The aim of this work was to evaluate the potential of hemolymph utilization as a culture medium supplement to cultivate the animal cell CHO-K1. For this purpose 1% v/v of hemolymph was added to DMEM medium containing 10% v/v of FBS and 1 or 4.5 g/L of glucose. The culture was grown in spinner flasks incubated in a 10% v/v CO2 environment, at 37ºC, with the Cytodex 1 microcarrier. Comparing the results obtained from the culture with hemolymph against those without hemolymph, a positive influence of the hemolymph was observed, as the experiment with hemolymph presented a 52% higher cell concentration and a higher productivity of up to 40%.Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB é uma das grandes prioridades de pesquisa em desenvolvimento de processos com célula animal. O objetivo do presente trabalho foi realizar uma análise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da célula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37ºC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influência positiva da hemolinfa no cultivo, já que o ensaio com hemolinfa apresentou uma concentração máxima de células 52% maior e uma produtividade máxima de até 40% maior.

  9. Radiobiologic response of CHO-KI cells treated with vitamin A

    International Nuclear Information System (INIS)

    Treatment of CHO-KI cells with vitamin A altered their response to subsequent gamma irradiation. In general longterm preincubation with low doses of the vitamin caused a relative increase in the number of cells surviving a given radiation dose. The effect resulted in an increase in the D0 of the survival curve. Long or short term exposure to high concentrations of the vitamin caused a decrease in the number of surviving cells leading to a decrease in the extrapolation number of the survival curve. Recovery of cells from radiation damage, assessed using the split dose technique, was also impaired by vitamin A pretreatment. A mechanism involving repair of potentially lethal damage may explain the protective effect of vitamin A since this was highly dependent on the cell density of cultures at the time of irradiation. However, in view of the data showing that the vitamin A concentrations necessary to alter the radiation survival curve shoulder caused a significant release of sialic acid into the medium, a mechanism involving membrane stability may account for both the reduction in repair/recovery capacity of the treated cells and the radioprotective effect. (orig.)

  10. Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Fan, Yuzhou; Wagtberg Sen, Jette;

    2016-01-01

    Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterogonous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this...

  11. Induction of proline prototrophs in CHO-K1 cells by heavy ions

    International Nuclear Information System (INIS)

    Using an established mammalian cell line, Chinese hamster ovary cells (CHO-K1), we have observed the induction of prototrophs by various heavy ions. This cell line requires proline for normal growth in medium with low serum concentration. X-rays, three types of heavy particles (600 MeV/u iron, 670 MeV/u neon, and 320 MeV/u silicon ions), ethylmethane sulphonate and 5-azacytidine were used to induce revertants which were proline independent. Log-phase cells treated with 5-azacytidine showed a very high reversion frequency. The induction frequency per viable cell appears to be dose dependent for these four types of radiation, and the dose-response curves are approximately linear. Our results also indicate that the effectiveness of high-LET particles in inducing proline prototrophs is much greater than that of low-LET radiation. The RBE value for the induction of prototrophs was calculated for neon, silicon, and iron particles and found to be about 1.3, 1.7 and 4.5, respectively. At equal survival level, the reversion frequency for X-rays and EMS was about the same. (author)

  12. A kinetic-metabolic model based on cell energetic state: study of CHO cell behavior under Na-butyrate stimulation.

    Science.gov (United States)

    Ghorbaniaghdam, Atefeh; Henry, Olivier; Jolicoeur, Mario

    2013-04-01

    A kinetic-metabolic model approach describing and simulating Chinese hamster ovary (CHO) cell behavior is presented. The model includes glycolysis, pentose phosphate pathway, TCA cycle, respiratory chain, redox state and energetic metabolism. Growth kinetic is defined as a function of the major precursors for the synthesis of cell building blocks. Michaelis-Menten type kinetic is used for metabolic intermediates as well as for regulatory functions from energy shuttles (ATP/ADP) and cofactors (NAD/H and NADP/H). Model structure and parameters were first calibrated using results from bioreactor cultures of CHO cells expressing recombinant t-PA. It is shown that the model can simulate experimental data for all available experimental data, such as extracellular glucose, glutamine, lactate and ammonium concentration time profiles, as well as cell energetic state. A sensitivity analysis allowed identifying the most sensitive parameters. The model was then shown to be readily adaptable for studying the effect of sodium butyrate on CHO cells metabolism, where it was applied to the cases with sodium butyrate addition either at mid-exponential growth phase (48 h) or at the early plateau phase (74 h). In both cases, a global optimization routine was used for the simultaneous estimation of the most sensitive parameters, while the insensitive parameters were considered as constants. Finally, confidence intervals for the estimated parameters were calculated. Results presented here further substantiate our previous findings that butyrate treatment at mid-exponential phase may cause a shift in cellular metabolism toward a sustained and increased efficiency of glucose utilization channeled through the TCA cycle. PMID:22976819

  13. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells

    OpenAIRE

    M. Rezaei; Zarkesh-Esfahani, S. H.; Gharagozloo, M.

    2013-01-01

    Cell lines derived from mammalian are dominant systems for the production of recombinant therapeutic proteins because of their capacity for correct protein folding, assembly and post-translational modification. In the search of an efficient method for the production of a recombinant protein using animal cell culture, we investigated the effects of different treatment including fetal calf serum concentration, glycerol and culture temperature on a Chinese hamster ovary (CHO) cell line on the pr...

  14. Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells.

    Science.gov (United States)

    Delafosse, Laurence; Xu, Ping; Durocher, Yves

    2016-06-10

    Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression. PMID:27085888

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

    Science.gov (United States)

    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

  16. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    International Nuclear Information System (INIS)

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, 60Co x-ray-, and 212Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of γ rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the γ-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The α-radiation, however, tended to produce larger intragenic deletions that γ radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to α radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect α-induced mutation spectra

  17. A lack of Adriamycin (ADR) resistance in Chinese hamster ovary (CHO) cells overexpressing P-glycoprotein (Pgp) following in vitro exposure to fractionated X-irradiation

    International Nuclear Information System (INIS)

    Using x-ray pretreated CHO cells, the authors demonstrated differing accumulation of adriamycin and vincristine in cells overexpressing P-glycoprotein. Response was also varied by the addition of calcium channel antagonist verapamil. (author)

  18. Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification.

    Science.gov (United States)

    Orlova, N A; Kovnir, S V; Vorobiev, I I; Yuriev, A S; Gabibov, A G; Vorobiev, A I

    2012-01-01

    Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma-derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies. PMID:22708069

  19. Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoyan; WANG Hao; YANG Yang; TAN Min; XUE Jingya; NI Haidong; GUO Yajun

    2006-01-01

    Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

  20. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  1. Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines.

    Science.gov (United States)

    Balasubramanian, Sowmya; Rajendra, Yashas; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-06-01

    Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc. PMID:26616356

  2. Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells.

    Science.gov (United States)

    Meuwly, F; von Stockar, U; Kadouri, A

    2004-09-01

    In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel((R)) disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5x10(7) cell ml(-1) of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a -25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operations. PMID:19003257

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

    Science.gov (United States)

    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

  4. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

    OpenAIRE

    Shimada, T.; Inokuchi, K; Nienhuis, A W

    1987-01-01

    The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites ...

  5. Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

    OpenAIRE

    Kawabe, Yoshinori; Makitsubo, Hirokatsu; Kameyama, Yujiro; Huang, Shuohao; Ito, Akira; Kamihira, Masamichi

    2011-01-01

    We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor ...

  6. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    International Nuclear Information System (INIS)

    Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity. Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR

  7. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  8. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

    Science.gov (United States)

    Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas D.

    2015-01-01

    Essentiality (ES) and Synthetic Lethality (SL) information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells) using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR) leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion) maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models. PMID:26426067

  9. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

    Directory of Open Access Journals (Sweden)

    Ratul Chowdhury

    2015-09-01

    Full Text Available Essentiality (ES and Synthetic Lethality (SL information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models.

  10. Effect of Caspase Inhibitor Ac-DEVD-CHO on Apoptosis of Vascular Smooth Muscle Cells Induced by Artesunate

    Directory of Open Access Journals (Sweden)

    Jingwen Zhang

    2014-05-01

    Full Text Available Numerous studies have shown that the proliferation and apoptosis of vascular smooth muscle cells play a key role in restenosis. Artesunate is a triterpenoid with a peroxide structure and its antimalarial, antitumor, and antiangiogenetic activities can inhibit the proliferation and apoptosis of multifarious cells. Apoptosis is caused by the activation of a series of intracellular proteolytic enzymes, among which caspase-dependent apoptosis was the earliest to be recognized. The purpose of this article is to study the effects of caspase-3 inhibitor Ac-DEVD-CHO on proliferation and apoptosis of vascular smooth muscle cells induced by Artesunate and to explore the mechanism of Artesunate-induced apoptosis of vascular smooth muscle cells. By using the method based on methyl thiazolyl tetrazolium to observe the effects of Artesunate on the growth and proliferation of vascular smooth muscle cells; observing the change in cell shape before and after Artesunate administration by transmission electron microscopy; detecting the changes in cell cycle and apoptosis rates before and after drug administration by flow cytometry; detecting the activity of caspase-3 in the caspase apoptosis pathway by the Western Blot method, we found that Artesunate inhibits the growth and proliferation of vascular smooth muscle cells in a dose- and time-dependent manner within the concentration range of 7.5–120 μg/mL, and the inhibition rate of Artesunate can be as high as 89.49 % at a concentration of 120 μg/mL after acting for 72 hours; vascular smooth muscle cells show a typical apoptosis peak due to the effects of higher concentration of Artesunate. Compared with the control group, the higher-concentration group shows major variability, Ac-DEVD-CHO, however, can significantly decrease this induction; it has been detected by Western Blot that Artesunate can induce caspase-3 activity dramatically in vascular smooth muscle cells, but this activation may be remarkably

  11. Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation.

    Science.gov (United States)

    Yang, M; Butler, M

    2000-05-20

    The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans

  12. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

    International Nuclear Information System (INIS)

    Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer. A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment. Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected. We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation

  13. The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells

    International Nuclear Information System (INIS)

    Insulin is the only hormone required for continued growth of Chinese hamster ovary CHO-K1 cells in the defined medium M-F12. When CHO-K1 cells are incubated in M-F12 without insulin for 48-72 hr, the cells accumulate in G1. In response to physiological concentrations of insulin an 18-fold increase in rate of DNA synthesis occurs due to cells entering S phase after an 8- to 10-hr lag; cell division begins after 24 hr. The inhibitory effect of actinomycin D and 5,6-dichlorobenzimidazole riboside indicates that RNA synthesis is required for progression to S phase. CHO-K1 cells possess insulin receptors, and the insulin effect results from insulin binding to its own receptor: (i) binding occurs at physiological insulin concentrations with a half-maximal stimulation at ∼ 14 ng/ml. (ii) At insulin concentrations used, insulin-like growth factor I and II (IGF-I and IGF-II) have little or no effect. (iii) Scatchard analysis of 125I-labeled insulin binding shows the curvilinear response typical of insulin. (iv) The Kd for the so-called high-affinity binding site and the Ke are characteristic of the insulin receptor. (v) At the minimal insulin concentrations that stimulate growth, IGF-I and IGF-II compete poorly with insulin for insulin binding, insulin competes poorly with IGF-I for IGF-I binding, and affinity labeling with 125I-labeled insulin identifies as polypeptide typical of the α subunit of the insulin receptor

  14. Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

    International Nuclear Information System (INIS)

    Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively. (Auth.)

  15. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    Energy Technology Data Exchange (ETDEWEB)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: kokazaki@ipen.br; Tsutsumi, Shiguetoshi [Amazon Food Ltd., Tokyo (Japan)], e-mail: fwip5138@mb.infoweb.ne.jp

    2009-07-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by {sup 60}Co {gamma}-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 {mu}g/ml), 1 h before irradiation, with 1 Gy of {gamma} radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  16. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    International Nuclear Information System (INIS)

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by 60Co γ-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 μg/ml), 1 h before irradiation, with 1 Gy of γ radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  17. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  18. Modification of radiation response of CHO cells by methyl-isobutyl xanthine. II. Increase in extrapolation number

    International Nuclear Information System (INIS)

    CHO cells exposed for several hours prior to irradiation to 0.2 mM 1-methyl-isobutyl xanthine (MIX) show a more that twofold increase in postirradiation survival over a wide dose range. Increase in radioresistance was shown to be independent of cell-cycle perturbation produced by MIX but was concomitant with MIX-induced morphological conversion of cells from an epithelial form to an elongated, fibroblast-like form. MIX added early in G1 protected cells irradiated subsequently in G1 and to a lesser extent in S. No enhancement of postirradiation survival was seen, however, if addition of MIX was delayed until after the commencement of S. Although MIX-treated cells appeared to accumulate more sublethal damage than did controls, the capacity to repair this damage during the interval between split doses was no greater in MIX-treated cells than in controls

  19. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    International Nuclear Information System (INIS)

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/106 clonable cells. As with HGPRT- mutants, XGPRT- mutants can be selected in 6-thioguanine. AS52 (XGPRT+) and wild type CHO (HGPRT+) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT- to HGPRT- mutants induced per unit dose (J/m2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT- AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

  20. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    International Nuclear Information System (INIS)

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed 2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV

  1. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing α2,6 sialyltransferase

    International Nuclear Information System (INIS)

    A CHO cell line, previously genetically modified by the introduction of rat α2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of α2,3- and 39% of α2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 μM methotrexate, presented a secretion level of ∼2 μg hTSH/106 cells/day, useful for product purification and characterization. The relative molecular masses (Mr) of the heterodimer and of the α- and β-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  2. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells.

    Science.gov (United States)

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  3. Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells.

    Science.gov (United States)

    Galbraith, Douglas J; Tait, Andrew S; Racher, Andrew J; Birch, John R; James, David C

    2006-01-01

    In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39

  4. Investigation of the mutagenic specificity of x-ray using a retroviral shuttle vector in CHO cells

    International Nuclear Information System (INIS)

    In some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. Moreover, while the point mutations are often randomly distributed, some systems exhibit hot spots. Retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. We have studied the mutational specificity of x-rays in a Chinese hamster ovary cell line (CHO) containing a stably integrated retroviral shuttle vector, carrying the CHO aprt cDNA as the genetic target. Cells were irradiated with 7 Gy using a 180 kVp x-ray source. The predominant mutation (87% of all APRT mutants), as determined by Southern analysis, was the complete deletion of the shuttle vector construct. In addition, 23 APRT mutants, carrying an apparently intact shuttle vector, were characterized at the sequence level: 5 were transitions, 9 were transversions, 3 were small deletions or insertions, 4 were frameshifts, and 2 were small rearrangements. Although the type and the location of the point mutations characterized appeared largely random, small deletions, insertions, and frameshifts were frequently associated with direct sequence repeats. 23 refs., 4 figs., 2 tabs

  5. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  6. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Science.gov (United States)

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  7. Productivity Studies Utilizing Recombinant CHO Cells In Stirred-Tank Bioreactors: A Comparative Study Between The Pitch-Blade And The Packed-Bed Bioreactor Systems

    OpenAIRE

    Hatton, Taylor Stephen

    2012-01-01

    A recombinat Chinese Hamster Ovary (rCHO) cell line designated as CHO SEAP was utilized in this investigation to optimize protein production. Two bench top stirred-tank bioreactors, namely a pitched-blade and a packed-bed basket bioreactor, were utilized for a comparative study to determine which bioreactor would produce the best results in terms of protein production. The objective of this research project was to provide basic data that shows cells cultured in a packed-bed basket bioreactor ...

  8. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

    OpenAIRE

    Przemysław Sitarek; Ewa Skała; Halina Wysokińska; Marzena Wielanek; Janusz Szemraj; Monika Toma; Tomasz Śliwiński

    2015-01-01

    Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased ex...

  9. Protective activity of a novel resveratrol analogue, HS-1793, against DNA damage in 137Cs-irradiated CHO-K1 cells

    International Nuclear Information System (INIS)

    Resveratrol has received considerable attention as a polyphenol with anti-oxidant, anti-carcinogenic, and anti-inflammatory effects. Radiation is an important component of therapy for a wide range of malignant conditions. However, it causes damage to normal cells and, hence, can result in adverse side effects. This study was conducted to examine whether HS-1793, a novel resveratrol analogue free from the restriction of metabolic instability and the high dose requirement of resveratrol, induces a protective effect against radiation-induced DNA damage. HS-1793 effectively scavenged free radicals and inhibited radiation-induced plasmid DNA strand breaks in an in vitro assay. HS-1793 significantly decreased reactive oxygen species and cellular DNA damage in 2 Gy-irradiated Chinese hamster ovary (CHO)-K1 cells. In addition, HS-1793 dose-dependently reduced the levels of phosphorylated H2AX in irradiated CHO-K1 cells. These results indicate that HS-1793 has chemical radioprotective activity. Glutathione levels and superoxide dismutase activity in irradiated CHO-K1 cells increased significantly following HS-1793 treatment. The enhanced biological anti-oxidant activity and chemical radioprotective activity of HS-1793 maintained survival of irradiated CHO-K1 cells in a clonogenic assay. Therefore, HS-1793 may be of value as a radioprotector to protect healthy tissue surrounding tumor cells during radiotherapy to obtain better tumor control with a higher dose. (author)

  10. Analysis of native cellular DNA after heavy ion irradiation: DNA double-strand breaks in CHO-K1 cells

    International Nuclear Information System (INIS)

    A fast assay for the detection of DNA double-strand breaks was developed involving constant field gel electrophoresis (Taucher-Scholz et al., 1994) and densitometric scanning of agarose gels stained with ethidium bromide. With this technique, DSB induction was investigated after irradiation of CHO cells with carbon ions with LET values between 14 keV/μm and 400 keV/μm. In parallel, a computer code was developed to simulate both the principle of the electrophoretic detection of DNA double-strand breaks and the action of radiations of different ionization density. The results of the experiments and the calculations are presented here and compared with each other. (orig./HSI)

  11. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing {alpha}2,6 sialyltransferase; Expressao estavel tireotrofina humana (r-hTSH) em celulas de mamifero (CHO) que expressam {alpha}2,6 sialiltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Damiani, Renata

    2009-07-01

    A CHO cell line, previously genetically modified by the introduction of rat {alpha}2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of {alpha}2,3- and 39% of {alpha}2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 {mu}M methotrexate, presented a secretion level of {approx}2 {mu}g hTSH/10{sup 6} cells/day, useful for product purification and characterization. The relative molecular masses (M{sub r}) of the heterodimer and of the {alpha}- and {beta}-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T{sub 4} release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  12. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  13. Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

    Institute of Scientific and Technical Information of China (English)

    Deng Wen LI; Qin YANG; Jia Tong CHEN; Hao ZHOU; Ru Ming LIU; Xi Tai HUANG

    2005-01-01

    The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determine its function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzed by indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylation begins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phosphoH3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms "sandwich-like structure" when the chromosomes condensed. With the cleavage progressing, the "ladders" of the histone contract into a bigger bright dot. Then the histone aggregates and some of compacted microtubules in the midbody region are composed into a "bar-like"complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the "bar",inside which locates microtubules and modified histones, to finish the cytokinesis and keep the "bar complex" out of the cells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may take part in the formation of midbody and play a crucial role in cytokinesis.

  14. Effects of BrdUrd incorporation on radiation sensitivity recovery from PLD and repair of DNA damage in CHO cells

    International Nuclear Information System (INIS)

    CHO cells grown in the presence of various concentrations (0-10 μM) of BrdUrd were exposed to X-rays either in the logarithmic or in the plateau-phase of growth and survival was measured either immediately after irradiation or 6 hr later. An increase in radiosensitivity (immediate plating) was observed in cells growing in the presence of BrdUrd that was similar for exponentially growing and plateau-phase cells when related to the amount of incorporated BrdUrd. The rate of induction of DNA damage as assayed by hydroxylapatite chromatography was similar for cells grown in the presence or absence of BrdUrd. This resulted in an increase in the amount of unrejoined breaks measured in BrdUrd-containing cells 1 hr after irradiation and which was more pronounced at higher radiation doses. These results are compared to similar results obtained with untransformed C/sub 3/H 10T1/2 cells, and implications on the mechanism of radiosensitization by BrdUrd are discussed

  15. Effects of γ (60Co) and β (90Sr) radiations in Chinese hamster ovarian cells (CHO-K1): induction of micronuclei and cell death

    International Nuclear Information System (INIS)

    Among various types of ionising radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequent increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of 90Sr, a pure, highly energetic beta source, on CHO-K1 cells and to compare them with data obtained with 60Co. CHO-K1 cells irradiated with different doses (1.0, 2.5, 5.0, 7.5 Gy) of 60Co (0.34 Gy.min-1) and 90Sr (0.23 Gy.min-1) were processed for analysis of clonogenic death, induction of micronuclei (MN) and necrotic and apoptotic death. The survival curves obtained for both types of radiation were better fitted by the linear-quadratic model and were similar. However, the cytogenetic results showed that both the proportion of micronucleated cells and the magnitude of radioinduced lesions demonstrated by the analysis of MN distribution were significantly higher in cells irradiated with 60Co than in cells irradiated with 90Sr, whereas 90Sr was more damaging than 60Co in terms of cell death induction. Necrosis was the major type of death observed in CHO-K1 cells. The data obtained suggest that the low incidence of micronucleated cells after exposure to 90Sr may be a consequence of selective elimination of severely damaged cells from the population by the necrotic process at a higher rate than observed with 60Co exposure. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics. (author)

  16. A rapid-mixing comparison of the mechanisms of radiosensitization by oxygen and misonidazole in CHO cells

    International Nuclear Information System (INIS)

    The mechanisms of hypoxic cell radiosensitization by oxygen and by the 2-nitroimidazole chemical sensitizer, misonidazole, were studied in CHO cells using rapid-mixing techniques. Both agents display a dose response of sensitivity in steady-state experiments which is satisfactorily fitted by a K function with maximum enhancement ratios (ER) of about 2.8. Rapid-mixing experiments demonstrated that oxygen sensitization in these cells is complete within about 10 msec even at concentrations as low as 2.6% oxygen, with no evidence for two time-resolved components as had been reported with V79 cells. Full sensitivity persists for at least 165 msec following an 8.4-fold dilution into hypoxic medium. No large temperature dependence was observed in the range 4 to 370C. Partial development of sensitization by misonidazole also occurs very quickly, within 25 msec with a 10mM drug concentration, but only to an ER of about 1.7, much below the level observed after many minutes incubation (approx.2.5) even at ice temperature. This development, too, has a very weak temperature dependence. It is suggested that although uptake of the drug is rapid, probably occurring by passive diffusion, an additional mechanism not involving gross metabolism and operating on the time scale of seconds to minutes is required to produce maximal sensitization in these cells

  17. A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells.

    Science.gov (United States)

    Zalai, Dénes; Hevér, Helga; Lovász, Krisztina; Molnár, Dóra; Wechselberger, Patrick; Hofer, Alexandra; Párta, László; Putics, Ákos; Herwig, Christoph

    2016-08-01

    The integration of physiological knowledge into process control strategies is a cornerstone for the improvement of biopharmaceutical cell culture technologies. The present contribution investigates the applicability of specific productivity as a physiological control parameter in a cell culture process producing a monoclonal antibody (mAb) in CHO cells. In order to characterize cell physiology, the on-line oxygen uptake rate (OUR) was monitored and the time-resolved specific productivity was calculated as physiological parameters. This characterization enabled to identify the tight link between the deprivation of tyrosine and the decrease in cell respiration and in specific productivity. Subsequently, this link was used to control specific productivity by applying different feeding profiles. The maintenance of specific productivity at various levels enabled to identify a correlation between the rate of product formation and the relative abundance of high-mannose glycoforms. An increase in high mannose content was assumed to be the result of high specific productivity. Furthermore, the high mannose content as a function of cultivation pH and specific productivity was investigated in a design of experiment approach. This study demonstrated how physiological parameters could be used to understand interactions between process parameters, physiological parameters, and product quality attributes. PMID:26910040

  18. SNARE-mediated trafficking of α5β1 integrin is required for spreading in CHO cells

    International Nuclear Information System (INIS)

    In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of α5β1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading

  19. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    International Nuclear Information System (INIS)

    Highlights: ► Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. ► These are due to increase in intracellular cAMP concentration. ► Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. ► The potencies of fatty acid moiety on LysoPS were oleoyl ⩾ stearoyl > palmitoyl. ► We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell–cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of Gαs protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with Gαs, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  20. Effects of hyperthermia and x irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    The BrdUrd labeling method was used to evaluate the effects of hyperthermia, x irradiation, and the combined treatment on the incidence of sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. Cells cultured in McCoy's 5A media containing 10 μM 5-bromodeoxyuridine were synchronized after one cell cycle by mitotic shake-off. Early-G1 cells were heated by submerging culture flasks in a 44 +- 0.050C water bath for periods of 20, 40, and 60 min. By the same method, other cultures were x irradiated at doses of 100, 200, 400, and 600 rad. A third protocol involved combined treatment of 20 min at 440C followed immediately by one of the above radiation doses. A fourth protocol reversed the sequence of the combined treatment applying x irradiation (200 or 400 rad) followed immediately by hyperthermia. The data showed that hyperthermia and x irradiation both elevated the frequency of SCEs significantly whether applied separately or together. The combined treatment (heat: 20 min at 440C plus varying x-radiation doses) produced results suggestive of a synergistic interaction. The sequence of the heat and x irradiation did not appear to have a significant effect on the production of SCE

  1. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    Science.gov (United States)

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering. PMID:27002234

  2. [Stable and efficient expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells].

    Science.gov (United States)

    Yang, Zhenxi; Li, Shichong; Liu, Hong; Zhang, Miao; Ye, Lingling; Wu, Yanzhuo; Xu, Mingbo; Chen, Zhaolie

    2013-12-01

    Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL. PMID:24660628

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

    Science.gov (United States)

    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.

  4. Correlation between hyperthermia-induced membrane blebbing and survival in G/sub 1/ CHO cells

    International Nuclear Information System (INIS)

    G/sub 1/ cells, obtained by mitotic selection, were incubated and heated in suspension culture at 45.50C for 3 - 20 min. Varying degrees of membrane blebbing were induced, ranging from nonblebbed cells indistinguishable from control cells to those with blebs larger than the cell itself. Both the proportion of cells exhibiting blebbing and the mean diameter of the blebs increased with duration of heating. A cell scoring system based on the ratio of the diameter of the largest bleb relative to that of the cell was established. Scoring was done within 30 min after heating, after which time blebs either were released from the cells or were reabsorbed. The percent of heated cells having blebs smaller than 50% of the cell diameter equalled the percent of cells survivivg. This relationship was confirmed by scoring single cells both for blebbing and for their ability to form colonies. Electron microscopy demonstrated that the subcellular organelles, except for ribosomes and microtubules, were absent from the blebs, and found within a juxtanuclear region in the blebbed cells. Freeze fracture replicas revealed no changes in membrane ultrastructure, either on the blebs or cell body. The only exception was a small number of blebs which contained bald patches devoid of membrane particles

  5. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  6. Lucifer Yellow uptake by CHO cells exposed to magnetic and electric pulses

    OpenAIRE

    Miklavčič, Damijan; Towhidi, Leila; Firoozabadi, S. M. P.; Mozdarani, Hossein

    2012-01-01

    Background The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Due to numerous applications in medicine, biology and biotechnology, the introduction of impermeant molecules into biological cells has drawn considerable attention in the past years. One of the most famous methods in this field is electroporation, in which electric pulses with high intensity and short duration are applied to the cells. The aim of our study was to investigate...

  7. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  8. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  9. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    Science.gov (United States)

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

  10. Expression of P-glycoprotein-mediated drug resistance in CHO cells surviving a single X-ray dose of 30 Gy

    International Nuclear Information System (INIS)

    The authors reported previously that Chinese hamster ovary (CHO) cells surviving exposure to repeated doses of 9 Gy of X-irradiation in vitro expressed a multiple drug resistance phenotype characterized by cross-resistance to epipodophyllotoxins and to Vinca alkaloids, and by P-glycoprotein (Pgp) overexpression. They now show that exposure of these CHO cells to a single 30-Gy X-ray dose similarly resulted in the survivors expressing resistance to vincristine and to etoposide and overexpressing Pgp. In agreement with data obtained on cells which received repeated X-ray exposures, this Pgp overexpression occurred in the absence of any significant elevation of Pgp mRNA. However, the reduced ability to accumulate rhodamine 123 identified in these sublines, and the ability of verapamil to reverse this accumulation defect, implies that the Pgp which was overexpressed was functional. (author)

  11. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Directory of Open Access Journals (Sweden)

    Jaime Mas-Oliva

    Full Text Available Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2 associated to a lethal influx of Ca(2+ in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  12. Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells

    DEFF Research Database (Denmark)

    Jiang, Xiumei; Miclăuş, Teodora; Wang, Liming;

    2015-01-01

    Toxicity of silver nanoparticles (Ag NPs) has been reported both in vitro and in vivo. However, the intracellular stability and chemical state of Ag NPs are still not very well studied. In this work, we systematically investigated the cellular uptake pathways, intracellular dissolution and chemical...... species, and cytotoxicity of Ag NPs (15.9 ± 7.6 nm) in Chinese hamster ovary cell subclone K1 cells, a cell line recommended by the OECD for genotoxicity studies. Quantification of intracellular nanoparticle uptake and ion release was performed through inductively coupled plasma mass spectrometry. X......-ray absorption near-edge structure (XANES) was employed to assess the chemical state of intracellular silver. The toxic potential of Ag NPs and Ag+ was evaluated by cell viability, reactive oxygen species (ROS) production and live–dead cell staining. The results suggest that cellular uptake of Ag NPs involves...

  13. Cross-scale predictive modeling of CHO cell culture growth and metabolites using Raman spectroscopy and multivariate analysis.

    Science.gov (United States)

    Berry, Brandon; Moretto, Justin; Matthews, Thomas; Smelko, John; Wiltberger, Kelly

    2015-01-01

    Multi-component, multi-scale Raman spectroscopy modeling results from a monoclonal antibody producing CHO cell culture process including data from two development scales (3 L, 200 L) and a clinical manufacturing scale environment (2,000 L) are presented. Multivariate analysis principles are a critical component to partial least squares (PLS) modeling but can quickly turn into an overly iterative process, thus a simplified protocol is proposed for addressing necessary steps including spectral preprocessing, spectral region selection, and outlier removal to create models exclusively from cell culture process data without the inclusion of spectral data from chemically defined nutrient solutions or targeted component spiking studies. An array of single-scale and combination-scale modeling iterations were generated to evaluate technology capabilities and model scalability. Analysis of prediction errors across models suggests that glucose, lactate, and osmolality are well modeled. Model strength was confirmed via predictive validation and by examining performance similarity across single-scale and combination-scale models. Additionally, accurate predictive models were attained in most cases for viable cell density and total cell density; however, these components exhibited some scale-dependencies that hindered model quality in cross-scale predictions where only development data was used in calibration. Glutamate and ammonium models were also able to achieve accurate predictions in most cases. However, there are differences in the absolute concentration ranges of these components across the datasets of individual bioreactor scales. Thus, glutamate and ammonium PLS models were forced to extrapolate in cases where models were derived from small scale data only but used in cross-scale applications predicting against manufacturing scale batches. PMID:25504860

  14. Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis

    International Nuclear Information System (INIS)

    Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60Gy. The repair curve obtained was biphasic with the respective half-times of τ1 = 3.8 ± 0.9 and τ2 = 118 ± 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells non-reparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial delections). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments ≤ 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells. (author)

  15. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    International Nuclear Information System (INIS)

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO2) and aluminium oxide (Al2O3) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 μg/mL TiO2 and 0.5-10 μg/mL Al2O3. SCE frequencies were higher for cells treated with 1-5 μg/mL TiO2. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO2. No SCE induction was achieved after treatment with 1-25 μg/mL Al2O3. In conclusion, findings showed cytotoxic and genotoxic effects of TiO2 and Al2O3 NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  16. Confocal microscopy study of pertussis toxin and toxoids on CHO-cells

    OpenAIRE

    Tan, Yajun; Fleck, Roland A.; Asokanathan, Catpagavalli; Yuen, Chun-Ting; Xing, Dorothy; Zhang, Shumin; Wang, Junzhi

    2013-01-01

    Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussi...

  17. Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

    OpenAIRE

    Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

    2014-01-01

    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific pro...

  18. Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks.

    Science.gov (United States)

    Meuwly, F; Loviat, F; Ruffieux, P-A; Bernard, A R; Kadouri, A; von Stockar, U

    2006-03-01

    Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions. PMID:16358288

  19. Quantitative mammalian cell mutagenesis and a preliminary study of the mutagenic potential of metallic compounds. [Cell system used was CHO/HGPRT

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, A.W.; Johnson, N.P.; Couch, D.B.; San Sebastian, J.R.; O' Neill, J.P.; Forbes, N.L.

    1978-01-01

    We have defined a set of stringent conditions required to quantify specific gene mutation in a mammalian cell system, CHO/HGPRT. Greater than 98% of the 6-thioguanine (TG)-resistant variants were shown to be deficient in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in Chinese hamster ovary (CHO) cells. The sensitive and quantitative nature of this assay was utilized to study the structure-activity (mutagenicity) relationship of various classes of chemicals. Mutagenicity as determined in the CHO/HGPRT assay, appears to correlate well (76/83 (92%)) with the reported animal carcinogenicity of 108 chemicals studied. The system also appears to be suitable for studying the mutagenicity and cytotoxicity of metallic compounds. We found that cis-dichlorodiammine Pt(II) (cis-Pt(NH/sub 3/)/sub 2/Cl/sub 2/) (cis-DDP), one of the widely used inorganic antitumor agents, is cytotoxic and mutagenic. Mutagenicity of cis-DDP correlates with its binding to DNA. However, trans-DDP, (Pt(NH/sub 3/)/sub 4/)Cl/sub 2/, and K/sub 2/(PtCl/sub 4/) exhibit greatly reduced biological activities. Among 14 other metals studied, we found that carcinogenic metallic compounds, such as MnCl/sub 2/, NiCl/sub 2/, and BeSO/sub 4/ are mutagenic, while non-carcinogenic compounds such as MgCl/sub 2/ and H/sub 2/SeO/sub 3/ are not. Determination of metal mutagenicity is apparently complicated by the ionic composition of the medium. This may account in part for varying results in studies of the mutagenicity of other metallic compounds. Further refinement of the assay conditions, especially with respect to the ionic environment necessary for quantifying mutagenesis of each metallic agent, is in progress.

  20. CHO Quasispecies—Implications for Manufacturing Processes

    Directory of Open Access Journals (Sweden)

    Florian M. Wurm

    2013-10-01

    Full Text Available Chinese hamster ovary (CHO cells are a source of multi-ton quantities of protein pharmaceuticals. They are, however, immortalized cells, characterized by a high degree of genetic and phenotypic diversity. As is known for any biological system, this diversity is enhanced by selective forces when laboratories (no sharing of gene pools grow cells under (diverse conditions that are practical and useful. CHO cells have been used in culture for more than 50 years, and various lines of cells are available and have been used in manufacturing. This article tries to represent, in a cursory way, the history of CHO cells, particularly the origin and subsequent fate of key cell lines. It is proposed that the name CHO represents many different cell types, based on their inherent genetic diversity and their dynamic rate of genetic change. The continuing remodeling of genomic structure in clonal or non-clonal cell populations, particularly due to the non-standardized culture conditions in hundreds of different labs renders CHO cells a typical case for “quasispecies”. This term was coined for families of related (genomic sequences exposed to high mutation rate environments where a large fraction of offspring is expected to carry one or more mutations. The implications of the quasispecies concept for CHO cells used in protein manufacturing processes are significant. CHO genomics/transcriptomics may provide only limited insights when done on one or two “old” and poorly characterized CHO strains. In contrast, screening of clonal cell lines, derived from a well-defined starting material, possibly within a given academic or industrial environment, may reveal a more narrow diversity of phenotypes with respect to physiological/metabolic activities and, thus, allow more precise and reliable predictions of the potential of a clone for high-yielding manufacturing processes.

  1. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

    DEFF Research Database (Denmark)

    Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel;

    2015-01-01

    the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant......-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the...

  2. Identification of an MRAP-Independent Melanocortin-2 Receptor: Functional Expression of the Cartilaginous Fish, Callorhinchus milii, Melanocortin-2 Receptor in CHO Cells

    OpenAIRE

    Reinick, Christina L.; Liang, Liang; Angleson, Joseph K.; Dores, Robert M.

    2012-01-01

    Phylogenetic analyses indicate that the genome of the cartilaginous fish, Callorhynchus milii (elephant shark), encodes a melanocortin-2 receptor (MC2R) ortholog. Expression of the elephant shark mc2r cDNA in Chinese hamster ovary (CHO) cells revealed that trafficking to the plasma membrane and functional activation of the receptor do not require coexpression with an exogenous melanocortin receptor-2 accessory protein (mrap) cDNA. Ligand selectivity studies indicated that elephant shark MC2R-...

  3. Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor.

    Science.gov (United States)

    Hammerschmidt, Nikolaus; Hintersteiner, Beate; Lingg, Nico; Jungbauer, Alois

    2015-08-01

    We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody. PMID:25781580

  4. Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

    Science.gov (United States)

    Chiverton, Lesley M; Evans, Caroline; Pandhal, Jagroop; Landels, Andrew R; Rees, Byron J; Levison, Peter R; Wright, Phillip C; Smales, C Mark

    2016-08-01

    There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs. PMID:27214759

  5. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

    Directory of Open Access Journals (Sweden)

    Przemysław Sitarek

    2016-01-01

    Full Text Available Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx. LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin, and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid. The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

  6. Condensation of interphase chromatin in nuclei of synchronized chinese hamster ovary (CHO-K1) cells.

    Science.gov (United States)

    Gacsi, Mariann; Nagy, Gabor; Pinter, Gabor; Basnakian, Alexei G; Banfalvi, Gaspar

    2005-01-01

    Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis. PMID:15684719

  7. Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

    DEFF Research Database (Denmark)

    Merz, G S; Benedikz, Eirikur; Schwenk, V;

    1997-01-01

    To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in...... the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of......, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through...

  8. Kinetics of DNA double-strand break repair throughout the cell cycle as assayed by pulsed field gel electrophoresis in CHO cells

    International Nuclear Information System (INIS)

    Repair of DNA double-strand breaks (dsb) was measured in exponentially growing, plateau-phase and synchronized G1, G1/S, early S, mid-S, late S, G2 + M amd mitotic CHO cells. Results suggest that the state of chromatin condensation has only a limited impact on the ability of the cells to rejoin dsb, and indicate that the cell cycle-dependent fluctuations in radiosensitivity cannot be explained by alterations in the rate of rejoining of dsb. Repair half-times of the slow component of dsb rejoining were similar to the half-times of rejoining of chromosome breaks as visualized by the technique of premature chromosome condensation, suggesting a cause-effect relationship between rejoining of this subject of dsb and rejoining of chromosome breaks. (author)

  9. X-ray sensitive strains of CHO cells show decreased frequency of stable transfection

    International Nuclear Information System (INIS)

    Six X-ray sensitive (xrs) strains of the Chinese hamster ovary cell line have previously been isolated and shown to have a defect in double strand break rejoining. In this study, these strains have been investigated for their ability to take up and integrate foreign DNA. All the xrs strains investigated so far have shown a decreased frequency of stable transfectants compared to their parent line, in experiments using the plasmid pSV2gpt, which contains the selectable bacterial gene, guanine phosphoribosyl transferase. This decreased frequency is observed over a wide range of DNA concentrations (0.1 to 20 μg DNA) but is more pronounced at higher DNA concentrations. In contrast, these xrs strains show the same level of transfection proficiency as the wild type parent using a transient transfection system with a plasmid containing the bacterial CAT (chloramphenicol acetyl transferase) gene. Since the level of CAT activity does not depend on integration of foreign DNA, this suggests that the xrs strains are able to take up the same amount of DNA as the parent strains, but have a defect in the integration of foreign DNA. Since this integration of foreign DNA probably occurs by non-homologous recombination, this may indicate a role of the xrs gene product in this process

  10. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Bolt, Gert; Hansen, Jens J;

    2015-01-01

    Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three...... orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII...

  11. Variation through the cell cycle in the dose-response of DNA neutral filter elution in X-irradiated synchronous CHO-cells

    International Nuclear Information System (INIS)

    Dose-response curves for DNA neutral (pH 9.6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of cell cycle. The dose response was similar in synchronized and plateau-phase G1 cells, as well as in cells arrested at the G1/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G1 cells, was observed as cells entered G2 phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S-700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (ssub(max) = 350S), indicating the formation of a DNA complex. DNA from G1, G1/S, or G2 + M cells sedimented as expected for free sedimenting molecules. (author)

  12. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO

    International Nuclear Information System (INIS)

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co γ radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400μ/ml) in PC3 cells irradiated with 5 Gγ. The survival curves obtained were adequately fitted by a linear-quadratic model, where the α coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a

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

    International Nuclear Information System (INIS)

    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 EC50 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Seishiro, E-mail: seishiro@nies.go.jp [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Fujitani, Yuji; Furuyama, Akiko [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Kanno, Sanae [Department of Legal Medicine, St. Marianna School of Medicine (Japan)

    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.

  15. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    Energy Technology Data Exchange (ETDEWEB)

    Di Virgilio, A.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata (1900) (Argentina); Reigosa, M. [Instituto Multidisciplinario de Biologia Celular (IMBICE), Calle 526 y Camino Gral. Belgrano (entre 10 y 11), La Plata 1900 (Argentina); Arnal, P.M. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina); Fernandez Lorenzo de Mele, M., E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina)

    2010-05-15

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO{sub 2}) and aluminium oxide (Al{sub 2}O{sub 3}) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 {mu}g/mL TiO{sub 2} and 0.5-10 {mu}g/mL Al{sub 2}O{sub 3}. SCE frequencies were higher for cells treated with 1-5 {mu}g/mL TiO{sub 2}. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO{sub 2}. No SCE induction was achieved after treatment with 1-25 {mu}g/mL Al{sub 2}O{sub 3}. In conclusion, findings showed cytotoxic and genotoxic effects of TiO{sub 2} and Al{sub 2}O{sub 3} NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  16. Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and (13)C-metabolic flux analysis.

    Science.gov (United States)

    Ahn, Woo Suk; Crown, Scott B; Antoniewicz, Maciek R

    2016-09-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in (13)C metabolic flux analysis ((13)C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-(13)C]glucose+[4,5,6-(13)C]glucose, [2-(13)C]glucose+[4,5,6-(13)C]glucose, and [3-(13)C]glucose+[4,5,6-(13)C]glucose and developed a new method to measure (13)C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells. PMID:27174718

  17. Stimulation of 125I-3-iodo-α-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

    International Nuclear Information System (INIS)

    Introduction: Transport of the amino acid analog 123I-3-iodo-α-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of 125I-3-iodo-α-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 deg. C or under ice-cold conditions. Results: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of L-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. Conclusions: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor

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

    Science.gov (United States)

    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

  19. Retroviral expression of a kinase-defective IGF-I receptor suppresses growth and causes apoptosis of CHO and U87 cells in-vivo

    International Nuclear Information System (INIS)

    Phosphatidylinositol-3,4,5-triphosphate (PtdInsP3) signaling is elevated in many tumors due to loss of the tumor suppressor PTEN, and leads to constitutive activation of Akt, a kinase involved in cell survival. Reintroduction of PTEN in cells suppresses transformation and tumorigenicity. While this approach works in-vitro, it may prove difficult to achieve in-vivo. In this study, we investigated whether inhibition of growth factor signaling would have the same effect as re-expression of PTEN. Dominant negative IGF-I receptors were expressed in CHO and U87 cells by retroviral infection. Cell proliferation, transformation and tumor formation in athymic nude mice were assessed. Inhibition of IGF-IR signaling in a CHO cell model system by expression of a kinase-defective IGF-IR impairs proliferation, transformation and tumor growth. Reduction in tumor growth is associated with an increase in apoptosis in-vivo. The dominant-negative IGF-IRs also prevented growth of U87 PTEN-negative glioblastoma cells when injected into nude mice. Injection of an IGF-IR blocking antibody αIR3 into mice harboring parental U87 tumors inhibits tumor growth and increases apoptosis. Inhibition of an upstream growth factor signal prevents tumor growth of the U87 PTEN-deficient glioma to the same extent as re-introduction of PTEN. This result suggests that growth factor receptor inhibition may be an effective alternative therapy for PTEN-deficient tumors

  20. Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality.

    Science.gov (United States)

    Meuwly, F; Weber, U; Ziegler, T; Gervais, A; Mastrangeli, R; Crisci, C; Rossi, M; Bernard, A; von Stockar, U; Kadouri, A

    2006-05-01

    During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed

  1. The Protective Effect of Rosemary Acid on DNA Damage of CHO Cells%迷迭香酸对CHO细胞DNA损伤的防护作用

    Institute of Scientific and Technical Information of China (English)

    夭建华; 高茜; 李雪梅; 黄海涛; 管莹; 米其利; 罗瑛

    2016-01-01

    采用高内涵分析技术测定DNA损伤/双键断裂的标志物γH2AX,来研究迷迭香酸对细胞DNA损伤的防护作用。即通过在体外对CHO细胞施予迷迭香酸,再诱导DNA损伤,对致损后的细胞进行γH2AX及Hoechst双荧光染色标记,最后通过分析γH2AX的荧光强度从而对细胞DNA损伤程度进行半定量检测。结果发现,施予迷迭香酸的细胞与对照相比较,DNA损伤程度降低,表明迷迭香酸对CHO细胞的DNA损伤具有防护作用。%To investigate the protective effect of rosemary acid on DNA damage,the DNA damage/double strand breaks biomarkerγH2AX was detected by using high content analysis technique. CHO cells were treated with rosemary acid, and then DNA damage was induced. Cells were double staining withγH2AX and Hoechst, andfinally the obtainedfluorescence intensity ofγH2AX was used to semi - quantitatively characterize the degree of DNA damage. The results showed that comparing with the control, CHO cells treated with rosemary acid had a lower degree of DNA damage, which indicated that the rosemary acid had protective effect on the DNA damage of CHO cells.

  2. Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy.

    Science.gov (United States)

    Opel, Cary F; Li, Jincai; Amanullah, Ashraf

    2010-01-01

    Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. PMID:20730773

  3. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  4. Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection.

    Science.gov (United States)

    Renois, Fanny; Hong, Saw-See; Le Naour, Richard; Gafa, Valérie; Talmud, Déborah; Andréoletti, Laurent; Lévêque, Nicolas

    2011-06-01

    The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors. PMID:21420451

  5. 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.

    Science.gov (United States)

    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 (15μg/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

  6. MeIQx-induced DNA adduct formation and mutagenesis in DNA repair deficient CHO cells expressing human CYP1A1 and rapid or slow acetylator NAT2

    OpenAIRE

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

    2007-01-01

    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). Nucleotide excision repair-deficient chinese hamster ovary (CHO) 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) alle...

  7. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    International Nuclear Information System (INIS)

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 430C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 370C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 109 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

  8. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-07-24

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43/sup 0/C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37/sup 0/C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 ..mu..g per 10/sup 9/ cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs.

  9. Detection of DNA double-strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis

    International Nuclear Information System (INIS)

    A pulsed field gel electrophoresis technique, asymmetric field inversion gel electrophoresis (AFIGE), was used to evaluate induction by X-rays of DNA damage in CHO cells. The fraction of DNA activity released from the plug (FAR) was used as a measure for the amount of radiation-induced DNA damage, predominantly DNA double-strand breaks (dsb) (Stamato and Denko 1990), and was determined at various stages of growth and phases of the cell cycle in a range of doses between zero and 70 Gy. It is concluded that caution needs to be exercised before differences observed in the FAR between different cell lines or between various phases of the cell cycle after exposure to a given dose of radiation are interpreted as suggesting differences in the induction of DNA dsb. (author)

  10. All colonies of CHO-K1 cells surviving γ-irradiation contain non-viable cells

    International Nuclear Information System (INIS)

    This paper addresses production of defective cells within clones arising from irradiated progenitor cells. It aims at answering the question whether lethal mutation result from a generalised effect which lowers the ability of all the progeny to divide successfully or whether it represents a late expressed but unique lethal defect induced by radiation which occurs in some cells only and which causes those cells only to cease dividing. Results obtained from autoradiographic analysis of cells within individual surviving colonies (i.e. containing more than 150 cells) suggests that some cells in all clones are not synthesizing DNA over a 9-h period and that the properties of non-synthesizing cells rises with increasing dose of radiation from less than 3% in controls to 80-85% after a progenitor dose of 12.5 Gy. because of the possibility that cells had longer division times post irradiation, these results were repeated using Ki67 antibody labelling, a technique that identifies cells which are in cycle. Results were similar. This suggests the non-labelled cells were not reproducing. Both techniques were also used to look at the % labelling of morphologically abnormal cells in colonies. Results suggested that up to 35% of these abnormal cells were actively cycling and about 20% were synthesizing DNA. Abnormal cells did not appear in subcultures of survivor progeny suggesting that they may have failed to replate successfully and may contribute to the lethally mutated population. The results are discussed in the context of their significance for survival curve analysis and for radiotherapy and radiation protection results. (author). 32 refs.; 6 figs.; 2 tabs

  11. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    Science.gov (United States)

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase. PMID:22251027

  12. Radiation-induced DNA breaks detected by immuno labelling of poly(ADP-ribose) in CHO cells. Standardization by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immuno-labelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation. (authors)

  13. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    Science.gov (United States)

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

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

    Science.gov (United States)

    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

  15. Radiation induced enhancement of the efficiency of DNA mediated gene transfer in UV and x-ray sensitive CHO mutant cell lines

    International Nuclear Information System (INIS)

    The authors present results of experiments studying the enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer in mutant CHO cell lines. The established cell lines UV-5, UV-20 and EM9 are transfected with the recombinant DNA plasmid, pSV2-gpt irradiated with either UV or X-rays and plated in MAX selective media. MAX-resistant colonies are the result of the integration of pSV2-gpt and the expression of the gene coding for the bacterial xanthine-guanine phosphoribosyl transferase. Dose response curves for UV and X-rays are generated for the frequency of MAX-resistant colonies/survivor. From these experiments, the authors hope to delineate the role of DNA repair enzymes in the phenomenon at plasmid DNA integration after DNA mediated gene transfer

  16. Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

    Science.gov (United States)

    López-Meza, Julián; Araíz-Hernández, Diana; Carrillo-Cocom, Leydi Maribel; López-Pacheco, Felipe; Rocha-Pizaña, María Del Refugio; Alvarez, Mario Moisés

    2016-08-01

    Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells-the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2-4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27-1.08 × 10(7) cell/mL and 0.72-2.79 × 10(6) cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking-Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10(-7) µg/cell and β = 7.68 × 10(-8) µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode. PMID:26091615

  17. In vitro genotoxic and cytotoxic effects of ivermectin and its formulation ivomec on Chinese hamster ovary (CHO{sub K1}) cells

    Energy Technology Data Exchange (ETDEWEB)

    Molinari, G.; Soloneski, S.; Reigosa, M.A. [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina); Larramendy, M.L., E-mail: m_larramendy@hotmail.com [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina)

    2009-06-15

    The effects of ivermectin (IVM) and its commercial formulation ivomec (IVM 1.0%) were studied on Chinese hamster ovary (CHO{sub K1}) cells by several genotoxicity [sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE)] and cytotoxicity [cell-cycle progression (CCP), mitotic index (MI), proliferative replication index (PRI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)] bioassays within the 1.0-250 {mu}g/ml concentration-range. While IVM and ivomec did not modified SCE frequencies, they induced DNA-strand breaks revealed by SCGE. An enhancement of slightly damaged cells and a decrease in undamaged cells were observed in IVM-treated cultures with 5.0-50.0 {mu}g/ml. In ivomec-treated cells, while an increase in slightly damaged cells was induced with 5.0-50.0 {mu}g/ml, the damaged and undamaged cells increased and decreased only with 50.0 {mu}g/ml. Both compounds exerted a delay in CCP and a reduction in PRI when 25.0 {mu}g/ml was employed whereas cytotoxicity was observed at higher concentration than 50.0 {mu}g/ml. No MI alteration was observed with 1.0-10.0 and 1.0-5.0 {mu}g/ml of IVM and ivomec, respectively. A concentration-related trend to an increase in MI was achieved within 1.0-10.0 {mu}g/ml. An increase in the MI was induced in 10.0 {mu}g/ml ivomec-treated cultures. A marked reduction of about 89% and 62% in regard to controls was observed with 25.0 {mu}g/ml of IVM and ivomec, respectively. NR and MTT assays revealed a cell growth inhibition when 0.25-250.0 {mu}g/ml of both compounds was employed. The results highlighted that IVM and ivomec exert both genotoxicity and cytotoxicity in mammalian cells in vitro, at least in CHO{sub K1} cells.

  18. A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor.

    Science.gov (United States)

    Wang, Ing-Kae; Hsieh, Sing-Ying; Chang, King-Ming; Wang, Yu-Chi; Chu, Andy; Shaw, Shyh-Yu; Ou, Jung-Jung; Ho, Lewis

    2006-02-10

    In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5-90 min), final cell densities of 1.6-4.0 x 10(9) have been obtained in 20 days while total angiostatin-human IgG production of 228-388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor. PMID:16162365

  19. [Construction of mammalian cell expression vector for pAcGFP-bFADD fusion protein and its expression in CHO-K1 cell].

    Science.gov (United States)

    Yang, Runjun; Xu, Shangzhong; Zhang, Lupei; Li, Junya; Gao, Xue

    2008-11-01

    Fas-associated death domain (FADD) is a signal connection protein in Fas/FasL apoptotic path which might play a key role on apoptosis by transferring apoptotic signal. To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in bovine ovary, we cloned FADD gene in bovine ovary tissue with RT-PCR, deleted the termination codon in its cDNA and directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-N1 including AcGFP, successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme Bgl II/EcoR I and sequencing, transfected pAcGFP-bFADD into CHO-K1 cell mediated by Lipofectamine 2000, observed the expression of AcGFP and detected the transcription and expression of FADD by RT-PCR and Western blotting. The results showed that the cattle FADD was successfully cloned, the pAcGFP-bFADD fusion protein recombinant plasmid was successfully constructed by introducing Bgl II, EcoR I cloning site at two ends of FADD open reading frame and inserting a Kozak sequence before start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 654 bp transcription was amplified by RT-PCR, and 51.4 kD target protein was detected by Western blotting. Construction of pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism of regulation of FADD on bovine oocytes formation and development. PMID:19256333

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

    Science.gov (United States)

    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

  1. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    Directory of Open Access Journals (Sweden)

    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

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

    Science.gov (United States)

    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

  3. Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

    Science.gov (United States)

    Chan, Kah Fai; Shahreel, Wahyu; Wan, Corrine; Teo, Gavin; Hayati, Noor; Tay, Shi Jie; Tong, Wen Han; Yang, Yuansheng; Rudd, Pauline M; Zhang, Peiqing; Song, Zhiwei

    2016-03-01

    Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies. PMID:26471004

  4. MeIQx-induced DNA adduct formation and mutagenesis in DNA repair deficient CHO cells expressing human CYP1A1 and rapid or slow acetylator NAT2

    Science.gov (United States)

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

    2007-01-01

    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). Nucleotide excision repair-deficient chinese hamster ovary (CHO) 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 CHO cell lines. CYP1A1 activity did not differ significantly (p > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of sulfamethazine N-acetyltransferase (p = 0.0001) and 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 (hprt) mutagenesis following MeIQx treatment. dG-C8-MeIQx was the primary DNA adduct formed and levels were dose-dependent in each cell line and in the order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 & NAT2*5B < transfected with CYP1A1 & 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 on MeIQx –induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiological studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators. PMID:17627018

  5. Effects of {gamma} ({sup 60}Co) and {beta} ({sup 90}Sr) radiations in Chinese hamster ovarian cells (CHO-K1): induction of micronuclei and cell death; Efeitos das radiacoes {gamma} ({sup 60}Co) e {beta} ({sup 90}Sr) em celulas de ovario de hamster chines (CHO-K1): inducao de micronucleos e morte celular

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Daniella

    2003-07-01

    Among various types of ionising radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequent increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of {sup 90}Sr, a pure, highly energetic beta source, on CHO-K1 cells and to compare them with data obtained with {sup 60}Co. CHO-K1 cells irradiated with different doses (1.0, 2.5, 5.0, 7.5 Gy) of {sup 60}Co (0.34 Gy.min{sup -1}) and {sup 90}Sr (0.23 Gy.min{sup -1}) were processed for analysis of clonogenic death, induction of micronuclei (MN) and necrotic and apoptotic death. The survival curves obtained for both types of radiation were better fitted by the linear-quadratic model and were similar. However, the cytogenetic results showed that both the proportion of micronucleated cells and the magnitude of radioinduced lesions demonstrated by the analysis of MN distribution were significantly higher in cells irradiated with {sup 60}Co than in cells irradiated with {sup 90}Sr, whereas {sup 90}Sr was more damaging than {sup 60}Co in terms of cell death induction. Necrosis was the major type of death observed in CHO-K1 cells. The data obtained suggest that the low incidence of micronucleated cells after exposure to {sup 90}Sr may be a consequence of selective elimination of severely damaged cells from the population by the necrotic process at a higher rate than observed with {sup 60}Co exposure. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics. (author)

  6. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO; Avaliacao do efeito radiomodificador da propolis em celulas de ovario de hamster chines (CHO-K1) e em celulas tumorais de prostata (PC3), irradiadas com CO-60

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Geyza Spigoti

    2011-07-01

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with {sup 60}Co {gamma} radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 {mu}g/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400{mu}/ml) in PC3 cells irradiated with 5 G{gamma}. The survival curves obtained were adequately fitted by a linear-quadratic model, where the {alpha} coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 {mu}g/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data

  7. Intracellular pH and 42.00 C heat response of CHO cells cultured at pH 6.6

    International Nuclear Information System (INIS)

    The authors previously reported that cells under chronic low pH (6.6) conditions have altered thermotolerance. They further characterized both the doubling time (t/sub d/) and the internal pH (pH/sub 1/) of CHO cells continuously cultured at pH 6.6 for times greater than one year. The following differences were noted: 1) A t/sub d/ of 16 hr compared to a t/sub d/ of 12 hr for cells at normal pH (7.3) and a t/sub d/ of 25 hr for the acute low pH cells (pH = 6.6; incubation time = 4 hr). 2) A pH/sub i/ 0.1-0.15 pH units > normal cells and 0.3 pH units > acute low pH cells. 3) Survival at 42.00C which differed from both normal and acute low pH cells. The chronic culture was still quite sensitive to 42.00C treatments during the first 5 hr, but developed tolerance at a higher level than cells under acute low pH conditions. The pH/sub i/ of the chronic culture responded to 42.00C heating in a manner similar to that for acute low pH cells. Whether this culture represents a normal response to long term low pH exposure, or was the response of a mutant population is at the present unknown

  8. Effect of ethanolic extract of propolis on cell viability of chinese hamster ovary cells (CHO-K1) irradiated with 60CO gamma-rays using differential staining technique

    International Nuclear Information System (INIS)

    The objective of present study was to assess the effect of Brazilian propolis (AF-08) on CHO-K1 cells irradiated with 60Co, through the differential staining technique, using acridine orange and ethidium bromide. The cells were pre-incubated with different concentrations of propolis (50, 100 and 200 μg/mL) for 24h and irradiated with 5 Gy, analyzed at 24 and 48h after exposure. This technique is based on the cell capacity to incorporate fluorescent DNA dyes, where the viable (green), apoptotic (orange/yellow) and necrotic (red) cells can be identified through fluorescence microscopy. Digital high-resolution images were acquired from at least 5 visualization fields, and cells were analyzed using ImageJ and Flowing software. This approach permitted to analyze a large number of cells/sample with the time reduction, much easier and faster, proportioning more statistical power of the technique. The treatment with propolis only was not cytotoxic at 24 and 48h, except for the higher concentration of 200 μg/mL associated or not with radiation, increasing apoptotic and mainly necrotic cells (p<0.001). The data showed a promising use of propolis as well as technique used, pointing out that 200 μg/mL of propolis was cytotoxic, but at lower one (50 μg/mL) presented a radioprotective effect in irradiated CHO-K1 cells. (author)

  9. Effect of ethanolic extract of propolis on cell viability of chinese hamster ovary cells (CHO-K1) irradiated with {sup 60}CO gamma-rays using differential staining technique

    Energy Technology Data Exchange (ETDEWEB)

    Castro, Marcos P.M. de; Castro, Renato F. de; Okazaki, Kayo; Vieira, Daniel P., E-mail: dpvieira@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    The objective of present study was to assess the effect of Brazilian propolis (AF-08) on CHO-K1 cells irradiated with {sup 60}Co, through the differential staining technique, using acridine orange and ethidium bromide. The cells were pre-incubated with different concentrations of propolis (50, 100 and 200 μg/mL) for 24h and irradiated with 5 Gy, analyzed at 24 and 48h after exposure. This technique is based on the cell capacity to incorporate fluorescent DNA dyes, where the viable (green), apoptotic (orange/yellow) and necrotic (red) cells can be identified through fluorescence microscopy. Digital high-resolution images were acquired from at least 5 visualization fields, and cells were analyzed using ImageJ and Flowing software. This approach permitted to analyze a large number of cells/sample with the time reduction, much easier and faster, proportioning more statistical power of the technique. The treatment with propolis only was not cytotoxic at 24 and 48h, except for the higher concentration of 200 μg/mL associated or not with radiation, increasing apoptotic and mainly necrotic cells (p<0.001). The data showed a promising use of propolis as well as technique used, pointing out that 200 μg/mL of propolis was cytotoxic, but at lower one (50 μg/mL) presented a radioprotective effect in irradiated CHO-K1 cells. (author)

  10. Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

    Science.gov (United States)

    Liu, Jintao; Wang, Jie; Fan, Li; Chen, Xinning; Hu, Dongdong; Deng, Xiancun; Poon, H Fai; Wang, Haibin; Liu, Xuping; Tan, Wen-Song

    2015-07-01

    Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system. PMID:25931375

  11. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  12. Yellow fluorescent protein-based assay to measure GABA(A channel activation and allosteric modulation in CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Teres Johansson

    Full Text Available The γ-aminobutyric acid A (GABA(A ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA(A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA(A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP-based assay to be able to study allosteric modulation of the GABA(A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA(A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA(A2 cells. As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA(A subunit composition and functionality. We found that the I(- concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS and could be used to discover novel modulators acting on GABA(A ion channels.

  13. Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy

    Science.gov (United States)

    Tuca, Silviu-Sorin; Badino, Giorgio; Gramse, Georg; Brinciotti, Enrico; Kasper, Manuel; Oh, Yoo Jin; Zhu, Rong; Rankl, Christian; Hinterdorfer, Peter; Kienberger, Ferry

    2016-04-01

    The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S 11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Y cell = 185 μS + j285 μS and Y bacteria = 3 μS + j20 μS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance-capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

  14. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    International Nuclear Information System (INIS)

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 μM concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 μM). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 μM. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 μM) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  15. Absence of mutagenic activity in Salmonella and of clastogenic activity in CHO cells of Caramel Colours I, II, III and IV.

    Science.gov (United States)

    Allen, J A; Brooker, P C; Jones, E; Adams, K; Richold, M

    1992-05-01

    A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed. PMID:1644380

  16. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Grillo, C.A. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Mirifico, M.V. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina); Morales, M.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Reigosa, M.A. [IMBICE (Instituto Multidisciplinario de Biologia Celular), CICPBA, CONICET, Calle 526 entre 10 y 11, 1900 La Plata (Argentina); Mele, M. Fernandez Lorenzo de, E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina)

    2009-10-30

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 {mu}M concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 {mu}M). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 {mu}M. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 {mu}M) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  17. Inhibition of topoisomerase IIα activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

    International Nuclear Information System (INIS)

    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

  18. Erythropoietin enhancer stimulates production of a recombinant protein by Chinese hamster ovary (CHO) cells under hypoxic condition

    OpenAIRE

    Moon, Sung-Kwon; Takeuchi, Shunsuke; Kambe, Taiho; Tsuchiya, Terumasa; Masuda, Seiji; Nagao, Masaya; Sasaki, Ryuzo

    1997-01-01

    Oxygen is a limiting nutrient in animal cell culture and its supply is still worthy of improvement for production of useful proteins with a high efficiency. From a different point of view, development of the system by which a high productivity can be maintained even under hypoxic condition as well as under normoxic condition may be important. A number of hypoxia-inducible genes have been found in eucaryotic cells and the induction in most cases, if not all, is due to hypoxic activation of the...

  19. Dynamic metabolic flux analysis using B-splines to study the effects of temperature shift on CHO cell metabolism

    Directory of Open Access Journals (Sweden)

    Verónica S. Martínez

    2015-12-01

    Full Text Available Metabolic flux analysis (MFA is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37 °C to 32 °C maintained a constant IgG volume-specific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32 °C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity.

  20. The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

    Directory of Open Access Journals (Sweden)

    Bogdanović Višnja

    2009-01-01

    Full Text Available Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC treatment on superoxide-dismutase (SOD activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH24 at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET. The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL after 3 and 24 h of cell incubation. Increasing of C60(OH24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL + MMC was noticed boost in total SOD activity in comparison with

  1. Six complementation classes of conditionally lethal protein synthesis mutants of CHO cells selected by 3H-amino acid

    International Nuclear Information System (INIS)

    Using a tritiated amino acid suicide procedure designed specifically to select conditional protein synthesis mutants, we have isolated and characterized a large number of such mutants of Chinese hamster ovary cells. All of the mutants are genetically stable and behave as recessives in somatic cell hybrids. Most of the new mutants are phenotypically dependent on the concentration of a specific amino acid as well as on temperature. In addition to identifying many additional leucyl- and asparagyl-tRNA synthetase mutants, complementation analysis has distinguished four new genetic classes representing methionine-, glutamine-, histidine-, and arginine-dependent mutants. Biochemical characterization of representative mutants from each of these six classes has identified the primary lesions as being defective aminoacyl-tRNA synthetases. Our slection results further demonstrate the high specificity of the 3H-amino acid procedure for isolating protein synthesis mutants. Reconstruction experiments performed with two representative mutants indicated a selection efficiency of approximately 10% under standard conditions

  2. Comparison of internal ribosome entry site (IRES and Furin-2A (F2A for monoclonal antibody expression level and quality in CHO cells.

    Directory of Open Access Journals (Sweden)

    Steven C L Ho

    Full Text Available Four versions of tricistronic vectors expressing IgG1 light chain (LC, IgG1 heavy chain (HC, and dihydrofolate reductase (DHFR in one transcript were designed to compare internal ribosome entry site (IRES and furin-2A (F2A for their influence on monoclonal antibody (mAb expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

  3. DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

    International Nuclear Information System (INIS)

    We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

  4. Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

    International Nuclear Information System (INIS)

    Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the β-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission

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

    Science.gov (United States)

    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,...

  6. 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

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, J.M. (Miami Univ., Oxford, OH (USA). 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.

  7. 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

    International Nuclear Information System (INIS)

    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

  8. Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Humans are exposed to arsenic and their organic derivatives, which are widely distributed in the environment, via food, water, and to a lesser extent, via air. Following uptake, inorganic arsenic undergoes biotransformation to mono- and dimethylated metabolites. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In the present study, the genotoxic effects and the cellular uptake of inorganic arsenic [arsenate, Asi(V); arsenite, Asi(III)] and the methylated arsenic species monomethylarsonic acid [[MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], trimethylarsenic oxide [TMAO(V)] were investigated in Chinese hamster ovary (CHO-9) cells. The chemicals were applied at different concentrations (0.1 μM to 10 mM) for 30 min and 1 h, respectively. Cytotoxic effects were investigated by the trypan blue extrusion test and genotoxic effects by the assessment of micronucleus (MN) induction, chromosome aberrations (CA), and sister chromatid exchanges (SCE). Intracellular arsenic concentrations were determined by ICP-MS techniques. Our results show that MMA(III) and DMA(III) induce cytotoxic and genotoxic effects to a greater extent than MMA(V) or DMA(V). Viability was significantly decreased after incubation (1 h) of the cells with ≥ 1 μM Asi(III), ≥ 1 μM Asi(V), ≥ 500 μM MMA(III), ≥ 100 μM MMA(V), and 500 μM DMA(V) and ≥ 0.1 μM DMA(III). TMAO(V) was not cytotoxic at concentrations up to 10 mM. A significant increase of the number of MN, CA and SCE was found for DMA(III) and MMA(III). Asi(III + V) induced CA and SCE but no MN. TMAO(V), MMA(V) and DMA(V) were not genotoxic in the concentration range tested (up to 5 mM). The nuclear division index (NDI) was not affected by any of the tested arsenic compounds after a recovery period of 14 to 35 h. When the uptake of the chemicals was measured by ICP-MS analysis, it was found that only 0

  9. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Kristensen, Claus; Betenbaugh, Michael J.;

    2015-01-01

    Background: The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins.  Results: Here we present the genomic sequen...

  10. Sister chromatid exchanges induced by two radiosensitizing platinum compounds (cis-dichloro-bis isopropylamine trans dihydroxy platinum IV (CHIP) and cis platinum metronidazole2Cl2(FLAP] in CHO cells in vitro.

    OpenAIRE

    Bocian, E; Laverick, M.; Nias, A H

    1983-01-01

    Sister chromatid exchange (SCE) induction by two radiosensitizing platinum compounds (cis-dichloro-bis isopropylamine trans dihydroxy platinum IV (CHIP) and cis-platinum metronidazole2 Cl2 (FLAP] was studied in CHO cells in vitro. Both drugs induced SCE in a dose dependent manner. CHIP was a much more potent inducer of SCE than FLAP and produced almost 4 times as many SCE as FLAP at equimolar concentrations and twice as many at equitoxic dosage. Induction of SCE by a component of the FLAP mol...

  11. Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

    OpenAIRE

    Zhang, Taylor; Huang, Yungfu; Chamberlain, Scott; Romeo, Tony; Zhu-Shimoni, Judith; Hewitt, Daniel; Zhu, Mary; Katta, Viswanatham; Mauger, Brad; Kao, Yung-Hsiang

    2012-01-01

    We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal s...

  12. The emerging CHO systems biology era: harnessing the ‘omics revolution for biotechnology

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan;

    2013-01-01

    line was recently sequenced. Now, the CHO systems biology era is underway. Critical ‘omics data sets, including proteomics, transcriptomics, metabolomics, fluxomics, and glycomics, are emerging, allowing the elucidation of the molecular basis of CHO cell physiology. The incorporation of these data sets...... into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As ‘omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein......Chinese hamster ovary (CHO) cells are the primary factories for biopharmaceuticals because of their capacity to correctly fold and post-translationally modify recombinant proteins compatible with humans. New opportunities are arising to enhance these cell factories, especially since the CHO-K1 cell...

  13. Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

    Science.gov (United States)

    Zhang, Taylor; Huang, Yungfu; Chamberlain, Scott; Romeo, Tony; Zhu-Shimoni, Judith; Hewitt, Daniel; Zhu, Mary; Katta, Viswanatham; Mauger, Brad; Kao, Yung-Hsiang

    2012-01-01

    We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations. PMID:23018810

  14. Effects of the radioprotector WR-1065 on aspects of DNA metabolism and cell cycle progression in CHO AA8 and human HSF4 cells

    International Nuclear Information System (INIS)

    The radioprotector WR-1065 (2-[(aminopropyl) amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radiation exposure, but the exact mechanisms involved in this protection are not fully known. The effect of WR-1065 on a variety of cellular processes in two cell lines was examined to determine how it may provide protection. Incubation of Chinese hamster ovary AA8 cells in 4 mM WR-1065 did not significantly affect the DNA synthetic rate. Autoradiographic analysis of heavily labeled nuclei of AA8 cells showed no significant difference in the size of the S phase population of WR-1065 treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy of gamma-rays, showed no difference between treated and sham treated cells for up to 2 h exposure time. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3h. WR-1065 concentrations ranging from 0.5 mM to 4 mM were not cytotoxic to normal human skin fibroblast cells (HSF4) for exposure to 137Cs gamma-rays resulted in a protection factor of 3.5, almost twice that observed for AA8 cells with 4 mM Wr-1065. Growth of AA8 cells in either alpha-minimal essential medium or McCoy's 5 a medium did not affect the alteration in cell cycle progression observed. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065 by allowing more time for the repair of DNA damage prior to division

  15. The levels of DNA polymerase alpha and beta during the cell cycle and their role in heat radiosensitization in CHO cells

    International Nuclear Information System (INIS)

    The levels of DNA polymerase alpha and beta were measured during the cell cycle using a whole cell assay technique. The results indicate a decrease in the levels of both enzymes during the G/sub 1/ phase and a gradual increase as cells enter the S phase. The recovery of the DNA polymerases was measured after heating for 10 minutes at 45.50C during G/sub 1/ phase or S phase. The activity of DNA polymerase beta recovers fully during 20-25 hours after heating for both G/sub 1/ phase or S phase cells. There is no recovery of the activity of the DNA polymerase alpha during this time. Survival was also measured when cells were irradiated (4 GY) at various times after hyperthermia (10 min at 45.50C), and for both G/sub 1/ and S phase the interaction between heat and x-ray disappeared fully after 20-25 hours following heating and was parallel to recovery of DNA polymerase beta. Furthermore, treatment with cyclohexamide inhibited protein synthesis and prevented recovery from heat damage assayed in terms of both cell survival and beta polymerase. These results, in addition to experiments with heat sensitization at low pH and heat protection with glycerol, indicate that beta polymerase is probably involved in repairing x-ray induced damage resulting in cell lethality

  16. Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5

    International Nuclear Information System (INIS)

    The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared to the parental CHO-K1 cells. Metaphase chromosomes from xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part, the radiation sensitivity of xrs-5 cells

  17. Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells

    International Nuclear Information System (INIS)

    The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. The results suggest all major techniques currently used for assaying rejoining of DNA dsb give similar results, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established. (author)

  18. Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells)

    International Nuclear Information System (INIS)

    Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of 14C(U)-L-tyrosine (14C-Tyr), 125I-4-iodo-L-meta-tyrosine (4-125I-mTyr), 125I-6-iodo-L-meta-tyrosine (6-125I-mTyr), 125I-3-iodo-α-methyl-L-tyrosine (125I-IMT) and 125I-3-iodo-L-tyrosine (3-125I-Tyr) using Chinese hamster ovary cells (CHO-K1). Methods: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na+ at 37oC or 4oC. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na+-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na+-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN3 and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. Results: 14C-Tyr exhibited affinity for systems L, A and ASC. 4-125I-mTyr and 3-125I-Tyr exhibited high specificity for system L, whereas 6-125I-mTyr and 125I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-125I-mTyr was markedly reduced by incubation at 4 oC, and was not significantly inhibited by NaN3, DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-125I-mTyr. Conclusions: 4-125I-mTyr exhibited the greatest system L specificity (93.46±0.13%) of all of the tested amino acids.

  19. Evaluation of genotoxic effect of prozac (fluoxetine without and with addition of vitamins A and C by means of the comet assay in culture of CHO-K1 cells

    Directory of Open Access Journals (Sweden)

    Noélle Giacomini Lemos

    2005-12-01

    Full Text Available The fluoxetine, commercially named Prozac, is efficient against depression and anxiety, with lower risk of collateral effects. However, the possible genotoxic effects are still unknown. The use of vitamins as protectors against damages on cells and DNA has been evaluated, mainly for vitamins A and C. Furthermore, the associative effect of vitamins with several medicines demands studies. The evaluations of genotoxic effect of Prozac and vitamins A and C protective effect were carried out in culture of Chinese hamster ovary cells, CHO-K1, by means of the comet test. The Prozac was used, in liquid formulation, diluted in 5µg, 1µg and 0.2 µg/mL of culture medium. The vitamins were used, in liquid formulation, at the concentrations of 3µg and 880,5 µg/mL of culture medium to vitamins A and C, respectively. The treatments were carried out during 1 hour. The obtained data demonstrated that only the highest concentration of Prozac (5 µg is genotoxic and both vitamins A and C reduced such genotoxicity. The data suggest a follow-up on patients who use Prozac and the possibility of vitamins A and C association in order to minimize the collateral genotoxic effects.

  20. 长效人促卵泡激素CHO细胞株的构建及其体内活性%Expression of human long-acting FSH in CHO cell and its bioactivity in vivo

    Institute of Scientific and Technical Information of China (English)

    黄晓平; 王晓; 杨春雪; 贾东方; 林俊生; 刁勇

    2014-01-01

    促卵泡激素(FSH)是具有促进卵泡与睾丸发育作用的一种垂体糖蛋白激素.因其体内半衰期较短,临床上需要连续10d以上每天注射,病人使用非常不方便.本文旨在通过提高糖基化程度,研制一种长效FSH.通过一段含有两个N-糖基化位点的连接序列,将人FSHα链与β链cDNA融合,并插入pcDNA3.1(+)表达载体.表达载体转染CHO-K1细胞后,通过G418筛选得到阳性单克隆细胞,并经PCR和Western blotting证实.该重组FSH为单链蛋白,分子量约为49 kDa.经无血清培养,工程细胞株培养上清液中重组FSH的表达量可达3 mg/L.单次注射该重组FSH能够促进大鼠卵巢发育与卵泡成熟,且药效与连续8次注射Folltropin-V的相近.实验结果显示,本研究已成功获得一种长效重组FSH.%Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules.The relatively short half-life of FSH in vivo requires daily injections for more than 10 days that is inconvenient and possibly contribute to the stress perceived by the patients.The goal of the present study was to increase FSH glycosylation,in order to develop a long-acting recombinant FSH.The cDNA of native α and β subunit of human FSH was linked by a sequence with two N-linked glycosylation sites,and the resulted DNA was inserted into pcDNA3.1 vector to generate a recombinant vector of pcDNA3.1-FSH.The pcDNA3.1-FSH was linearized and transfected into CHO-K1,positive transformants were selected by G418 and confirmed by PCR and Western blotting.A single chain recombinant FSH was expressed,with molecular weight of about 49 kDa.The recombinant FSH expression level in CHO-K1 cell strain in serum-free culture was 3 mg/L.Single injection of this recombinant FSH could induce folliculogenesis and ovulation in rats,the efficacy was similar with the commercially available FSH preparation (Folltropin

  1. The Effect of UDP-glucuronosyltransferase 1A1 Expression on the Mutagenicity and Metabolism of the Cooked-Food Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4-5,b]pyridine in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Malfatti, M A; Wu, R W; Felton, J S

    2004-08-13

    UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies UGT1A1 has been implicated in the detoxification of certain food-borne-carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDPglucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neogene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of eleven clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52 kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining 4 clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10{sup 4}-10{sup 5} fold higher relative to the UV5P3 parental cells. One clone (No.14) had a 10 fold higher increase in expression at 1.47 x 10{sup 5} over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D{sub 50} values, the cytotoxic effect of PhIP was decreased {approx}350 fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition no significant increase in mutation frequency was observed in the

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

    International Nuclear Information System (INIS)

    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

  3. Establishment of a Stable CHO-677 Cell Line Expressing Murine αvβ6 Integrin%稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建

    Institute of Scientific and Technical Information of China (English)

    朱志坚; 连凯琪; 杨帆; 张伟; 郑海学; 杨孝朴

    2015-01-01

    口蹄疫是由口蹄疫病毒(FMDV)引起的一种高度接触性传染病,主要侵害偶蹄动物.乳鼠常作为一种重要的实验动物模型用于FMDV的研究;整联蛋白αvβ6是FMDV的重要受体之一.为深入研究整联蛋白αvβ6在FMDV感染乳鼠中所发挥的作用,克隆了乳鼠整联蛋白αvβ6的两个亚基,并将其导入中国仓鼠卵巢细胞(Chinese hamster ovary,CHO-677)基因组中,构建了稳定表达乳鼠整联蛋白αV和β6亚基的细胞系CHO-677-mαvβ6,并分别选用两种不同血清型的野生型FMDV毒株Asia1/HN/CHA/06和O/BY/CHA/2010感染细胞系来分析细胞系对FMDV的易感性.首先通过PCR和间接免疫荧光试验证明了细胞系中整联蛋白αvβ6在基因水平成功导入,在蛋白水平成功表达.然后,通过实时荧光定量RT-PCR检测病毒RNA拷贝数,并结合TCID50试验测定了代表毒株在两个细胞上的生长曲线.结果表明,与亲本细胞CHO-677相比,细胞系CHO-677-mαvβ6对FMDV更易感,从αvβ6的功能性上进一步验证了细胞系被成功构建.

  4. Effect of deficiencies in DNA repair on the toxicity of mitomycin C and porfiromycin to CHO cells under aerobic and hypoxic conditions.

    Science.gov (United States)

    Hughes, C S; Irvin, C G; Rockwell, S

    1991-02-01

    A wild type Chinese hamster cell line (AA8) and three repair-deficient sublines of AA8 (EM9, UV4, and UV5) were used to study the nature of the cytotoxic lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin under aerobic and hypoxic conditions. The sensitivities of the repair-deficient sublines to the drugs varied markedly: EM9 was similar to AA8, whereas UV4 was exquisitely sensitive and UV5 was of intermediate sensitivity. Moreover, both the relative toxicities of the two drugs and the relative toxicities of each drug under aerobic and hypoxic conditions varied for the different cell lines. These data suggest that there are differences in the spectra of toxic lesions produced by mitomycin and porfiromycin and that there are differences in the lesions produced by these drugs under aerobic and hypoxic conditions. PMID:1899798

  5. The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy

    KAUST Repository

    Jourdain, P.

    2013-12-11

    The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

  6. A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian;

    2015-01-01

    analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early...... phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed...

  7. Binding, distribution, and clearance of 3H-benzo(a)pyrene-7,8-diol-9,10-epoxide from chromosomes of CHO cells

    International Nuclear Information System (INIS)

    The purpose of this study was to determine if the binding of a chemical carcinogen was random both within and between chromosomes. Chinese hamster ovary cells were exposed to 3H-labeled +/- anti[G-3H]-benzo(a)pyrene-7,8-diol-9,10-epoxide (3H-BPDE) and serially harvested. Autoradiographs were prepared and the distribution of silver grains over metaphase chromosomes was determined. The 3H-BPDE was randomly distributed down the length of the chromosome. The 3H-BPDE was rapidly removed from the chromosomes with 6% of the initial label remaining at 9 h after exposure. The distribution of silver grains between chromatids was nonrandom at 3 and 6 h after exposure, suggesting a differential removal of 3H-BPDE. 4 references, 4 figures

  8. Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors.

    Science.gov (United States)

    Schweitzer, C; Kratzeisen, C; Adam, G; Lundstrom, K; Malherbe, P; Ohresser, S; Stadler, H; Wichmann, J; Woltering, T; Mutel, V

    2000-07-24

    The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors. PMID:10884552

  9. Dicty_cDB: CHO586 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO586 (Link to dictyBase) - - - Contig-U11594-1 | Contig-U15357-1 CHO586P ... vl**khlvinfcy*qn**inn*yinvstttkksktm*kycttwllsk ***mri *srintsattttttttttttttttttttttttttttttttkf--- ---lm ...

  10. Dicty_cDB: CHO806 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ylknihiiinkneri*knyii*ffsffnfmfcktisfr*pfnn fnnnnnsncttndigsinskgnnnnhnyritsinhnyrstninyrsininyrstiiiifi e*nnsncntn*ktcntnt...ive seq. ID CHO806P (Link to Original site) Representative DNA sequence >CHO806 (CHO806Q) /CSM/CH/CHO8-A/CHO...2.25 Homology vs DNA Score E Sequences producing significant alignments: (bits) V...0 |AC116960.2 Dictyostelium discoideum chromosome 2 map complement(1004496-821614) strain AX4, complete sequen...ce. 36 0.003 13 dna update 2005. 9.10 Homology vs Protein Score E Sequences producing significant alignment

  11. A proteomic study of cMyc improvement of CHO culture

    Directory of Open Access Journals (Sweden)

    Dunn Michael J

    2010-03-01

    Full Text Available Abstract Background The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. Results Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS. Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin, protein biosysnthesis (eIF6 and energy metabolism (ATP synthetase, and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion. Conclusion These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

  12. Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells%抗VEGFR-2全人源IgG1抗体的构建及其在CHO-k细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    李致科; 何远; 张娟; 解伟; 曹婉璐; 王泽根; 王旻

    2013-01-01

    本文在实验室构建的单链抗体-Fc融合抗体[scFv(AK404R)-Fc]的基础上构建抗VEGFR-2全人源IgG1样全长抗体(Mab-04).利用重叠PCR,获得Mab-04的轻链和重链的核酸序列后分别克隆到真核表达载体pcDNA3.1,获得重组质粒.脂质体法将重组质粒转染至CHO-k细胞,经ProteinA柱纯化细胞培养上清液获得目的蛋白,利用Western blotting检测目的蛋白,ELISA检测Mab-04与抗原亲和力.测序表明重组质粒构建成功,Westem blotting检测显示目的蛋白成功表达(1μg.mL-1),ELISA检测阐明该抗体能与抗原结合并呈浓度依赖性(IC50为50 nmol.L-1),表明Mab-04成功表达并正确装配,为进一步大量制备该抗体及其活性研究打下基础.

  13. Dicty_cDB: CHO734 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO734 (Link to dictyBase) - - - Contig-U16427-1 CHO734P (Link to Original ... yfg*ytlyiyiiq*nviffrtdfrkdgfhr*ry*ihgns*sck*irkryf*ngs ... nl*k*ncykiisins*fsk*csrkcc*mfrfin*ks*rftsnrny*yiik ... ixsi Homology vs CSM-cDNA Score E Sequences producing s ignificant alignments: (bits) Value CHO734 (CHO734 ... 004.12.25 Homology vs DNA Score E Sequences producing s ignificant alignments: (bits) Value N AC116956 |AC ... 9. 6 Homology vs Protein Score E Sequences producing s ignificant alignments: (bits) Value (Q86KD1) RecNa ...

  14. CHO immobilization in alginate/poly-l-lysine microcapsules: an understanding of potential and limitations

    OpenAIRE

    Breguet, Véronique; Gugerli, Raphaël; von Stockar, Urs; Marison, Ian William

    2007-01-01

    Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-l-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 μm liquid core microcapsules, (b) 500 μm liquid core microcapsules, (c) 880 μm liquid core microcapsules with a double PLL membrane and (d) 7...

  15. Dicty_cDB: CHO229 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO229 (Link to dictyBase) - - - Contig-U15356-1 | Contig-U15914-1 CHO229P ... a novel protein, a ribosomal protein L29 (RPL29) (HIP , HUMRPL29) pseudogene and a CpG island. 38 0.047 7 ...

  16. Dicty_cDB: CHO188 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO188 (Link to dictyBase) - - - Contig-U11087-1 CHO188P (Link to Original ... 2-28.g_054.ab1 Whole-genome shotgun library of the elephant ... shark (aka elephant ... fish) Callorhinchus milii geno ...

  17. Dicty_cDB: CHO410 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO410 (Link to dictyBase) - - - Contig-U11087-1 CHO410P (Link to Original ... 2-28.g_054.ab1 Whole-genome shotgun library of the elephant ... shark (aka elephant ... fish) Callorhinchus milii geno ...

  18. Dicty_cDB: CHO122 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO122 (Link to dictyBase) - - - Contig-U16351-1 CHO122P (Link to Original ... 3P4. 38 7e-04 12 DD041861 |DD041861.1 Light-driven energy ... generation using proteorhodopsin. 36 0.006 8 CR382 ...

  19. Dicty_cDB: CHO432 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO432 (Link to dictyBase) - - - Contig-U16524-1 CHO432P (Link to Original ... .1 Influenza A virus (swine/Cotes d'Armor/1515/99 (H1N1 )) partial N1NA gene for Neuraminidase, genomic RNA ...

  20. Dicty_cDB: CHO361 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO361 (Link to dictyBase) - G21912 DDB0185980 Contig-U11792-1 CHO361P (Lin ... gcy*krlattldesidiid*nlkdixht --- ---kg*kre*ke*nr*r*dft *rfngvfnvllkynhtitklyfq*yrcydtngiwyl*ls ngngytnlkev ...

  1. Dicty_cDB: CHO822 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO822 (Link to dictyBase) - - - Contig-U16405-1 CHO822P (Link to Original ... ikyc*glyi*cfgfsilsksiqfei**ktkniw*ryshywli fkiiwws*fpy *fh*ck*tfcc--- ---lqhslefvqdqvqh*pi*nq*fqkfqplkiklk ...

  2. Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-yan; SHI Wei-wei; WANG Hao; LI Bo-hua; YANG Yang; TAN Min; XUE Jing-ya; GUO Ya-jun

    2005-01-01

    Objective: To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results:rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

  3. Experimental verification for in vitro technique confirmation of bystander effect induced by gamma radiation in CHO-K1 cell line; Verificacao experimental para confirmacao da tecnica in vitro do efeito bystander induzido por radiacao gama na linhagem celular CHO-K1

    Energy Technology Data Exchange (ETDEWEB)

    Viana, P.H.L.; Goes, A.M.; Gomes, D.A., E-mail: pedroleroybio@hotmail.com [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento Bioquimica e Imunologia. Lab. de Imunologia Celular e Molecular; Grynberg, S.E., E-mail: seg@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2013-08-15

    The bystander effect refers to biological responses detected in cells not directly irradiated but influenced, somehow, by signals transmitted from neighboring irradiated cells. These biological responses include sister chromatid exchange, mutations, micronucleus formation, chromosomal aberrations, carcinogenesis, apoptosis and necrosis. Although its existence is unquestionable, the mechanisms involved on triggering the bystander effect are not yet completely elucidated. Previous studies have shown that the bystander effect depends on a large variety of parameters including the radiation dose, the dose rate, the type of radiation and type of cells or tissue. This study aims to confirm the technique previously used in the literature in human cell lines for the bystander effect verification. The results suggest that the working conditions adopted by the group show technical efficiency and enables the reproduction of the bystander effect. (author)

  4. The Products of the Thermal Decomposition of CH3CHO

    Energy Technology Data Exchange (ETDEWEB)

    Vasiliou, AnGayle; Piech, Krzysztof M.; Zhang, Xu; Nimlos, Mark R.; Ahmed, Musahid; Golan, Amir; Kostko, Oleg; Osborn, David L.; Daily, John W.; Stanton, John F.; Ellison, G. Barney

    2011-04-06

    We have used a heated 2 cm x 1 mm SiC microtubular (mu tubular) reactor to decompose acetaldehyde: CH3CHO + DELTA --> products. Thermal decomposition is followed at pressures of 75 - 150 Torr and at temperatures up to 1700 K, conditions that correspond to residence times of roughly 50 - 100 mu sec in the mu tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: VUV photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH3CHO, we have studied three isotopologues, CH3CDO, CD3CHO, and CD3CDO. We have identified the thermal decomposition products CH3(PIMS), CO (IR, PIMS), H (PIMS), H2 (PIMS), CH2CO (IR, PIMS), CH2=CHOH (IR, PIMS), H2O (IR, PIMS), and HC=CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH3CHO: Radical decomposition: CH3CHO + DELTA --> CH3 + [HCO] --> CH3 + H + CO Elimination: CH3CHO + DELTA --> H2 + CH2=C=O. Isomerization/elimination: CH3CHO + DELTA --> [CH2=CH-OH] --> HC=CH + H2O. Both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH2=C:, as an intermediate in the decomposition of vinyl alchohol: CH2=CH-OH + DELTA --> [CH2=C:] + H2O --> HC=CH + H2O.

  5. Dicty_cDB: CHO519 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO519 (Link to dictyBase) - - - Contig-U14432-1 - (Link to Original site) ... KXXFFFFFXKK--- Translated Amino Acid sequence (All Frames ) Frame A: fffxxfffffffffffffffffffff*kkkkkxxkkkkkk ...

  6. Dicty_cDB: CHO672 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO672 (Link to dictyBase) - - - Contig-U16426-1 - (Link to Original site) ... NNNIN*kkikk--- Translated Amino Acid sequence (All Frames ) Frame A: NNKIIITHSLIIIYRKKEKKEKRKKKKEKFLNNNIN*kki ...

  7. Dicty_cDB: CHO769 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO769 (Link to dictyBase) - - - Contig-U16371-1 CHO769P (Link to Original ... Oryzias latipes DNA, clone: ola1-209J09.R, genomic survey ... sequence. 54 0.004 1 DE086252 |DE086252.1 Oryzias ... latipes DNA, clone: ola1-188M19.F, genomic survey ... sequence. 54 0.004 1 DE075571 |DE075571.1 Oryzias ...

  8. Dicty_cDB: CHO558 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO558 (Link to dictyBase) - - - Contig-U16298-1 | Contig-U16523-1 CHO558P ... .10.25 Translated Amino Acid sequence *qykffkylfif*tnt *yfh*lif*YFQIKMIPKKQKGFNGEPKSHQEIDRSQSPLKQRT NNNNYQ ... Q YFQPRQTQNAWGKQNNSIPLEEQFQEVKPITQPQTPETTTTTTTTTTTTTNTTNT NLLVP RIS--- ---DMRGHSVVGFVATIDDKFQKYFSRSFVQKRPG ... VRVPATCQFAHKMAFLIGRTVFADAAPQ Frame B: *qykffkylfif*tnt *yfh*lif*YFQIKMIPKKQKGFNGEPKSHQEIDRSQSPLKQRT NNNNYQ ... Q YFQPRQTQNAWGKQNNSIPLEEQFQEVKPITQPQTPETTTTTTTTTTTTTNTTNT NLLVP RIS--- ---i*eviq*ldslqlsminfksisqdllfksvlv ...

  9. Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay

    International Nuclear Information System (INIS)

    Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125I decay accumulation, under conditions preventing repair, based on the observation that each 125I decay in the DNA produces approximately one dsb. Results suggest that that observed fluctuations in the fraction of DNA activity released (FAR) per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. (author)

  10. Differential mutant quantitation at the mouse lymphoma tk and CHO hgprt loci.

    Science.gov (United States)

    Moore, M M; Harrington-Brock, K; Doerr, C L; Dearfield, K L

    1989-09-01

    Recent reports by several laboratories indicate that not all non-essential target loci are equally capable of detecting chromosomal mutations. The present study was undertaken to determine if both the tk locus in mouse lymphoma cells and the hgprt locus in Chinese hamster ovary (CHO) cells can be used to quantitate chromosomal mutations. Seven known mutagens for the tk locus were selected. These compounds were evaluated in the mouse lymphoma assay and in a suspension adapted CHO assay for their mutagenicity. In addition to the specific locus mutagenesis analysis, mouse lymphoma and CHO cells were evaluated for the frequency of gross chromosome aberrations. From these investigations, it appears that only those compounds [2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl] aminopropylamino)-acridine-dihydrochloride (ICR 170), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)] that induce significant numbers of large-colony thymidine kinase (TK) mutants also induce significant numbers of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mutants. The four acrylates evaluated (methyl acrylate, ethyl acrylate, trimethylolpropane triacrylate and tetraethyleneglycol diacrylate) induced almost exclusively small-colony TK mutants and very few if any HGPRT mutants. Aberration analysis revealed that both the mouse lymphoma and CHO cells responded to the clastogenicity of the compounds (except for ICR 170 which was not positive in CHO cells) and that neither cell line was clearly more sensitive than the other to the clastogens tested. It is significant that the four acrylates give little or no evidence of genotoxicity when evaluated using selection for HGPRT-deficient mutants, yet are clearly clastogenic to the same cells in the same experiment. These results are consistent with the hypothesis that the hgprt locus may not be useful as a marker to evaluate the clastogenic component of a genotoxic compound. The present study adds to the increasing number of studies

  11. Determination of IGF-1-Producing CHO-K1 Growth Phases Using GCMS-Based Global Metabolite Analysis

    OpenAIRE

    S. E. M. SABERI; Y.Z.H-Y Hashim; V. Packeer Mohamad; M. Mel; R. Ahmad-Raus; and A. Amid

    2011-01-01

    Mammalian cell lines, in particular CHO-K1 is vital for the multibillion dollar biotechnology industry. The majority of large scale bioprocessing of commercially valuable protein biopharmaceuticals is produced using this type of cell. An ideal mammalian cell system as host for biologics production should retain efficient use of energy sources in order to boost productivity at minimum cost. Various analyses such as cell counting and monitoring of specific biochemical responses are used to prov...

  12. CHO immobilization in alginate/poly-L: -lysine microcapsules: an understanding of potential and limitations.

    Science.gov (United States)

    Breguet, Véronique; Gugerli, Raphaël; von Stockar, Urs; Marison, Ian William

    2007-04-01

    Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-L: -Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 mum liquid core microcapsules, (b) 500 mum liquid core microcapsules, (c) 880 mum liquid core microcapsules with a double PLL membrane and (d) 740 mum semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2-3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 x 10(7) cell mL(-1), corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (varphi = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization. PMID:19003193

  13. Study on the immuno-effects and influencing factors of Chinese Hamster Ovary (CHO) cell hepatitis B vaccine among adults, under different dosages%不同剂量国产重组酵母乙型肝炎疫苗成年人免疫效果及影响因素研究

    Institute of Scientific and Technical Information of China (English)

    张卫; 马建新; 张海燕; 王晨; 杨鹏; 李辉; 孙美平; 贺雄; 庞星火; 林长缨; 韩莉莉; 李立秋; 高培; 林晖; 龚晓红; 黄芳; 唐雅清

    2010-01-01

    Objective To evaluate the immuno-effect and related influencing factors on 10 μg and 20 μg Chinese hamster ovary (CHO) cell hepatitis B vaccine, using the randomized double-blind controlled trials in adult population. Methods A total of 642 adults aged 18-45 years old, non-vaccinated against hepatitis B, and negative on five blood indicators for hepatitis B, were selected as the study objects from four districts in Beijing. The study objects were randomly divided into two groups, and then accepted 10 tg and 20 μg recombinant CHO hepatitis B vaccination by 0-1-6 month schedule. Influencing factors were investigated by means of questionnaire. Blood samples were collected one month after the third dose of vaccination. Anti-HBs level was detected by Abott chemiluminescence detection method. For the anti-HBs negative person, fluorescent quantitative PCR method was used to find out if the person had been infected with HBV. Logistic regression analysis was used to find out the influencing factors of anti-HBs seroconversion on every studied subject. Results The anti-HBs seroconversion rates on 10 μg and 20 μg dose groups were 88.8%(95%CI: 85.4%-92.2%) and 95.3%(95%CI: 93.0%-97.6%)respectively. Taking the anti-HBs level<100 mIU/ml as the low/non-response standard, the low response and non-response rates were 34.3% and 17.4% respectively. The geometric mean titers(GMT)of anti-HBs were 173.42 mIU/ml for the 10 μg dose group and 588.51 mIU/ml for the 20 μg dose group. Data from the Multivariate analysis showed that: diabetes, spouses infected with hepatitis B virus and old age were unfavorable factors for anti-HBs Seroconversion. 20 μg dose of the vaccine was conducive to seroconversion.Conclusion 20 μg CHO hepatitis B vaccine seemed better than 10 μg CHO hepatitis B vaccine while many factors need to be taken into account for evaluation on hepatitis B vaccines.%目的 通过随机双盲对照试验,评价10μg和20μg国产重组酵母(CHO)乙型肝炎(乙肝)疫

  14. Tracking dipeptides at work-uptake and intracellular fate in CHO culture.

    Science.gov (United States)

    Sánchez-Kopper, Andres; Becker, Max; Pfizenmaier, Jennifer; Kessler, Christian; Karau, Andreas; Takors, Ralf

    2016-12-01

    Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like L-alanine-L-glutamine (AQ) or glycyl-L-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production. PMID:27447702

  15. Dicty_cDB: CHO796 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO796 (Link to dictyBase) - - - Contig-U15285-1 - (Link to Original site) ... CV462822 |CV462822.1 CS_hyp_49g10_M13Reverse Blue crab ... hypodermis, normalized Callinectes sapidus cDNA cl ...

  16. Dicty_cDB: CHO553 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO553 (Link to dictyBase) - - - Contig-U15936-1 - (Link to Original site) ... nsert in 3'HPRT insertion targeting and chromosome engineering ... clone MHPP373m12. 60 5e-06 2 AC116305 |AC116305.2 ...

  17. Dicty_cDB: CHO467 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO467 (Link to dictyBase) - - - Contig-U14004-1 - (Link to Original site) ... nsert in 3'HPRT insertion targeting and chromosome engineering ... clone MHPP354d09. 34 0.008 2 AZ589684 |AZ589684.1 ...

  18. Dicty_cDB: CHO266 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO266 (Link to dictyBase) - - - Contig-U16405-1 - (Link to Original site) ... *ikyc*glyi*cfgfsilsksiqfei**ktkniw*ryshywlifkiiwws*fpy * fh*ck*tfcc*r*fl*sif*l**yyc--- Frame B: slf*fsfiki ...

  19. Evaluation of genotoxic effect of prozac (fluoxetine) without and with addition of vitamins A and C by means of the comet assay in culture of CHO-K1 cells

    OpenAIRE

    Noélle Giacomini Lemos; Mário Sérgio Mantovani; Veronica Elisa Pimenta Vicentini

    2005-01-01

    The fluoxetine, commercially named Prozac, is efficient against depression and anxiety, with lower risk of collateral effects. However, the possible genotoxic effects are still unknown. The use of vitamins as protectors against damages on cells and DNA has been evaluated, mainly for vitamins A and C. Furthermore, the associative effect of vitamins with several medicines demands studies. The evaluations of genotoxic effect of Prozac and vitamins A and C protective effect were carried out in cu...

  20. CHO immobilization in alginate/poly-l-lysine microcapsules: an understanding of potential and limitations

    Science.gov (United States)

    Breguet, Véronique; Gugerli, Raphaël; von Stockar, Urs

    2007-01-01

    Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-l-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 μm liquid core microcapsules, (b) 500 μm liquid core microcapsules, (c) 880 μm liquid core microcapsules with a double PLL membrane and (d) 740 μm semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2–3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 × 107 cell mL-1, corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (ϕ = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization. PMID:19003193

  1. Degradable Dextran Nanopolymer as a Carrier for Choline Kinase (ChoK siRNA Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Zhihang Chen

    2016-02-01

    Full Text Available Although small interfering RNA (siRNA therapy has proven to be a specific and effective treatment in cells, the delivery of siRNA is a challenge for the applications of siRNA therapy. We present a degradable dextran with amine groups as an siRNA nano-carrier. In our nano-carrier, the amine groups are conjugated to the dextran platform through the acetal bonds, which are acid sensitive. Therefore this siRNA carrier is stable in neutral and basic conditions, while the amine groups can be cleaved and released from dextran platform under weak acid conditions (such as in endosomes. The cleavage and release of amine groups can reduce the toxicity of cationic polymer and enhance the transfection efficiency. We successfully applied this nano-carrier to deliver choline kinase (ChoK siRNA for ChoK inhibition in cells.

  2. Dicty_cDB: CHO244 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO244 (Link to dictyBase) - - - Contig-U16708-1 - (Link to Original site) ... AV065281 |AV065281.1 Mus musculus adult male small intestine ... cDNA, partial sequence, clone 2010015E09. 40 7.2 1 ... AV062105 |AV062105.1 Mus musculus adult male small intestine ... cDNA, partial sequence, clone 2010002D17. 40 7.2 1 ...

  3. The Products of the Thermal Decomposition of CH3CHO

    OpenAIRE

    Vasiliou, AnGayle

    2011-01-01

    We have used a heated 2 cm x 1 mm SiC microtubular (mu tubular) reactor to decompose acetaldehyde: CH3CHO + DELTA --> products. Thermal decomposition is followed at pressures of 75 - 150 Torr and at temperatures up to 1700 K, conditions that correspond to residence times of roughly 50 - 100 mu sec in the mu tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: VUV photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscop...

  4. Dicty_cDB: CHO559 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHO559 (Link to dictyBase) - - - Contig-U13910-1 - (Link to Original site) ... 6 (A8DZH4) RecName: Full=Xenotropic and polytropic retrovirus ... rece... 87 4e-16 AL844505_73( AL844505 |pid:none) ... 4 (Q9TU72) RecName: Full=Xenotropic and polytropic retrovirus ... rece... 81 2e-14 protein update 2009. 5.20 PSORT p ...

  5. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  6. Perspectives on UV light mutagenesis: Investigation of the CHO aprt gene carried on a retroviral shuttle vector

    International Nuclear Information System (INIS)

    The extent to which the cellular processing of shuttle vector-carried genes reflects that of endogenous chromosomal loci has been a subject of considerable controversy. In order to address this issue, we have developed a retroviral-based shuttle vector carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene stably integrated into the genome to be used for studying mutational specificity in mammalian cells. Initially, we have characterized a collection of UV-induced mutants in a CHO cell background. We have therefore been able to directly compare this shuttle vector data to that previously obtained for UV-induced mutation at the endogenous CHO (aprt) locus. Although some potential differences between the two spectra have been noted, there appears to be a remarkable similarity in the distribution and site specificity of UV-induced mutations. These similarities extend to extrachromosomal shuttle vectors as well and consolidate the role of shuttle vectors as powerful analytical tools for studying mechanisms of point mutagenesis in mammalian cells

  7. Determination of IGF-1-Producing CHO-K1 Growth Phases Using GCMS-Based Global Metabolite Analysis

    Directory of Open Access Journals (Sweden)

    S. E. M. SABERI

    2011-12-01

    Full Text Available Mammalian cell lines, in particular CHO-K1 is vital for the multibillion dollar biotechnology industry. The majority of large scale bioprocessing of commercially valuable protein biopharmaceuticals is produced using this type of cell. An ideal mammalian cell system as host for biologics production should retain efficient use of energy sources in order to boost productivity at minimum cost. Various analyses such as cell counting and monitoring of specific biochemical responses are used to provide data to enable bioprocess control in order to achieve the ideal system. Our study aimed to see whether global metabolite analysis using Gas Chromatography Mass Spectrometry (GCMS would be a potential alternative approach in providing data for bioprocess control. In this study, we analyzed metabolites of CHO-K1 cells at different growth phases using GCMS. CHO-K1 cells producing insulin like growth factor-I (IGF1 were obtained from ATCC. Cells were grown in T-flask and incubated at 37°C/ 5% CO2 until 70-80% confluent in RPMI 1640 media. Samples (cells and spent/conditioned media were taken at designated intervals for routine cell counting (Trypan Blue dye exclusion method; glucose, glutamine and lactate determination (YSI 2700; IGF-1 production (ELISA kit R&D Sstems, Inc; and global metabolite analysis (GCMS. Conditioned media from each time point were spun down before subjecting into GCMS. Data from GCMS was then transferred to SIMCA P+12.0 for chemometric evaluation using Principal Component Analysis (PCA. The first component, PC1 results was able to explain 36% of the variation of the data with clear separation between exponential phase and other phases (initial and death phase. This suggests that GCMS-based global metabolite analysis has the ability to capture cell growth behaviour and offered insights of factors that may influence the biological system.ABSTRAK: Produk yang berupa sel kekal mamalia, terutamnya CHO-K1 adalah penting dan menguntungkan

  8. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    Science.gov (United States)

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. PMID:26676266

  9. Measurements of the absorption cross section of (13)CHO(13)CHO at visible wavelengths and application to DOAS retrievals.

    Science.gov (United States)

    Goss, Natasha R; Waxman, Eleanor M; Coburn, Sean C; Koenig, Theodore K; Thalman, Ryan; Dommen, Josef; Hannigan, James W; Tyndall, Geoffrey S; Volkamer, Rainer

    2015-05-14

    The trace gas glyoxal (CHOCHO) forms from the atmospheric oxidation of hydrocarbons and is a precursor to secondary organic aerosol. We have measured the absorption cross section of disubstituted (13)CHO(13)CHO ((13)C glyoxal) at moderately high (1 cm(-1)) optical resolution between 21 280 and 23 260 cm(-1) (430-470 nm). The isotopic shifts in the position of absorption features were found to be largest near 455 nm (Δν = 14 cm(-1); Δλ = 0.29 nm), whereas no significant shifts were observed near 440 nm (Δν DOAS) in a series of sensitivity tests using synthetic spectra, and laboratory measurements of mixtures containing (12)C and (13)C glyoxal, nitrogen dioxide, and other interfering absorbers. We find the changes in apparent spectral band shapes remain significant at the moderately high optical resolution typical of CE-DOAS (0.55 nm fwhm). CE-DOAS allows for the selective online detection of both isotopes with detection limits of ∼200 pptv (1 pptv = 10(-12) volume mixing ratio), and sensitivity toward total glyoxal of few pptv. The (13)C absorption cross section is available for download from the Supporting Information. PMID:25551419

  10. Effects of Supplementation of Various Medium Components on Chinese Hamster Ovary Cell Cultures Producing Recombinant Antibody

    OpenAIRE

    Kim, Do Yun; Lee, Joon Chul; Chang, Ho Nam; Oh, Duk Jae

    2005-01-01

    Thirteen vitamins, twenty amino acids, hormones, inorganic salts, and other chemical agents, which constitute typical serum-free media, were evaluated for the development of fortified medium to enhance cell growth and productivity of recombinant antibody in the cultures of the recombinant Chinese hamster ovary (rCHO) cells. Two different rCHO cell lines, rCHO-A producing recombinant antibodies against the human platelet and rCHO-B secreting recombinant antibodies against the S surface antigen...

  11. Dicty_cDB: CHO546 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available kkpikixkm Frame B: sklmenfqdplihilcvalvitvilsfvgyaewfegvgiasavflatfvstyseyknens fqelqekasrvkcnvfrngshisevygf...8 5.2 2 CN880849 |CN880849.1 010305AASA001459HT (AASA) Royal Gala 10 DAFB fruit Malus x domestica cDNA clone...D CHO546 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15543-1 Original sit...icus clone CH230-58P22, WORKING DRAFT SEQUENCE, 1 ordered piece. 42 1.5 6 AE014818 |AE014818.1 Plasmodium fal...a: 0.00 m1b: 0.00 m2 : 0.00 mNt: 0.00 m3a: 0.00 m3b: 0.00 m_ : 1.00 48.0 %: cytoplasmic 36.0 %: nuclear 4.0 %: mitochondrial

  12. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

    OpenAIRE

    Karentz, D; Cleaver, J.E.

    1986-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Cont...

  13. Man Made: Seung Hui Cho and the Deconstruction of Asian American Masculinity and Violence

    OpenAIRE

    Rhee, Margaret

    2008-01-01

    Within hours of the Virginia Tech tragedy, the late Seung Hui Cho identified as the school shooter, became the most “famous” Asian American with his images, digital films, and stories on every website, television news program, and radio segment transnationally. While mainstream newspaper articles on Cho provoked specific discourse around mental health, gun control, and ethnicity; a critical lens of race, sexuality, and ethnicity was seemingly absent from the media blitz. Analysis of mainstre...

  14. Interview with Dr. Seokhee CHO About Gifted Education and Its Future

    OpenAIRE

    Hüseyin MERTOL

    2014-01-01

    The purpose of this study, which is a major name in the education of gifted Dr. Cho 's about gifted education is to put forward their views. Dr. Seokhee Cho is a Professor at the School of Education, St. John’s University. She is currently conducting three research projects funded by US Department of Education: Project HOPE as a Principal Investigator, Project WIN and Project LEADER as a research director.

  15. CHO: A Benchmark Suite for OpenCL-based FPGA Accelerators

    OpenAIRE

    Ndu, Geoffrey; Lujan, Mikel; Navaridas, Javier

    2014-01-01

    Programming FPGAs with OpenCL-based high-level synthesis frameworks is gaining attention with a number of commercial and research frameworks announced. However, there are no benchmarks for evaluating these frameworks. To this end, we present CHO benchmark suite an extension of CHStone, a commonly used C-based high-level synthesis benchmark suite, for OpenCl. We characterise CHO at various levels and use it to investigate compiling non-trivial software to FPGAs.

  16. Cultivos de células CHO-K1

    Directory of Open Access Journals (Sweden)

    M.C. Nóvoa-Valiñas

    2005-01-01

    Full Text Available El uso de determinados metales pesados y pesticidas es la estrategia más empleada para el control de plagas. Estas sustancias, una vez aplicadas a los cultivos, pueden pasan al medio ambiente, permaneciendo en él como xenobióticos que van a afectar, en mayor o menor medida, a los seres vivos. En el presente estudio se ha evaluado la toxicidad basal de un metal, cobre, y un pesticida organoclorado, lindano, así como mezclas de ambos a distintas concentraciones. Para llevar a cabo este trabajo se ha utilizado la línea celular CHO-K1 (células epiteliales de ovario de hamster, usándose como criterio de citotoxicidad la muerte celular, determinada mediante la técnica del rojo neutro. Las concentraciones iniciales fueron: 0,03; 0,06 y 0,9 mM de cobre y 0,01; 0,03 y 0,1 mM de lindano. Y en las mezclas, las concentraciones estuvieron comprendidas entre 0,01-0,9 de cobre y 0,001-0,1de lindano. Como resultados, la citotoxicidad del cobre y lindano fue dosis-dependiente. En las exposiciones a mezclas se observa que a concentraciones fijas de lindano, la viabilidad desciende al aumentar la concentración de cobre, mientras que, dentro de un cierto rango, a concentraciones fijas de cobre, la viabilidad celular se incrementa al aumentar la concentración de lindano

  17. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang; Nagarajan, Harish; Yerganian, George; O'Brien, Edward; Bordbar, Aarash; Roth, Anne M; Rosenbloom, Jeffrey; Bian, Chao; Xie, Min; Chen, Wenbin; Li, Ning; Baycin-Hizal, Deniz; Latif, Haythem; Förster, Jochen; Betenbaugh, Michael; Famili, Iman; Xu, Xun; Wang, Jun; Palsson, Bernhard O

    2013-01-01

    Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been...... stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This...... analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this...

  18. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan; 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; Zhang, Hui; Betenbaugh, Michael

    2012-01-01

    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......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...... 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...

  19. Transient recombinant protein expression in mammalian cells: the role of mRNA level and stability

    OpenAIRE

    Wulhfard, Sarah

    2009-01-01

    Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt wa...

  20. ERCC2/XPD基因缺失对短波紫外线所致CHO细胞DNA损伤修复的影响%Study on the effect of deficient ERCC2/XPD gene on the repair of DNA damage induced by UVC in CHO cell line

    Institute of Scientific and Technical Information of China (English)

    关阳阳; 肖莎; 李丹丹; 薛萍; 张国培; 刘秋芳; 靳翠红; 杨敬华; 巫生文

    2014-01-01

    目的 探讨核苷酸切除修复交叉互补基因2 (excision repair cross complementation group 2/Xeroderma pigmentosum D,ERCC2/XPD)在短波紫外线(UCC)所致细胞DNA损伤与修复过程中的作用.方法 采用中国仓鼠卵巢细胞系(CHO)野生型AA8及ERCC2蛋白表达缺失型UV5构建细胞对照模型.MTT法比较两种细胞经UVC处理后细胞抑制率的差别;彗星试验与Rad51免疫荧光试验检测经不同照射强度的UVC处理后修复1、3、6和24 h,不同细胞对UVC所致DNA损伤修复能力的差异.结果 与野生型AA8相比,UV5对UVC所致DNA损伤更加敏感,细胞存活率降低(P<0.05).彗星试验和Rad51免疫荧光试验结果显示,UV5细胞DNA损伤程度较AA8严重,并且修复UVC所致DNA损伤的能力降低(P<0.05).结论 ERCC2/XPD基因缺失细胞DNA损伤修复能力下降,说明ERCC2/XPD修复酶在UVC所致DNA损伤的切除修复过程中发挥重要作用.

  1. Gas phase UV and IR absorption spectra of CxF2x+1CHO (x=1-4)

    DEFF Research Database (Denmark)

    Hashikawa, Y; Kawasaki, M; Waterland, RL;

    2004-01-01

    The UV and IR spectra of CxF2x+1 CHO (x = 1-4) were investigated using computational and experimental techniques. CxF2x+1CHO (x = 1-4) have broad UV absorption features centered at 300-310 nm. The maximum absorption cross-section increases significantly and shifts slightly to the red with increas...

  2. A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, R.; Verma, P.; Deobagkar, D.

    % Composition of amino acid from amino acid sequence of xylanase enzyme from Bacillus subtilis Cho40 Amino acid composition Alanine (Ala (A) 15 7.1% Arginine (Arg) (R) 6 2.8% Asparagine (Asn) (N) 19 9.0% Aspartic acid (Asp) (D...

  3. Radiation enhancement of the efficiency of DNA-mediated gene transfer in CHO UV-sensitive mutants

    International Nuclear Information System (INIS)

    We have employed the Chinese hamster ovary (CHO) UV-sensitive mutant cell lines, UV5 and UV20, to determine whether ionizing and ultraviolet irradiation enhance the efficiency of DNA-mediated gene transfer in cells deficient in excision repair. Confluent AA8 (wild type), UV5, and UV20 cells were transfected (via polybrene and dimethyl sulfoxide treatments) with the recombinant DNA plasmid, pSV2-gpt, trypsinized, irradiated with either X rays or ultraviolet in suspension, and then plated into flasks. After a 48-h expression time, cells were trypsinized, counted, and plated in XMAT media to select for pSV2-gpt transformation. We report that X-ray irradiation enhances gene transfer in wild-type AA8 and in both UV-sensitive cell lines. Ultraviolet irradiation enhances gene transfer in AA8 and UV20, but not in UV5. Since both UV20 and UV5 are deficient in excision repair, we suggest that ultraviolet-enhanced gene transfer may involve a postreplication repair mechanism deficient in UV5

  4. Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2.

    Science.gov (United States)

    Siebert, Nikolai; Eger, Christin; Seidel, Diana; Jüttner, Madlen; Zumpe, Maxi; Wegner, Danilo; Kietz, Silke; Ehlert, Karoline; Veal, Gareth J; Siegmund, Werner; Weiss, Michael; Loibner, Hans; Ladenstein, Ruth; Lode, Holger N

    2016-04-01

    Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients. PMID:26785755

  5. Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression

    OpenAIRE

    Haredy, Ahmad M.; Nishizawa, Akitoshi; Honda, Kohsuke; Ohya, Tomoshi; Ohtake, Hisao; Omasa, Takeshi

    2013-01-01

    To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host c...

  6. Pyrolysis of the Simplest Carbohydrate, Glycolaldehyde (CHO-CH2OH), and Glyoxal in a Heated Microreactor.

    Science.gov (United States)

    Porterfield, Jessica P; Baraban, Joshua H; Troy, Tyler P; Ahmed, Musahid; McCarthy, Michael C; Morgan, Kathleen M; Daily, John W; Nguyen, Thanh Lam; Stanton, John F; Ellison, G Barney

    2016-04-14

    Both glycolaldehyde and glyoxal were pyrolyzed in a set of flash-pyrolysis microreactors. The pyrolysis products resulting from CHO-CH2OH and HCO-CHO were detected and identified by vacuum ultraviolet (VUV) photoionization mass spectrometry. Complementary product identification was provided by argon matrix infrared absorption spectroscopy. Pyrolysis pressures in the microreactor were about 100 Torr, and contact times with the microreactors were roughly 100 μs. At 1200 K, the products of glycolaldehyde pyrolysis are H atoms, CO, CH2═O, CH2═C═O, and HCO-CHO. Thermal decomposition of HCO-CHO was studied with pulsed 118.2 nm photoionization mass spectrometry and matrix infrared absorption. Under these conditions, glyoxal undergoes pyrolysis to H atoms and CO. Tunable VUV photoionization mass spectrometry provides a lower bound for the ionization energy (IE)(CHO-CH2OH) ≥ 9.95 ± 0.05 eV. The gas-phase heat of formation of glycolaldehyde was established by a sequence of calorimetric experiments. The experimental result is ΔfH298(CHO-CH2OH) = -75.8 ± 1.3 kcal mol(-1). Fully ab initio, coupled cluster calculations predict ΔfH0(CHO-CH2OH) of -73.1 ± 0.5 kcal mol(-1) and ΔfH298(CHO-CH2OH) of -76.1 ± 0.5 kcal mol(-1). The coupled-cluster singles doubles and noniterative triples correction calculations also lead to a revision of the geometry of CHO-CH2OH. We find that the O-H bond length differs substantially from earlier experimental estimates, due to unusual zero-point contributions to the moments of inertia. PMID:26979134

  7. Proteomic analysis of Chinese hamster ovary cells.

    Science.gov (United States)

    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

  8. Some Applications of Differential Subordination of p-Valent Functions Associated with Cho-Kwon-Srivastava Operator

    Institute of Scientific and Technical Information of China (English)

    A. O. MOSTAFA; M.K. AOUF

    2009-01-01

    Making use of the principle of differential subordination, we investigate some inclusion relationships of certain subclasses of p-valent analytic functions which are defined by Cho-Kwon-Srivastava Operator.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael;

    2015-01-01

    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 t...

  11. Direct Dynamics Study on CH2O + CH·3 → CHO + CH4 Reaction

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    It is still a formidable challenge to study CH2O + CH·3 → CHO + CH4 reaction in the gas phase by traditional dynamics, because of the large number of freedom degrees for the system.In this paper, direct dynamics, in which trajectories were run directly on the DFT potential energy surface, have been applied to the reaction, which gave a direct look in the reaction processes.Two sets of trajectories at different initial orientations of reactants and temperature have been simulated. And the detailed reaction mechanisms have been described.

  12. Citotoxicidad del fungicida mancozeb en cultivos de CHO-K1

    OpenAIRE

    A.E. Bayoumi; Ordóñez, C.; Y. Pérez Pertejo; R. Balaña Fouce; Ordóñez, D.

    2002-01-01

    Se ha determinado la citotoxicidad del fungicida ditiocarbámico mancozeb, en cultivos celulares de ovario de hámster (CHO-K1), usando los bioensayos estandarizados de incorporación de rojo neutro (RN) y del contenido total de proteínas (PT). Las dos técnicas mostraron ser comparables en la determinación del efecto citotóxico, mostrando valores de RN50 menores de 15 mg/ml después de 24 h de exposición al plaguicida. La citotoxicidad fue mayor cuanto mayor fue el tiempo ...

  13. Design, synthesis, crystallization and biological evaluation of new symmetrical biscationic compounds as selective inhibitors of human Choline Kinase α1 (ChoKα1)

    Science.gov (United States)

    Schiaffino-Ortega, Santiago; Baglioni, Eleonora; Mariotto, Elena; Bortolozzi, Roberta; Serrán-Aguilera, Lucía; Ríos-Marco, Pablo; Carrasco-Jimenez, M. Paz; Gallo, Miguel A.; Hurtado-Guerrero, Ramon; Marco, Carmen; Basso, Giuseppe; Viola, Giampietro; Entrena, Antonio; López-Cara, Luisa Carlota

    2016-03-01

    A novel family of compounds derivative of 1,1‧-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bispyridinium or –bisquinolinium bromide (10a-l) containing a pair of oxygen atoms in the spacer of the linker between the biscationic moieties, were synthesized and evaluated as inhibitors of choline kinase against a panel of cancer-cell lines. The most promising compounds in this series were 1,1‧-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1‧-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before.

  14. Differential cytotoxic effects of arsenic on human and animal cells.

    OpenAIRE

    Lee, T C; Ho, I C

    1994-01-01

    Human fibroblasts (HFW) were 10-fold more susceptible than Chinese hamster ovary (CHO-K1) cells to sodium arsenite. Comparison of cellular antioxidant enzyme activities showed that CHO-K1 cells contained 3- and 8-fold more glutathione-peroxidase and catalase activities, respectively, than HFW cells. Since vitamin E, methylamine, and benzyl alcohol could prevent, in part, the arsenite-induced killing of HFW cells, we suggest that arsenite can induce oxidative damage in HFW cells. We have also ...

  15. Cross-cultural adaptation of the CHO-KLAT for boys with hemophilia in rural and urban china

    Directory of Open Access Journals (Sweden)

    Wu Runhui

    2012-09-01

    Full Text Available Abstract Background Quality of life (QoL is increasingly recognized as an important outcome measure in clinical trials. The Canadian Hemophilia Outcomes-Kids Life Assessment Tool (CHO-KLAT shows promise for use in China. Objective To adapt the CHO-KLAT version 2.0 for use in clinical trials in China. Methods Forward and back translations of the CHO-KLAT2.0 were completed in 2008. Between October 2009 and June 2010, a series of 3 focus groups were held with 20 boys and 31 parents in rural and urban China to elicit additional concepts, important to their QoL, for the Chinese CHO-KLAT2.0. All of the items identified by boys and parents were reviewed by a group of experts, resulting in a Chinese version of the CHO-KLAT2.0. This version underwent a detailed cognitive debriefing process between October 2010 and June 2011. Thirteen patient-parent pairs participated in this cognitive debriefing process until a stable and clearly understood Chinese version of the CHO-KLAT2.0 was obtained. Results The initial back translation of the Chinese CHO-KLAT2.0 was slightly discrepant from the original English version on 12 items. These were all successfully adjudicated. The focus groups identified 9 new items that formed an add-on Socio-Economic Context (SEC module for China. Linguistic improvements were made after the 2nd, 5th, 7th and 13th cognitive debriefings pairs and affected a total of 18 items. The result was a 35 item CHO-KLAT2.0 and a SEC module in Simplified Chinese, both of which have good content validity. Conclusion This detailed process proved to be extremely valuable in ensuring the items were accurately interpreted by Chinese boys with hemophilia ages ≤18 years. The need for the additional SEC module highlighted the different context that currently exists in China with regard to hemophilia care as compared to many Western countries, and will be important in tracking progress within both rural and urban China over time. Changes based on the

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

    OpenAIRE

    Ye Zhang; Véronique Chotteau

    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.

  17. H-atom bombardment of CO2, HCOOH and CH3CHO containing ices

    CERN Document Server

    Bisschop, S E; Van Dishoeck, E F; Linnartz, H

    2007-01-01

    Context: Hydrogenation reactions are expected to be among the most important surface reactions on interstellar ices. However, solid state astrochemical laboratory data on reactions of H-atoms with common interstellar ice constituents are largely lacking. Aims: The goal of our laboratory work is to determine whether and how carbon dioxide (CO2), formic acid (HCOOH) and acetaldehyde (CH3CHO) react with H-atoms in the solid state at low temperatures and to derive reaction rates and production yields. Methods: Pure CO2, HCOOH and CH3CHO interstellar ice analogues are bombarded by H-atoms in an ultra-high vacuum experiment. The ices are monitored by reflection absorption infrared spectroscopy and the reaction products are detected in the gas phase through temperature programmed desorption to determine the destruction and formation yields as well as the corresponding reaction rates. Results: Within the sensitivity of our set-up we conclude that H-atom bombardment of pure CO2 and HCOOH ice does not result in detecta...

  18. Novel Stable Compounds in the C-H-O Ternary System at High Pressure.

    Science.gov (United States)

    Saleh, Gabriele; Oganov, Artem R

    2016-01-01

    The chemistry of the elements is heavily altered by high pressure, with stabilization of many new and often unexpected compounds, the emergence of which can profoundly change models of planetary interiors, where high pressure reigns. The C-H-O system is one of the most important planet-forming systems, but its high-pressure chemistry is not well known. Here, using state-of-the-art variable-composition evolutionary searches combined with quantum-mechanical calculations, we explore the C-H-O system at pressures up to 400 GPa. Besides uncovering new stable polymorphs of high-pressure elements and known molecules, we predicted the formation of new compounds. A 2CH4:3H2 inclusion compound forms at low pressure and remains stable up to 215 GPa. Carbonic acid (H2CO3), highly unstable at ambient conditions, was predicted to form exothermically at mild pressure (about 1 GPa). As pressure rises, it polymerizes and, above 314 GPa, reacts with water to form orthocarbonic acid (H4CO4). This unexpected high-pressure chemistry is rationalized by analyzing charge density and electron localization function distributions, and implications for general chemistry and planetary science are also discussed. PMID:27580525

  19. Metabolism Kinetics of Glucose in Anchorage-dependent Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    孙祥明; 张元兴

    2001-01-01

    The kinetic model of glucose metabolism was established and successfully applied to batchcultures of rCHO and rBHK cells. It was found that a large amount of glucose was utilized for cellmaintenance, and the overwhelming majority of maintenance energy from glucose was by its anaerobicmetabolism in both rBHK and rCHO cell cultures. The overall maintenance coefficients from aerobicmetabolism were 1.9×10-13 mmol/(cell.h) for rCHO cells and 7×10-13 mmol/(cell.h) for rBHK cells. Inaddition, all Go/T and Eo/T gradually increased with the same trend as the cell growth in the culture ofboth rCHO and rBHK cells. The overall molecule yield coefficients of lactate to glucose were 1.61 for rCHO cells and 1.38 for rBHK cells. The yield coefficients of cell to glucose were 4.5×108 cells/mmol for rCHO cells and 1.9 × 108 cells/mmol for rBHK cells, respectively.

  20. Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells

    OpenAIRE

    Hornigold, David C; Mistry, Rajendra; Raymond, Pamela D; Blank, Jonathan L; John Challiss, R A

    2003-01-01

    We have examined possible mechanisms of cross-talk between the Gq/11-linked M3 muscarinic acetylcholine (mACh) receptor and the Gi/o-linked M2 mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P3) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, ex...

  1. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin; Influencia do hospedeiro (Cho) e da estrategia de cultivo nas estruturas glicidicas e propriedades moleculares da tireotrofina humana

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Joao Ezequiel de

    2007-07-01

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of {approx} 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO{sub 2} conditions from 5% CO{sub 2} to air environment (0.03% CO{sub 2}). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen{sup R}) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing {approx} 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO{sub 2} and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 {+-} 10% was in the {alpha} 2,6 and 32 {+-} 10% in the {alpha}2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5

  2. CHO cell death, strand break damage, and repair due to combination radiation and hyperthermia

    International Nuclear Information System (INIS)

    Previous reports have suggested a relationship between the hyperthermia induced changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity. In this investigation, the level of initial strand break damage, DNA strand rejoining kinetics, DNA/protein ratios, and residual DNA damage were measured following combined hyperthermia and radiation treatments in an attempt to further understand these relationships

  3. Differential phospholipid-labeling suggests two subtypes of phospholipase D in rat Leydig cells

    DEFF Research Database (Denmark)

    Lauritzen, L.; Hansen, Harald S.

    1995-01-01

    Cho). The [H] phosphatidylethanol formation in response to 4ß-phorbol 12-myristate 13-acetate (PMA), sphingosine, or Ca-ionophore A23187, was lower when Leydig cells were labeled with 1-O-[H]alkyl lysoPtdCho compared with the responses when [H]myristic acid was employed. In contrast, the results...

  4. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin

    International Nuclear Information System (INIS)

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of ∼ 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO2 conditions from 5% CO2 to air environment (0.03% CO2). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (ThyrogenR) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing ∼ 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 ± 10% was in the α 2,6 and 32 ± 10% in the α2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39 - 7.35 pl range. A considerably different distribution

  5. Kinetic differences between fed and starved Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    When Chinese hamster ovary (CHO) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. A manuscript has recently been published which draws attention to the important, and perhaps unexpected, differences in the kinetic status of different types of plateau-phase CHO cells (Nelson, Todd and Metting 1984). The essence of this manuscript and the most significant findings are summarized in this paper

  6. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

    OpenAIRE

    Rosenwald, A G; Stanley, P; Krag, S S

    1989-01-01

    A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Sta...

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

    Science.gov (United States)

    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. Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event

    OpenAIRE

    1989-01-01

    Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate ...

  9. Report on energy saving vision in Santo-cho region; Santocho chiiki sho energy vision hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-02-01

    An energy-saving vision was decided on in Santo-cho region in Hyogo Prefecture, with its outline reported. This town is such that about 80% of the region is mountains, forests and wilderness and that aging is advancing at the rate above that of Hyogo Prefecture or the national average. Nearly entire energy of the town is dependent on the supply from outside. The energy consumption is somewhat increasing as a whole, with that of the people's livelihood/domestic sector and of transportation sector are rising. In the classification of fuels, electricity is growing in consumption. As an energy-saving vision, it aimed principally at personal surroundings in which every one got into the habit of saving energy continuously without being forced. The basic plan for the energy conservation drive consisted of inducement to an energy-saving life style, energy conservation to be spread by the next generation children, continuation of energy saving activity rooted in the region, and promotion of energy conservation as a basis for introducing new energy. The diffusion and enlightenment for children destined to lead the next generation were defined as a particularly important assignment, as was the promotion of energy conservation and environmental education. (NEDO)

  10. The CH...O hydrogen bonds in biodegradable polyhydroxyalkanoate studied by Raman and infrared spectroscopy and quantum chemical calculation

    Czech Academy of Sciences Publication Activity Database

    Sato, H.; Dybal, Jiří; Murakami, R.; Hirose, F.; Senda, K.; Noda, I.

    Gold Coast : Queensland University of Technology, 2004, 051/1-051/2. ISBN 0-643-09122-X. [International Conference on Raman Spectroscopy. Gold Coast (AU), 08.08.2004-13.08.2004] R&D Projects: GA AV ČR KSK4050111 Institutional research plan: CEZ:AV0Z4050913 Keywords : CH...O interactions * infrared spectroscopy * quantum chemical calculation Subject RIV: CD - Macromolecular Chemistry

  11. Regulation of glucose transport and c-fos and egr-1 expression in cells with mutated or endogenous growth hormone receptors

    DEFF Research Database (Denmark)

    Gong, T W; Meyer, D J; Liao, J;

    1998-01-01

    To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cel...

  12. Understanding and Controlling Sialylation in a CHO Fc-Fusion Process.

    Science.gov (United States)

    Lewis, Amanda M; Croughan, William D; Aranibar, Nelly; Lee, Alison G; Warrack, Bethanne; Abu-Absi, Nicholas R; Patel, Rutva; Drew, Barry; Borys, Michael C; Reily, Michael D; Li, Zheng Jian

    2016-01-01

    A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability. PMID:27310468

  13. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    International Nuclear Information System (INIS)

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia

  14. Task-based detectability comparison of exponential transformation of free-response operating characteristic (EFROC) curve and channelized Hotelling observer (CHO)

    Science.gov (United States)

    Khobragade, P.; Fan, Jiahua; Rupcich, Franco; Crotty, Dominic J.; Gilat Schmidt, Taly

    2016-03-01

    This study quantitatively evaluated the performance of the exponential transformation of the free-response operating characteristic curve (EFROC) metric, with the Channelized Hotelling Observer (CHO) as a reference. The CHO has been used for image quality assessment of reconstruction algorithms and imaging systems and often it is applied to study the signal-location-known cases. The CHO also requires a large set of images to estimate the covariance matrix. In terms of clinical applications, this assumption and requirement may be unrealistic. The newly developed location-unknown EFROC detectability metric is estimated from the confidence scores reported by a model observer. Unlike the CHO, EFROC does not require a channelization step and is a non-parametric detectability metric. There are few quantitative studies available on application of the EFROC metric, most of which are based on simulation data. This study investigated the EFROC metric using experimental CT data. A phantom with four low contrast objects: 3mm (14 HU), 5mm (7HU), 7mm (5 HU) and 10 mm (3 HU) was scanned at dose levels ranging from 25 mAs to 270 mAs and reconstructed using filtered backprojection. The area under the curve values for CHO (AUC) and EFROC (AFE) were plotted with respect to different dose levels. The number of images required to estimate the non-parametric AFE metric was calculated for varying tasks and found to be less than the number of images required for parametric CHO estimation. The AFE metric was found to be more sensitive to changes in dose than the CHO metric. This increased sensitivity and the assumption of unknown signal location may be useful for investigating and optimizing CT imaging methods. Future work is required to validate the AFE metric against human observers.

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

    Science.gov (United States)

    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

  16. Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

    OpenAIRE

    Parham, Janet H.; Iannone, Marie A.; Overton, Laurie K.; Hutchins, Jeff T.

    1998-01-01

    The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and ...

  17. Women in church and society: Report of research done by a research team at the PU vir CHO

    Directory of Open Access Journals (Sweden)

    F.J. van Rensburg

    2002-08-01

    Full Text Available The research project “Women in Church and Society” was conducted under the auspices of one of the focus areas for research and postgraduate education at the Potchefstroomse Universiteit vir Christelike Hoër Onderwys: “Reformed Theology and the Development of the South African Society”. This focus area is based in the Faculty of Theology (PU vir CHO and is directed by Herrie van Rooy. Project 2 of this focus area is “The socio-historic context of the Bible and its implications for the development of South African Society” and is under the leadership of Fika J. van Rensburg. The first sub-project of Project 2 to be completed is “Women in Church and Society”. It commenced in 2000 and had its fourth and final workshop in September 2002. It was managed by a five-person executive committee and had the following categories of collaborators: 16 PU vir CHO researchers, 10 researchers from other South African universities, 6 international researchers, 19 masters’ and doctoral students, and 21 researchers with special expertise in relevant areas. In total 48 papers1 were read and discussed at the four workshops; and most of them have either been published or are in the process of being published as articles in accredited journals. This article is a report on the activities and outcome of the research project.

  18. Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

    International Nuclear Information System (INIS)

    Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results reported for human cells, UV irradiation of transfecting DNA did not stimulate genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. Transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. The authors conclude responses of recipient cells to UV-damaged transfecting plasmids depend on type of recipient cell and characteristics of the genetic sequence used for transfection. (author)

  19. Reproductive and interphase death of CHO fibroblasts after X-ray microbeam irradiation

    International Nuclear Information System (INIS)

    Subject of the investigation was the topologic differentiation in the cell of the radiosensitive sites for production of cell death. Circular radiation cross sections of alternatively 27, 105 and 275 μm in diameter were centered to the nucleus, the cell or one to three cells respectively. 0.07 mmAl filtered 50 kVp X-rays were collimated with lead glass capillaries of the mentioned diameters. Dosimetry was performed by comparison of the results of measurements with a Geiger-Mueller counter and a microscope fluorescence dosemeter, both of which had been gauged absolutely with an ionization chamber dosemeter. Cells were grown and irradiated in growth chambers of a modified Rose type, which had walls of polystyrol foils with a thickness of 75 μm. Before and after irradiation in the dose range between 385 and 20 000 rds spreading, migration and division of the irradiated cell and its unirradiated sister as control and of the daughter cells were recorded up to 60 hs by time lapse cinemicrography. 82 pedigrees of irradiated and for control 75 pedigrees of unirradiated cells were analysed. As a result a 50% fraction of radiation damage required 2 000 rds for reproductive death at all beam diameters and 16 000 rds for interphase death at 275 μm only. The results are discussed in terms of chromosome aberrations and membrane damage as causes of reproductive resp. interphase death. (orig.)

  20. A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    Rocha H.A.O.

    2001-01-01

    Full Text Available Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1 and the mutant type deficient in xylosyltransferase (CHO-745. The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5(and CHO-745 (2 x 10(5 and 5 x 10(5 cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

  1. Postural instability at a simulated altitude of 5,000 m before and after an expedition to Mt. Cho-Oyu (8,201 m).

    Science.gov (United States)

    Hoshikawa, Masako; Hashimoto, Shiori; Kawahara, Takashi; Ide, Rika

    2010-10-01

    To clarify the effects of altitude acclimatization on postural instability at altitudes, six female climbers stood with their eyes open or closed on a force-measuring platform under normoxia (NC) and hypobaric hypoxia, equivalent to a 5,000 m altitude (HC), before and after an expedition to Mt. Cho-Oyu (8,201 m). The expedition extended over 84 days. We recorded sways in the center of foot pressure, electromyograms (EMGs) of lower-leg muscles, blood components and arterial oxygen saturation (SpO(2)). Before the expedition, the maximum amplitude of sway with the eyes open and integrated EMG from the medial gastrocnemius increased for HC. After the expedition, red blood cell (from 423.4 ± 15.4 to 498.0 ± 24.5 × 10(4) μl(-1)), hemoglobin content (from 12.6 ± 0.32 to 14.5 ± 1.00 g/dl) and 2,3-diphosphoglycerate (from 1.93 ± 0.21 to 2.24 ± 0.34 μmol/ml) increased. The SpO(2) under HC increased from 69.2 ± 9.6 to 77.2 ± 10.0%. The maximum amplitude of sway with the eyes open decreased for HC. No difference in the sway path length and integrated EMGs was observed between NC and HC. These results suggest that acclimatization can improve the impaired postural stability on initial arrival at altitudes. However, it is still unclear how long acclimatization period is needed. Further studies are needed to reveal this point. PMID:20549513

  2. Study on Aldol Condensation of HCHO and CH3CHO over MgO Catalysts Modified by Lanthanum and Cerium

    Institute of Scientific and Technical Information of China (English)

    王幸宜; 李春香; 杨菊群

    2004-01-01

    Aldol condensation of HCHO and CH3CHO over MgO, modified MgO and Al2O3 with rare earth oxides, was studied. The measurement of adsorption of pyrrole on catalysts by in-situ FT-IR and NH3 TPD indicated that the addition of elements La or Ce into MgO increased the acidity of the solid. In-situ FT-IR showed that the activation of-C=O in HCHO adsorbed on CeO-MgO and La2O3-MgO occurred. The measurement of catalytic activity implied that the modified catalysts can promote the formation of pentaerythritol, dipentaerthritol and tripentaerythritol.

  3. Phylosenetic identification and microbial diversity in snow of the summit (8201 m) of Cho Oyu Mountain,Tibet

    Institute of Scientific and Technical Information of China (English)

    TONG XiaoMei; WANG Jian; CHEN Fang; YU Jun; HUA Sang; ASAN Ciren; LUOSANG JiangBai; WANG Wei; YU Liang; ZHENG XiaoGuang

    2008-01-01

    The bacterial diversity and abundance in snow of the summit (8201 m) of Cho Oyu mountain, Tibet,were analyzed by 16S rRNA gene sequencing followed by scanning electronic microscopy analysis. Most of bacteria were found to be of spherical or oval shape (>95%). Bacterial 16S rDNA sequences were classified into 5 genera (Caulobacter, Ralstonia, Cupriavidus, Pelomonas and Pseudornonas).Gammaproteobacteria were the most abundant (91.25%) among the library that consists of 594 clones. The sequences found in this study are highly similar to those previously retrieved from other cold en-vironments, such as ice core, sea ice, permafrost and snow. The results showed that the cold and barren environments strongly influence the survival of bacteria. The high similarity among sequences retrieved from snow sample and other places, such as ocean, soil and water, suggested that the bacte-ria in snow, soil and water environments have the same origin.

  4. Characterization of Chinese Hamster Ovary Cells Producing Coagulation Factor VIII Using Multi-omics Tools

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder

    The first public draft of a genome from Chinese hamster ovary (CHO) cells was published in 2011, an entire decade after the first draft of the human genome. This publication of a relevant CHO reference genome, in combination with the fact that the cost for DNA sequencing has dropped more than 10,...... for analysis and engineering of industrially relevant CHO cells. Full implementation of such tools for generating specifically engineered CHO production cell lines may allow significant cost-reductions in production of complex biopharmaceuticals such as FVIII....... using omics tools. A wide range of methods were applied including whole-genome sequencing, targeted genome sequencing, mRNA sequencing, miRNA sequencing and mass spectrometry based shotgun proteomics on a number of clones in order to get a more holistic picture of the inner workings of these CHO...... further lead to degradation of FVIII by the endoplasmic-reticulum-associated protein degradation pathway. This is to our knowledge, the first time that such extensive omics tools have been applied to a broad panel of CHO cells producing a very complex protein. The holistic view obtained for the FVIII...

  5. Radiation-induced mutagenicity in repair deficient Chinese hamster ovary (CHO) mutants

    International Nuclear Information System (INIS)

    To determine if there is a relationship between DNA double-strand break repair and mutagenicity the authors utilized two x-ray sensitive mutants of Chinese hamster ovary cells along with the parental line K1. The two mutant lines xrs-5 and xrs-6, which have different DSB repair capabilities, were used to determine cell killing and 6-thioguanine resistance (6TG/sup r/) mutation frequencies induced by either x-rays of α-particles, x-ray survival data indicated the two mutant lines have similar sensitivity and are 5-7 fold more sensitive than the parental line K1. The mutant lines are also sensitive to α-particles but to a lesser extent. The authors' 6TG mutation data indicated that the two mutant lines are hypermutable. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in mutant cell population than in parental K1 cells. Their results support the notion that repair of DSB play an important role in the expression of radiation-induced cell killing and mutagenicity

  6. Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

    OpenAIRE

    Mondal, Mousumi; Mesmin, Bruno; Mukherjee, Sushmita; Maxfield, Frederick R.

    2009-01-01

    The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocyti...

  7. AGT-181: Expression in CHO Cells and Pharmacokinetics, Safety, and Plasma Iduronidase Enzyme Activity in Rhesus Monkeys

    OpenAIRE

    Boado, Ruben J.; Hui, Eric K.-W.; Lu, Jeff Zhiqiang; Pardridge, William M.

    2009-01-01

    Enzyme replacement therapy is not effective for the brain, owing to the lack of transport of the enzyme across the blood-brain barrier (BBB). Recombinant proteins such as the lysosomal enzyme, iduronidase, can penetrate the human BBB, following the re-engineering of the protein as an IgG fusion protein, where the IgG moiety targets an endogenous BBB transport system. The IgG acts as a molecular Trojan horse to ferry the fused protein into brain. AGT-181 is a genetically engineered fusion prot...

  8. Modeling of the gas-phase chemistry in C-H-O gas mixtures for diamond chemical vapor deposition

    Science.gov (United States)

    Petherbridge, James R.; May, Paul W.; Ashfold, Michael N. R.

    2001-05-01

    The boundaries of the diamond deposition region in the C-H-O (Bachmann) atomic phase composition diagram have been reproduced successfully for 38 different C, H, and O containing gas mixtures using the CHEMKIN computer package, together with just two criteria—a minimum mole fraction of methyl radicals [CH3] and a limiting value of the [H]/[C2H2] ratio. The diamond growth/no-growth boundary coincides with the line along which the input mole fractions of C and O are equal. For every gas mixture studied, no-growth regions are found to coincide with a negligible (<10-10) mole fraction of CH3 radicals, while for gas mixtures lying within the diamond growth region the CH3 mole fraction is ˜10-7. Each no-growth→diamond growth boundary is seen to be accompanied by a 2-3 order of magnitude step in CH3 mole fraction. The boundary between diamond and nondiamond growth is less clearly defined, but can be reproduced by assuming a critical, temperature dependent [H]/[C2H2] ratio (0.2, in the case that Tgas=2000 K) that reflects the crucial role of H atoms in the etching of nondiamond phases. The analysis allows prediction of the composition process window for good quality diamond growth for all stable input gas mixtures considered in this study.

  9. Linear antenna microwave plasma CVD diamond deposition at the edge of no-growth region of C-H-O ternary diagram

    Czech Academy of Sciences Publication Activity Database

    Potocký, Štěpán; Babchenko, Oleg; Hruška, Karel; Kromka, Alexander

    2012-01-01

    Roč. 249, č. 12 (2012), s. 2612-2615. ISSN 0370-1972 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GAP205/12/0908 Institutional research plan: CEZ:AV0Z10100521 Keywords : C-H-O phase diagram * nanocrystalline diamond * plasma enhanced CVD * Raman spectroscopy * SEM Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.489, year: 2012

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

    Science.gov (United States)

    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

  11. Simultaneous hydrogenation and UV-photolysis experiments of NO in CO-rich interstellar ice analogues; linking HNCO, OCN-, NH2CHO and NH2OH

    Science.gov (United States)

    Fedoseev, G.; Chuang, K.-J.; van Dishoeck, E. F.; Ioppolo, S.; Linnartz, H.

    2016-05-01

    The laboratory work presented here, simulates the chemistry on icy dust grains as typical for the "CO freeze-out stage" in dark molecular clouds. It differs from previous studies in that solid-state hydrogenation and vacuum UV-photoprocessing are applied simultaneously to co-depositing molecules. In parallel, the reactions at play are described for fully characterized laboratory conditions. The focus is on the formation of molecules containing both carbon and nitrogen atoms, starting with NO in CO-, H2CO-, and CH3OH-rich ices at 13 K. The experiments yield three important conclusions. 1. Without UV-processing hydroxylamine (NH2OH) is formed, as reported previously. 2. With UV-processing (energetic) NH2 is formed through photodissociation of NH2OH. This radical is key in the formation of species with an N-C bond. 3. The formation of three N-C bearing species, HNCO, OCN- and NH2CHO is observed. The experiments put a clear chemical link between these species; OCN- is found to be a direct derivative of HNCO and the latter is shown to have the same precursor as formamide (NH2CHO). Moreover, the addition of VUV competing channels decreases the amount of NO molecules converted into NH2OH by at least one order of magnitude. Consequently, this decrease in NH2OH formation yield directly influences the amount of NO molecules that can be converted into HNCO, OCN- and NH2CHO.

  12. Dynamized Preparations in Cell Culture

    OpenAIRE

    Girija Kuttan; Korengath Chandran Preethi; Ramadasan Kuttan; Ellanzhiyil Surendran Sunila

    2009-01-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The ...

  13. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  14. Stable expression of human opioid receptor-like 1 in chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Objective: To stably transfect human opioid receptor-like 1 (hORL1) in Chinese hamster ovary (CHO) cells. Methods: pcDNA3.1 (+) -hORL1 was stably transfected into CHO cells by a lipofectamine based method. Transfected CHO cells were selected in culture medium containing G418. Radioligand receptor binding assay and [35S] GTPSγ binding assay were used to determine densities and functions of the expressed receptors. Results and Conclusion: hORL1 was expressed in the CHO cells. The Kd and Bmax were (0.44 ± 0.21) nmol/L and (0.35 ± 0.06) pmol/mg protein, respectively, for hORL1 in [3H] nociceptin binding assay. The EC50 of nociceptin was 3.45 nmol/L in stimulation of [35S] GTPγS binding by nociceptin.A model system of CHO cells with stable expression of hORL1 is established. (authors)

  15. Expression of UV-irradiated adenovirus in normal and UV-sensitive Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    The chinese hamster ovary (CHO) cell mutants UV-20, UV-24, and UV-41 are abnormally sensitive to UV and harbour various defects lin their ability to repair cellular DNA. This study has examined the expression of UV-irradiated AD2 in these cells. HCR of UV-irradiated Ad2, as measured by viral structural antigen (Vag) formation or progeny production, was found to be similar for the normal and the UV-sensitive CHO strains. UV-irradiation of Ad2 (1200 J/m/sup 2/) resulted in a delay of Vag expression of 18 hours in normal human fibroblasts, which is thought to reflect the time required for removal of UV-induced lesions from the DNA before viral DNA synthesis can proceed. However, a similar UV-irradiation of Ad2 did not result in a delay of Vag expression for infection of CHO cells, suggesting that UV-induced lesions in Ad2 DNA do not inhibit its replication in CHO cells. These results indicate a fundamental difference in the processing of UV-irradiated AD2-DNA in CHO as compared to human cells

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

    Science.gov (United States)

    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

  17. Inhibition of Tumor Growth in Mice by Endostatin Derived from Abdominal Transplanted Encapsulated Cells

    Institute of Scientific and Technical Information of China (English)

    Huaining TENG; Ying ZHANG; Wei WANG; Xiaojun MA; Jian FEI

    2007-01-01

    Endostatin, a C-terminal fragment of collagen 18a, inhibits the growth of established tumors and metastases in vivo by inhibiting angiogenesis. However, the purification procedures required for largescale production and the attendant cost of these processes, together with the low effectiveness in clinical tests, suggest that alternative delivery methods might be required for efficient therapeutic use of endostatin.In the present study, we transfected Chinese hamster ovary (CHO) cells with a human endostatin gene expression vector and encapsulated the CHO cells in alginate-poly-L-lysine microcapsules. The release of biologically active endostatin was confirmed using the chicken chorioallantoic membrane assay. The encapsulated endostatin-expressing CHO cells can inhibit the growth of primary tumors in a subcutaneous B16 tumor model when injected into the abdominal cavity of mouse. These results widen the clinical application of the microencapsulated cell endostatin delivery system in cancer treatment.

  18. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

    International Nuclear Information System (INIS)

    We reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine. In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively

  19. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    International Nuclear Information System (INIS)

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves' patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves' disease will be elucidated. (author). 25 refs

  20. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  1. Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor

    OpenAIRE

    John, Scott A.; Ottolia, Michela; James N Weiss; Ribalet, Bernard

    2007-01-01

    To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600µM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (τ =5 to 15 s) in COS and C2C12 cells and slow (τ =20 to 40 s) in HEK and CHO cells. Upon ...

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

    Science.gov (United States)

    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.

  3. Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Larraza, Daniel M. [IMBICE, C.C. 403, 1900 La Plata (Argentina)]. E-mail: danielop@imbice.org.ar; Padron, Juan [IMBICE, C.C. 403, 1900 La Plata (Argentina); Ronci, Natalia E. [IMBICE, C.C. 403, 1900 La Plata (Argentina); Vidal Rioja, Lidia A. [IMBICE, C.C. 403, 1900 La Plata (Argentina)

    2006-08-30

    Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 {sup o}C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.

  4. A newly identified type of attachment cell is critical for normal patterning of chordotonal neurons.

    Science.gov (United States)

    Halachmi, Naomi; Nachman, Atalya; Salzberg, Adi

    2016-03-01

    This work describes unknown aspects of chordotonal organ (ChO) morphogenesis revealed in post-embryonic stages through the use of new fluorescently labeled markers. We show that towards the end of embryogenesis a hitherto unnoticed phase of cell migration commences in which the cap cells of the ventral ChOs elongate and migrate towards their prospective attachment sites. This migration and consequent cell attachment generates a continuous zigzag line of proprioceptors, stretching from the ventral midline to a dorsolateral position in each abdominal segment. Our observation that the cap cell of the ventral-most ChO attaches to a large tendon cell near the midline provides the first evidence for a direct physical connection between the contractile and proprioceptive systems in Drosophila. Our analysis has also provided an answer to a longstanding enigma that is what anchors the neurons of the ligamentless ventral ChOs on their axonal side. We identified a new type of ChO attachment cell, which binds to the scolopale cells of these organs, thus behaving like a ligament cell, but on the other hand exhibits all the typical features of a ChO attachment cell and is critical for the correct anchoring of these organs. PMID:26794680

  5. Expression of Bacterial β-Galactosidase in Animal Cells

    OpenAIRE

    An, Gynheung; Hidaka, Katsuhiko; Siminovitch, Louis

    1982-01-01

    A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.

  6. Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

    OpenAIRE

    Ebina, Y; Edery, M; Ellis, L; Standring, D; Beaudoin, J; Roth, R A; Rutter, W J

    1985-01-01

    We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growt...

  7. Toward genome-scale models of the Chinese hamster ovary cells: incentives, status and perspectives

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Fan, Yuzhou; Weilguny, Dietmar; Kristensen, Claus; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2014-01-01

    Bioprocessing of the important Chinese hamster ovary (CHO) cell lines used for the production of biopharmaceuticals stands at the brink of several redefining events. In 2011, the field entered the genomics era, which has accelerated omics-based phenotyping of the cell lines. In this review we...

  8. Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells.

    OpenAIRE

    Voelker, D R

    1989-01-01

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with [3H]serine, and the synthesis of phosphatidyl[3H]ethanolamine from phosphatidyl[3H]serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 micrograms of saponin per ml, there was no significant turnover of nascent phosphatidyl[3H]serine to form phosphatidyl[3H]ethanolamine during an ensuing chase. Saponin trea...

  9. Communication: Photodissociation of CH3CHO at 308 nm: Observation of H-roaming, CH3-roaming, and transition state pathways together along the ground state surface

    International Nuclear Information System (INIS)

    Following photodissociation of acetaldehyde (CH3CHO) at 308 nm, the CO(v = 1–4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH3CO core and CH3-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH3CO. By analyzing the CH4 emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH3-roamings. A branching fraction of H-roaming/CH3-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface

  10. Communication: Photodissociation of CH3CHO at 308 nm: Observation of H-roaming, CH3-roaming, and transition state pathways together along the ground state surface

    Science.gov (United States)

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen

    2015-01-01

    Following photodissociation of acetaldehyde (CH3CHO) at 308 nm, the CO(v = 1-4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH3CO core and CH3-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH3CO. By analyzing the CH4 emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH3-roamings. A branching fraction of H-roaming/CH3-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  11. Evaluación Genotóxica del D-003 en el ensayo de aberraciones cromosómicas en cultivo de células CHO

    Directory of Open Access Journals (Sweden)

    Rafael Gámez

    2010-01-01

    Full Text Available El D-003 es una mezcla de ácidos alifáticos primarios de alto peso molecular purificada de la cera de caña, con efectos antiosteoporóticos, hipolipemiantes y antioxidantes. El objetivo de este estudio fue investigar si el D-003 induce aberraciones cromosómicas en cultivo de células CHO. Diferentes concentraciones de D-003 (50, 500, 1000, 2500 y 5000¿g/ml se añadieron a los cultivos en presencia o ausencia de activación metabólica, incluyéndose controles positivos y negativos. Se examinaron 200 metafases por grupo, cuantificando el número de aberraciones cromosómicas por metafases y las frecuencias de células con aberraciones. El D-003 añadido a dosis de hasta 5000¿g/mL a los cultivos de células CHO no indujo aberraciones cromosómicas ni citotoxicidad en presencia o ausencia de activación metabólica respecto al control negativo, ni tampoco se apreciaron tendencias con las dosis. El índice mitótico no se modificó con el tratamiento, lo que indica que el D-003 no afectó la proliferación celular.

  12. Mechanism of action of cis-dichloro-bis (isopropylamine) trans dihydroxyplatinum IV (CHIP) on Chinese hamster and C3H mouse tumor cells and its interaction with x-irradiation

    International Nuclear Information System (INIS)

    CHIP acts as a hypoxic cell sensitizer when used in conjunction with x-irradiation. A change in the extrapolation number of the radiation survival curve of Chinese hamster ovary (CHO) cells is observed but the exponential slope (D0) remains the same. Chinese hamster ovary cells appear to be less sensitive than C3H mouse mammary tumor cells to CHIP. The type and level of chromosome aberrations observed in CHO cells indicate that the mechanism of action of CHIP is similar to that seen with bifunctional alkylating agents

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

    Science.gov (United States)

    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

  14. Electrophoretic shift mutants in Chinese hamster ovary cells: evidence for genetic diploidy

    International Nuclear Information System (INIS)

    Electrophoretic shift mutants induced in Chinese hamster ovary (CHO) cells indicate that these cells are not extensively functionally hemizygotic. Therefore, effective haploidy is unsatisfactory as a general theory to explain the frequency of recessive mutants in this cell line. CHO cells were screened for electrophoretic shift variants of enzymes coded by approximately 40 genetic loci. Clones isolated after exposure to ultraviolet radiation were examined by starch gel and Cellogel electrophoresis. Shift variants were recovered for enzymes representing 11 different loci. Variant clones were subcloned to demonstrate the heritability of the variations. Mutants at nine loci produced multiple-banded patterns consistent with the patterns expected of genes at loci represented twice (diploid). Chromosome localization of these diploid loci in other mammalian species where they have been mapped, suggests that they represent a random sample of CHO genes. Chromosome analysis of mutant subclones indicated that the variation did not take place in tetraploid cells. The data indicate that the quasi-diploid CHO cells appear only as functionally hemizygous as would be expected of a slightly hypodiploid cell line derived from an organism in which the haploid number is 11

  15. A human cell factor is essential for HIV-1 Rev action.

    OpenAIRE

    Trono, D; Baltimore, D

    1990-01-01

    To examine the restriction of HIV growth in murine cells, we infected NIH 3T3 cells with HIV pseudotyped by Moloney murine leukemia virus. The virus, which carried a dominant selectable marker under the control of the HIV LTR, gave large numbers of resistant clones, showing that murine cells are permissive for HIV uncoating, reverse transcription, nuclear transport and integration. However, we found that several murine cell lines, as well as CHO cells, could not support the function of rev, t...

  16. Cholesterol-Dependent Energy Transfer between Fluorescent Proteins—Insights into Protein Proximity of APP and BACE1 in Different Membranes in Niemann-Pick Type C Disease Cells

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    2012-11-01

    Full Text Available Förster resonance energy transfer (FRET -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP and amyloid precursor protein-mRFP (APP-mRFP in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer’s disease (AD pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1−/−, exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC, were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT and CHO-NPC1−/− cells (EPI-illumination microscopy, as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM, were performed. Additionally, generalized polarization (GP measurements of CHO-WT and CHO-NPC1−/− cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1−/− cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1−/−. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

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

    Science.gov (United States)

    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

  18. Protective effect of butylated hydroxytoluene (BHT) against the clastogenic acitivity of cadmium chloride and potassium dichromate in hamster ovary cells

    OpenAIRE

    Grillo Claudia A.; Seoane Analía I.; Dulout Fernando N.

    1999-01-01

    The effect of butylated hydroxytoluene (BHT), a widely used food additive, on chromosomal alterations induced by cadmium chloride (CC) and potassium dichromate (PD) in Chinese hamster ovary (CHO) cells was studied both at metaphase and anaphase-telophase. CHO cells were cultured for 15-16 h in the presence of PD (6.0, 9.0 or 12.0 mM), BHT (1.0 mg/ml), or PD plus BHT as well as CC (0.5, 1.0 and 2.0 mM), BHT or CC plus BHT for the analysis of chromosomal aberrations. To perform the anaphase-tel...

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

    International Nuclear Information System (INIS)

    The subject matter of this investigation concerns the killing and mutagenic effects induced by far-UV radiation and broad spectra of black, white and gold lights. Applying radiation directly on CHO (Chinese hamster ovary) 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 (6TG), ouabain and diptheria toxin (DT). Cells in the G1/early S boundary are the most sensitive to far-UV or unfiltered fluorescent lights. When synchronous cells are irradiated with moderate doses of far-UV or unfiltered broad spectra of black light, mutations to 6-TG and ouabain resistance are slightly higher in early S period than in the remaining parts of the cell cycle. Mutation induction of 6-TG, ouabain or DT resistance is increased in the split-dose samples of the asynchronous and synchronous CHO cells. CHO cells predominantly express an error-prone repair mechanism after photoirradiation

  20. Vitamin K metabolism in Chinese Hamster Ovary cells

    International Nuclear Information System (INIS)

    Recent investigations suggest that vitamin K may have functions other than in blood coagulation and calcification. The present study was undertaken to investigate this hypothesis using cells in culture. Chinese Hamster Ovary (CHO) cells were chosen due to their active metabolism and growth and lack of similarity to liver and bone cells, in which vitamin K metabolism is well known. Cells were adapted to serum-free media, incubated in media containing the appropriate concentrations of vitamin K for specified times, scraped from plates, pelleted, extensively washed to remove adhering vitamin K, extracted with chloroform:methanol (2:1, v/v) and analyzed on C18 HPLC columns. Uptake of vitamin K by CHO cells follows saturation kinetics at vitamin K concentrations up to 25 μ M and is transported into cells at the rate of 10 pmol/min. 106 cells. After 24 hours, 3H vitamin K is metabolized by CHO cells to several compounds, the major of which was isolated and identified as vitamin K epoxide. In 3 experiments, after 24 hours, the average cellular uptake of vitamin K was 8% with approximately half being metabolized to vitamin K epoxide. These results demonstrate that vitamin K is metabolized in cells with widely different functions and suggest a generalized function for vitamin K which has yet to be elucidated

  1. Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: Correlation with various leukemias and abnormal megakaryocytopoiesis

    International Nuclear Information System (INIS)

    To study the yet unknown role of the ubiquitous family of cholinesterases (ChoEases) in developing blood cells, the recently isolated cDNAs encoding human acetylcholinesterase and butyrylcholinesterase were used in blot hybridization with peripheral blood DNA from various leukemic patients. Hybridization signals and modified restriction patterns were observed with both cDNA probes in 4 of the 16 leukemia DNA preparations examined. These reflected the amplification of the corresponding AcCho-Ease and BtChoEase genes (ACHE and CHE) and alteration in their structure. Parallel analysis of 30 control samples revealed nonpolymorphic, much weaker hybridization signals for each of the probes. In view of previous reports on the effect of acetylcholine analogs and ChoEase inhibitors in the induction of megakaryocytopoiesis and production of platelets in the mouse. The authors further searched for such phenomena in nonleukemic patients with platelet production disorders. Amplifications of both ACHE and CHE genes were found in 2 of the 4 patients so far examined. Pronounced coamplification of these two related but distinct genes in correlation with pathological production of blood cells suggests a functional role for members of the ChoEase family in megakaryocytopoiesis and raises the question whether the coamplification of these genes could be casually involved in the etiology of hemocytopoietic disorders

  2. Photodissociation of CH3CHO at 248 nm by time-resolved Fourier-transform infrared emission spectroscopy: Verification of roaming and triple fragmentation

    Science.gov (United States)

    Hung, Kai-Chan; Tsai, Po-Yu; Li, Hou-Kuan; Lin, King-Chuen

    2014-02-01

    By using time-resolved Fourier-transform infrared emission spectroscopy, the HCO fragment dissociated from acetaldehyde (CH3CHO) at 248 nm is found to partially decompose to H and CO. The fragment yields are enhanced by the Ar addition that facilitates the collision-induced internal conversion. The channels to CH2CO + H2 and CH3CO + H are not detected significantly. The rotational population distribution of CO, after removing the Ar collision effect, shows a bimodal feature comprising both low- and high-rotational (J) components, sharing a fraction of 19% and 81%, respectively, for the vibrational state v = 1. The low-J component is ascribed to both roaming pathway and triple fragmentation. They are determined to have a branching ratio of 0.06, respectively, relative to the whole v = 1 population. The CO roaming is accompanied by a highly vibrational population of CH4 that yields a vibrational bimodality.

  3. Méthode impact-écho basse fréquence pour la détection de vide dans les gaines de précontrainte

    OpenAIRE

    ABRAHAM, O; Cote, P.

    2002-01-01

    Un des problèmes majeurs des ouvrages d'art est la détection de vide dans les gaines de précontrainte. Les méthodes couramment utilisées pour leur détection sont la radiographie X et la gammagraphie. Ces techniques requièrent malheureusement des zones de protection étendues contre les radiations. Depuis la fin des années 1980, la méthode impact-écho est aussi proposée. Deux indicateurs de présence de vide sont décrits dans la littérature : le changement de la fréquence de résonance liée à la ...

  4. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    Energy Technology Data Exchange (ETDEWEB)

    Komatsuzaki, Shunsaku; Horii, Hideyuki (Dept. of Civil Engineering, Univ. of Tokyo (Japan)); Saigo, Takahiro (Mitsubishi Research Institute, Inc. (Japan))

    2010-09-15

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  5. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    International Nuclear Information System (INIS)

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  6. Atmospheric chemistry of C2F5CHO: reaction with Cl atoms and OH radicals, IR spectrum of C2F5C(O)O2NO2

    DEFF Research Database (Denmark)

    Andersen, Mads Peter Sulbæk; Hurley, MD; Wallington, TJ;

    2003-01-01

    Smog chamber/FTIR techniques were used to measure k(Cl + C2F5CHO) = (1. 96 +/- 0.28) X 10(-12) and k(OH + C2F5CHO) = (5.26 +/- 0.80) x 10(-13) cm(3) molecule(-1) s(-1) in 700 Torr of N-2 or air at 296 +/- 2 K. The C1 initiated oxidation Of C2F5CHO in the presence of NO in air diluent gave COF2, CF3......ONO2, and C2F5C(O)O2NO2 in molar yields of 180 +/- 19 6.1 +/- 0.6 and 1.0 +/- 0.3 respectively. The IR spectrum for C2F5C(O)O2NO2 is reported. Results are discussed with respect to the potential for the atmospheric degradation of fluorinated aldehydes, CnF2n+1CHO, to contribute to the observed...... environmental burden of fluorinated carboxylic acids, CnF2n+1C(O)OH. (C) 2003 Elsevier B.V. All rights reserved....

  7. Nanosecond pulsed electric fields and the cell cycle

    Science.gov (United States)

    Mahlke, Megan A.

    Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when

  8. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

    OpenAIRE

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin; Denning, Chris

    2016-01-01

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have compl...

  9. In vitro metabolism study of normal and tumor cells when exposed to red LED light

    Science.gov (United States)

    Stolbovskaya, Olga V.; Khairullin, Radik M.; Saenko, Yuri V.; Krasnikova, Ekaterina S.; Krasnikov, Aleksandr V.; Fomin, Aleksandr A.; Skaptsov, Aleksandr A.

    2016-04-01

    This work presents the results of studying the mitochondrial membrane potential, intracellular ROS, peculiarities of the cell cycle of cancer cells HCT-116 and the normal line of CHO cells when exposed to the red LED light with a wavelength range of 0.620-0.680 μm. A dose-dependent increase in mitochondrial membrane potential and intracellular ROS concentration in cancer cells HCT-116 was established. In normal CHO cell line a dose-dependent reduction of mitochondrial membrane potential and dose-dependent increase in intracellular ROS occur. It has been shown that the sensitivity of the studied cell lines to the red light depends on the stage of the cell cycle.

  10. A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

    OpenAIRE

    Rocha H.A.O.; Franco C.R.C.; Trindade E.S.; Carvalho L.C.M.; Veiga S.S.; Leite E.L.; Dietrich C.P.; Nader H.B.

    2001-01-01

    Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion p...

  11. Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

    OpenAIRE

    Smith, Tim A. D.; Phyu, Su M.

    2016-01-01

    Introduction The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined. Methods MDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14...

  12. Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating

    International Nuclear Information System (INIS)

    A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [3H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment

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

    Science.gov (United States)

    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

  14. In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

    OpenAIRE

    Masuda, Seiji; Kada, Emi; Nagao, Masaya; Sasaki, Ryuzo

    1999-01-01

    In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in ...

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

    Science.gov (United States)

    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

  16. Refeeding alters superoxide dismutase activity in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    The authors previously showed superoxide dismutase (SOD) activity is increased in heat shocked Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells during the time period when thermotolerance (TT) is observed (Ca Res 45,3029). SOD is also increased in OvCa cells following transient exposure to ethanol, carbonyl cyanide-N-chlorophenyl-hydrazone, or hypoxia; all treatments which induce TT (1986 Rad Res Abstr Co-2). As these experiments involved refeeding of cell cultures, the authors examined the effect of refeeding on SOD in CHO cells. Refeeding confluent CHO cells with fresh McCoy's 5A medium containing 10% FCS decreased SOD 0 to 6 hours after refeeding, which was due to loss of the mitochondrial or Mn SOD. Addition of glucose to the medium at the concentration originally found in the medium caused a similar decline in SOD. At 6-24 hours after refeeding or the addition of glucose an increase in Mn SOD was observed. These results suggest metabolic status can affect Mn SOD in the cell. The possible role of metabolic regulation of SOD in heat sensitivity is being investigated

  17. Degradable Dextran Nanopolymer as a Carrier for Choline Kinase (ChoK) siRNA Cancer Therapy

    OpenAIRE

    Zhihang Chen; Balaji Krishnamachary; Zaver M. Bhujwalla

    2016-01-01

    Although small interfering RNA (siRNA) therapy has proven to be a specific and effective treatment in cells, the delivery of siRNA is a challenge for the applications of siRNA therapy. We present a degradable dextran with amine groups as an siRNA nano-carrier. In our nano-carrier, the amine groups are conjugated to the dextran platform through the acetal bonds, which are acid sensitive. Therefore this siRNA carrier is stable in neutral and basic conditions, while the amine groups can be cleav...

  18. An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells

    DEFF Research Database (Denmark)

    Hempel, Casper; Boisen, Ida M; Efunshile, Akinwale;

    2015-01-01

    BACKGROUND: Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an...... automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. METHODS: Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO...

  19. Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies.

    Science.gov (United States)

    Sengupta, Bhaswati; Narasimhulu, Chandrakala Aluganti; Parthasarathy, Sampath

    2013-12-01

    Generation of foam cells, an essential step for reverse cholesterol transport studies, uses the technique of receptor-dependent macrophage loading with radiolabeled acetylated LDL. In this study, we used the ability of a biologically relevant detergent molecule, lysophosphatidylcholine (lyso-PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabeled cholesterol/lyso-PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month of storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled, or radiolabeled)/lyso-PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by TLC. Such foam cells unloaded cholesterol when incubated with HDL but not with oxidized HDL. We propose that stable cholesterol or CE/lyso-PtdCho micelles would offer advantages over existing methods. PMID:24115226

  20. Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

    International Nuclear Information System (INIS)

    Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approx.50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to <10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with μM concentrations of BSO. Furthermore, BSO exposure also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells

  1. S-Sulfocysteine simplifies fed-batch processes and increases the CHO specific productivity via anti-oxidant activity.

    Science.gov (United States)

    Hecklau, Caroline; Pering, Sascha; Seibel, Ronja; Schnellbaecher, Alisa; Wehsling, Maria; Eichhorn, Thomas; Hagen, Joerg von; Zimmer, Aline

    2016-01-20

    Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule. PMID:26654938

  2. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  3. Targeted destruction of HIV-positive cells

    Directory of Open Access Journals (Sweden)

    Jyoti R Sharma

    2014-11-01

    Full Text Available Introduction: HIV/AIDS is now a global epidemic that has become the leading infectious killer of adults worldwide. Although antiretroviral (ARV therapy has dramatically improved the quality of life and increased the life expectancy of those infected with HIV but frequency of dosing and drug toxicity as well as the development of viral resistance pose additional limitations. The rapidly expanding field of nanotechnology has vast potential to radically advance the treatment and prevention of HIV/AIDS. Nanoparticles can provide improved drug delivery, by virtue of their small size, robustness, safety, multimodality or multifunctionality. Aims and objectives: Since HIV primarily infects CD4+ cells; we aim to use CD4 as a selectable target to deliver a pro-apoptotic protein to HIV-infected cells using nanoparticles as carriers. The aim of study was to develop a nanotechnology-based death inducing delivery system for the destruction of CD4+HIV infected cells through the activation of caspase-3. Methodology: A modified caspase-3 protein (Mut-3 was engineered, which is cleavable only by HIV-1 protease. Mut-3 can activate apoptosis in the presence of HIV-1 protease, consequently killing HIV-positive cells. Mut-3 protein was conjugated to gold nanoparticles together with a CD4-targeting peptide. The efficacy of the gold nanoparticles was tested on CHO cells that were genetically engineered to express GFP labelled CD4 and HIV-1 protease. Results: Mut-3 was expressed in bacterial cells and purified. CHO cells that stably over express CD4-GFP and HIV-1 protease were selected using Fluorescence Activated Cell Sorting. Dose response cell culture experiments showed that gold nanoparticles without Mut-3 and CD4-targeting peptide did not induce cell death in CHO cells, while gold nanoparticles that was conjugated with Mut-3 and the CD4-targeting peptide rapidly induced cell death in CHO cells. Conclusions: Our results suggest that gold nanoparticles conjugated

  4. Survival and kinetics of Chinese hamster ovary cell subpopulations induced by Adriamycin and radiation

    International Nuclear Information System (INIS)

    Mitotic selection of Chinese hamster ovary (CHO) cells, at 10 min intervals after the initiation of Adriamycin and/or x-ray treatment was used to measure the kinetics and survival of cells which progressed without delay, the ''refractory'' cells, the cells that reached mitosis only after recovery from the treatment-induced delay, the ''recovered'' cells, and the survival of the cells remaining attached to the flask 5 h after treatment. The cell kinetics were determined from the rate at which cells entered mitosis, and the reproductive integrity from the survival of the selected refractory, recovered and remaining (unselected) cells

  5. Linear antenna microwave plasma CVD diamond deposition at the edge of no-growth region of C-H-O ternary diagram

    International Nuclear Information System (INIS)

    The process parametric window for diamond deposition using the chemical vapor deposition at low pressures is quite limited where addition of oxygen in the gas phase broadens this window. The lower boundary of the lens-shaped domain in C-H-O ternary diagram concurs with the H2-CO tie-line (C/(C + O) = 0.5). In this work, we present the set of experiments where the ratio of C/(C + O) was kept at a constant value 0.385. The effect of hydrogen concentration (ratio O/(O + H) varied from 0.047 to 0.364) on plasma characteristics and deposited NCD films were investigated. Raman spectroscopy confirmed the diamond character of all deposited coatings while scanning electron microscopy showed transformation from not closed to continuous film and further decrease of grain size and finally growth of diamond nanowires while decreasing hydrogen concentration in a gas mixture. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  6. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    OpenAIRE

    Michał Arabski; Aneta Węgierek-Ciuk; Grzegorz Czerwonka; Anna Lankoff; Wiesław Kaca

    2012-01-01

    Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of sap...

  7. Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Tim A D Smith

    Full Text Available The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.MDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14C(U]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK, CTP:phosphocholine cytidylyl transferase (CCT and PtdCho-phospholipase C (PLC were also measured. [3H] Radiolabelled metabolites were determined using thin layer chromatography.Metformin-treated cells exhibited decreased formation of [3H]phosphocholine but increased accumulation of [3H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [14C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [14C(U]glucose.This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.

  8. Fluctuation of the dopamine uptake inhibition potency of cocaine, but not amphetamine, at mammalian cells expressing the dopamine transporter

    OpenAIRE

    Ukairo, Okechukwu T.; Ramanujapuram, Suneetha; Surratt, Christopher K.

    2006-01-01

    Cocaine, amphetamines and other psychostimulants inhibit synaptic dopamine uptake by interfering with dopamine transporter (DAT) function. The resultant potentiation of dopaminergic neurotransmission is associated with psychostimulant addiction. Fluctuations in dopamine uptake inhibition potency (DUIP) were observed for classical DAT blockers including cocaine, mazindol, methylphenidate (Ritalin™) and benztropine in CHO cells expressing wildtype DAT; cocaine potency also decreased in DAT-expr...

  9. Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining

    International Nuclear Information System (INIS)

    The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells. The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology. The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 'revertant' xrs-5 clones isolated following treatment with 5-azacytidine. Nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity. Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity. Radiation sensitivity did correlate with DNA double-strand break rejoining ability. The radioresistant clones showing the over-condensed xrs-5-like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins

  10. Survival and concanavalin-A-induced capping in CHO fibroblasts after exposure to hyperthermia, ethanol, and x irradiation

    International Nuclear Information System (INIS)

    Two parameters, concanavalin-A-induced capping and clonogenicity, were examined in HA-1 fibroblasts after exposure to heat, ethanol, x irradiation, and heat plus deuterium oxide. Capping and clonogenicity were shown to decrease over time in samples exposed to 43, 44, and 450C, while both parameters remained unaffected at 37, 41, and 420C. Ethanol exposure showed a survival decrease at a concentration of 8% but no significant effect on survival at 4, 6, and 7%. Capping, however, decreased over time at ethanol concentrations of 6, 7, and 8%. X irradiation had no effect on capping over a dose range of 0 to 1400 rad, while survival was markedly decreased. At 370C, exposure to 20, 40, and 80% deuterium oxide showed no effect on either survival or capping. However, at 430C, the same concentrations of deuterium oxide markedly increased both capping and clonogenicity over that of cells exposed to 430C without deuterium oxide. Potential mechanisms accounting for the heat- and ethanol-induced impairment of capping ability are inhibition of cellular energy metabolism and/or alteration of plasma membrane composition

  11. Report on new energy vision at Hinase Town area; 2001 nendo Hinase cho chiiki shin energy vision hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-03-01

    With an objective of accelerating introduction of and uplifting consciousness on new energies at Hinase Town in Hyogo Prefecture, investigations and discussions were given on the energy demand, quantity of new energies available, and the new energy introduction projects for the town, thus a new energy vision was established. The estimated amount of energy consumption in Hinase Town is 242,814,320,000 kcal in fiscal 2000. The amount of carbon dioxide discharge associated therewith is 76.57 t-CO2/year. Regarding the energy consumption by departments, the industrial department consumes the most, followed by the transportation department, civil household department, and civil business department. The new energy introduction projects have discussed servicing of the bio-gas plant utilizing organic wastes and cogeneration, the new energy introduction projects for the Natural Energy Island at Kakui Island and the ocean ranch consisted of the use of solar heat water warmer, solar cell powered street lights, photovoltaic power generation, and electric cars for sightseeing visitors, the Eco-school promotion project for primary schools, introduction of energy saving devices into the town office, and the new energy proliferation and enlightenment project. (NEDO)

  12. GeneOptimizer program-assisted cDNA reengineering enhances sRAGE autologous expression in Chinese hamster ovary cells.

    Science.gov (United States)

    Wei, Wen; Kim, Ji Min; Medina, Danny; Lakatta, Edward G; Lin, Li

    2014-03-01

    Soluble receptor for advanced glycation end products (sRAGE) is a secreted mammalian protein that functions as a decoy to counter-react RAGE signaling-resultant pathological conditions, and has high therapeutic potentials. Our prior studies showed that recombinant human sRAGE expressed in Chinese hamster, Ceanothus griseus, ovary (CHO) cells is modified by specific N-glycosylation, and exhibits higher bioactivity than that expressed in other host systems including insect Spodoptera frugiperda cells. Here, we show that GeneOptimizer software program-assisted, reengineered sRAGE cDNA enhances the recombinant protein expression in CHO cells. The cDNA sequence encoding human sRAGE was optimized for RNA structure, stability, and codon usages in CHO cells. We found that such optimization augmented sRAGE expression over 2 folds of its wild-type counterpart. We also studied how individual parameter impacted sRAGE autologous expression in CHO cells, and whether sRAGE bioactivity was compromised. We found that the enhanced expression appeared not to affect sRAGE N-glycosylation and bioactivity. Optimization of sRAGE expression provides a basis for future large-scale production of this protein to meet medical needs. PMID:24373844

  13. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    OpenAIRE

    Tetsushi Sakuma; Mitsumasa Takenaga; Yoshinori Kawabe; Takahiro Nakamura; Masamichi Kamihira; Takashi Yamamoto

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, includi...

  14. Expression in CHO Cells and Pharmacokinetics and Brain Uptake in the Rhesus Monkey of an IgG-Iduronate-2-Sulfatase Fusion Protein

    OpenAIRE

    Lu, Jeff Zhiqiang; Boado, Ruben J.; Hui, Eric K.-W.; Zhou, Qing-Hui; Pardridge, William M.

    2011-01-01

    Sulfatases are potential therapeutic biopharmaceuticals, as mutations in sulfatase genes leads to inherited disease. Mucopolysaccharidosis (MPS) Type II is caused by mutations in the lysosomal enzyme, iduronate 2-sulfatase (IDS). MPS-II affects the brain and enzyme replacement therapy is ineffective for the brain, because IDS does not cross the blood-brain barrier (BBB). To deliver IDS across the human BBB, the sulfatase has been re-engineered as an IgG-sulfatase fusion protein with a genetic...

  15. Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

    OpenAIRE

    Dahodwala, Hussain; Nowey, Mark; Mitina, Tatyana; Sharfstein, Susan T.

    2011-01-01

    The growth, metabolism, and productivity of five Chinese hamster ovary (CHO) clones were explored in response to stimulation with insulin (5 mg/L) and LONG®R3IGF-I (20 μg/L or 100 μg/L). All five clones were derived from the same parental CHO cell line (DG44) and produced the same recombinant monoclonal antibody, with varying specific productivities. There was no uniform response among the clones to stimulation with the different trophic factors. One of the high productivity clones (clone D) ...

  16. A New Practical Method to Simulate Flood-Induced Bridge Pier Scour—A Case Study of Mingchu Bridge Piers on the Cho-Shui River

    Directory of Open Access Journals (Sweden)

    Jian-Hao Hong

    2016-06-01

    Full Text Available The evolution of bridge pier scour is very important for bridge safety warning and assessment. During typhoon seasons in Taiwan, the torrential river flow often causes scour monitoring instruments to fail in their attempt to measure the temporal variations of pier scour depths. To better understand the scouring phenomenon during a large flood event, this study proposes a fast and reliable method for bridge pier scour simulation. The proposed method consists of two main components: (1 a robust finite-volume hydraulic model that simulates the flow depth and velocities; and (2 two scour depth computation algorithms that predict the temporal development of the general and local scour depths. The greatest advantage of this method is that it is very straight-forward and reliable, giving bridge managers sufficient time to make an informed decision for bridge safety warning. Only a few hydraulic flow conditions near the bridge are necessary to simulate the scour depth evolution. Moreover, the method can be applied for both general and local scour simulations. To demonstrate the accuracy of the proposed method, field data collected using the “numbered-brick” method were performed at the Mingchu Bridge, which is located along an incised channel reach of the Cho-Shui River in Taiwan. The simulated water levels and total scour depths are in good agreements with the field data. Finally, to help bridge authorities responsible for making a decision towards bridge scour warning, a bridge safety curve (scoured bed level-discharge relationship is proposed. Based on the results of two flood events, the study shows that the proposed method can quickly and accurately simulate the bridge pier scour.

  17. Induction of apoptotic cell death by putrescine

    DEFF Research Database (Denmark)

    Takao, Koichi; Rickhag, Karl Mattias; Hegardt, Cecilia;

    2006-01-01

    overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their...... growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication...... polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high....

  18. Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

    International Nuclear Information System (INIS)

    Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

  19. Macroscopic modelling of hybridoma cell fed-batch cultures with overflow metabolism: model-based optimization and state estimation

    OpenAIRE

    Amribt, Zakaria

    2014-01-01

    Monoclonal antibodies (MAbs) have an expanding market for use in diagnostic and therapeutic applications. Industrial production of these biopharmaceuticals is usually achieved based on fed-batch cultures of mammalian cells in bioreactors (Chinese hamster ovary (CHO) and Hybridoma cells), which can express different kinds of recombinant proteins. In order to reach high cell densities in these bioreactors, it is necessary to carry out an optimization of their production processes. Hence, macros...

  20. Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

    OpenAIRE

    Detjen, K.; Yang, J; Logsdon, C D

    1995-01-01

    Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in t...

  1. Accelerated Homology-Directed Targeted Integration of Transgenes in Chinese Hamster Ovary Cells Via CRISPR/Cas9 and Fluorescent Enrichment

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Pedersen, Lasse Ebdrup;

    2016-01-01

    Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of...

  2. Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: impact of the route of inoculation

    Science.gov (United States)

    In a companion study, we reported that the cellular Jumonji-C Domain containing Protein 6 (JMJD6) protein is involved in an alternate integrin- and HS-independent pathway of FMDV infection in CHO cells. Here, we investigated the JMJD6 localization in animal tissues from cattle infected with either ...

  3. In vitro evaluation of low-intensity light radiation on murine melanoma (B16F10) cells.

    Science.gov (United States)

    Peidaee, P; Almansour, N M; Pirogova, E

    2016-03-01

    Changes in the energy state of biomolecules induced by electromagnetic radiation lead to changes in biological functions of irradiated biomolecules. Using the RRM approach, it was computationally predicted that far-infrared light irradiation in the range of 3500-6000 nm affects biological activity of proto-oncogene proteins. This in vitro study evaluates quantitatively and qualitatively the effects of selected far-infrared exposures in the computationally determined wavelengths on mouse melanoma B16F10 cells and Chinese hamster ovarian (CHO) cells by MTT (thiazolyl blue tetrazolium bromide) cell proliferation assay and confocal laser-scanning microscopy (CLSM). This paper also presents the findings obtained from irradiating B16F10 and CHO cells by the selected wavelengths in visible and near-infrared range. The MTT results show that far-infrared wavelength irradiation induces detrimental effect on cellular viability of B16F10 cells, while that of normal CHO cells is not affected considerably. Moreover, CLSM images demonstrate visible cellular detachment of cancer cells. The observed effects support the hypothesis that far-infrared light irradiation within the computationally determined wavelength range induces biological effect on cancer cells. From irradiation of selected visible and near-infrared wavelengths, no visible changes were detected in cellular viability of either normal or cancer cells. PMID:26002595

  4. Isolation and partial characterisation of a mammalian cell mutant hypersensitive to topoisomerase II inhibitors and X-rays

    International Nuclear Information System (INIS)

    The authors have isolated, following one-step mutagenesis, a Chinese hamster ovary cell mutant hypersensitive to the intercalating agent, adriamycin. This agent exerts at least part of its cytotoxic action via inhibition of the nuclear enzyme, topoisomerase II. The mutant, designated ADR-3, showed hypersensitivity to all classes of topoisomerase II inhibitors, inlcuding actinomycin D, amsacrine (m-AMSA), etoposide (VP16) and mitoxantrone. ADR-3 cells also showed cross-sensitivity to ionizing radiation, but not no UV light. Topoisomerase II activity was elevated to a small but significant degree in ADR-3 cells, and this was reflected in a 1.5-fold higher level of topoisomerase II protein in ADR-3 than in CHO-K1 cells, as judged by Western blotting. ADR-3 cells were hypersensitive to cumene hydroperoxide but cross-resistant to hydrogen peroxide, suggesting possible abnormality in the detoxification of peroxides by glutathione peroxidase or catalase. Glutathione peroxidase activity against hydroperoxide was elevated to a small but significant extent in mutant cells. Catalase levels were not significantly different in ADR-3 and CHO-K1 cells. ADR-3 cells were recessive in hybrids with parental CHO-K1 cells with respect to sensitivity to topoisomerase II inhibitors and X-rays, and represent a different genetic complementation group from the previously reported adriamycin-sensitive mutant, ADR-1. (author). 34 refs.; 5 figs.; 3 tabs

  5. Heat shock response in Chinese hamster (DHFR/sup -/) cells transfected with mouse DHFR and drosophila HSP70 genes

    International Nuclear Information System (INIS)

    In order to understand the role of the M/sub r/ = 70,000 heat shock protein (hsp70) in mammalian cells in response to heat shock, the authors transfected CHO-DHFR/sup -/ cells with the recombinant vectors pSV2-DHFR (cell lines G and M) or with pSV2-DHFR-hsp70 (cell lines H and L). Both vectors express the DHFR gene, with the latter vector containing all the regulatory, coding and termination sequences for the gene coding for hsp70 from Drosophila melanogaster. The parental cell line (CHO-DHFR/sup -/) shows the induction of several hsps (including hsp70, hsp 68 and hsp110) after a 450C heat shock. Some transfected cell lines (M and L) showed a reduced level of hsp110 and failed to show any detectable hsp68 synthesis after a 450C heat shock. A non-tranfected, methotrexate-resistant CHO cell line that expresses high DHFR activity also fails to express or shows very reduced levels of hsp68 after a heat shock. The authors also tranfected cells (G and H) that do express hsp68 and hsp110 after a heat shock. They are currently investigating the effects of hsp68 and DHFR expression on growth and thermotolerance in these cell lines. The authors conclude that there are sequences present in the vector pSV2-DHFR (possibly the DHFR gene) that may be involved in hsp68 expression

  6. Influence of acute hypoxia and radiation quality on cell survival

    OpenAIRE

    Tinganelli, Walter; Ma, Ning-Yi; von Neubeck, Cläre; Maier, Andreas; Schicker, Corinna; Kraft-Weyrather, Wilma; Durante, Marco

    2013-01-01

    To measure the effect of acute oxygen depletion on cell survival for different types of radiation, experiments have been performed using Chinese hamster ovary (CHO) cells and RAT-1 rat prostate cancer cells. A special chamber has been developed to perform irradiations under different levels of oxygenation. The oxygen concentrations used were normoxia (air), hypoxia (94.5% N2, 5% CO2, 0.5% O2) and anoxia (95% N2, 5% CO2). Cells were exposed to X-rays and to C-, N- or O-ions with linear energy ...

  7. Metabotyping of docosahexaenoic acid - treated Alzheimer's disease cell model.

    Directory of Open Access Journals (Sweden)

    Priti Bahety

    Full Text Available BACKGROUND: Despite the significant amount of work being carried out to investigate the therapeutic potential of docosahexaenoic acid (DHA in Alzheimer's disease (AD, the mechanism by which DHA affects amyloid-β precursor protein (AβPP-induced metabolic changes has not been studied. OBJECTIVE: To elucidate the metabolic phenotypes (metabotypes associated with DHA therapy via metabonomic profiling of an AD cell model using gas chromatography time-of-flight mass spectrometry (GC/TOFMS. METHODS: The lysate and supernatant samples of CHO-wt and CHO-AβPP695 cells treated with DHA and vehicle control were collected and prepared for GC/TOFMS metabonomics profiling. The metabolic profiles were analyzed by multivariate data analysis techniques using SIMCA-P+ software. RESULTS: Both principal component analysis and subsequent partial least squares discriminant analysis revealed distinct metabolites associated with the DHA-treated and control groups. A list of statistically significant marker metabolites that characterized the metabotypes associated with DHA treatment was further identified. Increased levels of succinic acid, citric acid, malic acid and glycine and decreased levels of zymosterol, cholestadiene and arachidonic acid correlated with DHA treatment effect. DHA levels were also found to be increased upon treatment. CONCLUSION: Our study shows that DHA plays a role in mitigating AβPP-induced impairment in energy metabolism and inflammation by acting on tricarboxylic acid cycle, cholesterol biosynthesis pathway and fatty acid metabolism. The perturbations of these metabolic pathways by DHA in CHO-wt and CHO-AβPP695 cells shed further mechanistic insights on its neuroprotective actions.

  8. Detachment variants of Chinese hamster cells. Hyaluronic acid as a modulator of cell detachment

    Energy Technology Data Exchange (ETDEWEB)

    Barnhart, B.J.; Cox, S.H.; Kraemer, P.M.

    1979-01-01

    Variants of the Chinese hamster cell line CHO have been isolated and characterized with respect to attachment and trypsin- or EGTA-mediated detachment kinetics, cell morphologies, and the complex carbohydrates (labeled with (/sup 3/H)glucosamine) of the cell surface. The variant which was more readily detached from the substratum exhibited a more rounded cell shape and had three times more label as hyaluronic acid on the cell surface than the parental cell. The slowly detaching variant had a morphology similar to the parental cell but only half the radioactivity ascribable to hyaluronic acid. Endogenous levels of cAMP were unaltered in the variants. Exogenous db-cAMP caused the cells to elongate and flatten but did not alter the characteristic detachment kinetics. The role of hyaluronic acid as a modulator of the cell substratum interface is discussed.

  9. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

    International Nuclear Information System (INIS)

    Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32Pi and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells

  10. Sustained Uptake of a Hospital-Based Handwashing with Soap and Water Treatment Intervention (Cholera-Hospital-Based Intervention for 7 Days [CHoBI7]): A Randomized Controlled Trial.

    Science.gov (United States)

    George, Christine Marie; Jung, Danielle S; Saif-Ur-Rahman, K M; Monira, Shirajum; Sack, David A; Rashid, Mahamud-Ur; Mahmud, Toslim; Mustafiz, Munshi; Rahman, Zillur; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Sack, R Bradley; Alam, Munirul

    2016-02-01

    Diarrhea is the second leading cause of death in children under 5 years of age globally. The time patients and caregivers spend at a health facility for severe diarrhea presents the opportunity to deliver water, sanitation, and hygiene (WASH) interventions. We recently developed Cholera-Hospital-Based Intervention for 7 days (CHoBI7), a 1-week hospital-based handwashing with soap and water treatment intervention, for household members of cholera patients. To investigate if this intervention could lead to sustained WASH practices, we conducted a follow-up evaluation of 196 intervention household members and 205 control household members enrolled in a randomized controlled trial of the CHoBI7 intervention 6 to 12 months post-intervention. Compared with the control arm, the intervention arm had four times higher odds of household members' handwashing with soap at a key time during 5-hour structured observation (odds ratio [OR]: 4.71, 95% confidence interval [CI]: 2.61, 8.49) (18% versus 50%) and a 41% reduction in households in the World Health Organization very high-risk category for stored drinking water (OR: 0.38, 95% CI: 0.15, 0.96) (58% versus 34%) 6 to 12 months post-intervention. Furthemore, 71% of observed handwashing with soap events in the intervention arm involved the preparation and use of soapy water, which was promoted during the intervention, compared to 9% of control households. These findings demonstrate that the hospital-based CHoBI7 intervention can lead to significant increases in handwashing with soap practices and improved stored drinking water quality 6 to 12 months post-intervention. PMID:26728766

  11. Cell-cycle specific expression of a small proline-rich protein in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Squamous metaplasia of the bronchial epithelium is generally believed to be involved in the neoplastic progression toward squamous cell carcinomas. Thus, it is important to understand the mechanisms controlling this type of differentiation. The induction of two families of cDNAs encoding a small proline-rich protein (sPRP), sprI and sprII, was first identified in human keratinocytes exhibiting squamous differentiation. cDNAs similar to sprI have also been identified in cultured tracheal epithelial cells undergoing squamous differentiation. The first step during the squamous differentiation process is the inhibition of cell growth; it has also been noted that a sPRP mRNA in Chinese hamster ovary (CHO) cells is induced 10-fold just before the cultures reach confluence. Thus, sPRP may stop cell division in cells undergoing squamous differentation. In support of this possibility are the recent investigations correlating expression of sPRP with cell morphology. Specific immunoreactivity to sPRP, using affinity-purified antibodies, showed a strong immunostaining in cells with a round configuration, while less staining was observed in other cells. The major part of the CHO population showed no immunoreactivity. One interpretation of this observation is that the expression of sPRP may be cell-cyle regulated. The purpose of this investigation was to determine the phase of the cell cycle where induced synthesis of sPRP mRNA occurs

  12. Cloned s-Lap Gene Coding Area, Expression and Localizationof s-Lap/GFP Fusion Protein in Mammal Cells

    Institute of Scientific and Technical Information of China (English)

    SONG Yi-shu; SONG Zhi-yu; LI Hong-jun; Wu Yin; BAO Yong-li; TAN Da-peng; LI Yu-xin

    2005-01-01

    s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Sialylation patterns and branch location of dimeric N-acetyllactosamine units

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hokke, C.H.; Bergwerff, A.A.; Dedem, G.W.K. van; Kamerling, J.P.

    1995-01-01

    The N-linked carbohydrate chains of recombinant human erythropoietin expressed in CHO cells were quantitatively released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, separated from the remaining O-glycoprotein by gel-permeation chromatography, and subsequently fractionated via F

  16. Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of innoculation

    Science.gov (United States)

    In a companion study, we reported that the cellular Jumonji-C Domain containing Protein 6 (JMJD6) protein is involved in an alternate integrin- and HS-independent pathway of FMDV infection in CHO cells. Here, we investigated the JMJD6 localization in animal tissues from cattle infected with either ...

  17. Genome-wide DNA methylation profiling in cultured eutopic and ectopic endometrial stromal cells.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Yamagata

    Full Text Available The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa and without endometriosis (euESCb and ovarian endometrial cysts (choESC. Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2, which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.

  18. Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

    OpenAIRE

    Wileman, T; Kane, L. P.; Terhorst, C

    1991-01-01

    The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were in...

  19. Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

    DEFF Research Database (Denmark)

    Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie;

    2013-01-01

    of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for...... GPRC6A is thus useful in high-throughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor....

  20. Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

    OpenAIRE

    1980-01-01

    Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substra...

  1. Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents

  2. Sister-chromatid exchange induction in Chinese hamster ovary cells by 8-methoxypsoralen and brief pulses of laser light

    International Nuclear Information System (INIS)

    Brief (-sup(%) M 8-methoxypsoralen. Additional laser pulses caused less than proportional further increments in SCEs, although the total yield of 8-methoxypsoralen adducts in similarly exposed solutions of isolated DNA increased linearly. Higher numbers of laser pulses induced detectable amounts of 8-methoxypsoralen-DNA crosslinks and distorted the DNA flow histogram of CHO cells in a manner consistent with retarded cell cycle traverse. (orig./AJ)

  3. Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

    Directory of Open Access Journals (Sweden)

    C. Sternberg

    2010-10-01

    Full Text Available We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively. Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

  4. Study on Construction of Accelerating Ulcer Concrescence Cell/Hydrogel Compound and Its Biological Character%促溃疡愈合细胞/水凝胶三维复合物的初步构建及表征

    Institute of Scientific and Technical Information of China (English)

    董荣娜; 毋中明; 于德民

    2012-01-01

      目的:应用壳聚糖衍生物水凝胶负载表达人血小板衍化生长因子-BB 的 CHO 细胞系,初步构建促溃疡愈合复合物。方法:选择壳聚糖经 N-乙酰-L-半胱氨酸修饰后合成含巯基的双硫键壳聚糖,加水合成水凝胶,用 MTT 法检测生物相容性并负载转基因 CHO 细胞,构建促溃疡愈合复合物并进行表征。结果:该水凝胶材料与转基因 CHO 细胞系生物相容性良好。水凝胶呈网格结构,孔径一致、细胞黏附生长,负载转基因 CHO 细胞构成三维复合物。结论:CHO-rhPDGF-BB 细胞具有新的生物学功能,水凝胶与该细胞相容性良好,为研究糖尿病溃疡创面愈合提供了实验基础。%  Objective :Use natural macromolecule hydrogel material to compose 3D cell scaffolds loading transgenic cell that could stably express platelet-derived growth factor-BB (PDGF-BB) to construct accelerating ulcer concrescence compound.Method:Choosed chitosan and modified it by NAC to synthesize disulfide bond chitosan with sulfydryl,dissolved the compound into deionized water generating hydrogel,tested it’s cytotoxicity by MTT;used it loading transgenic CHO as 3D cell scaffolds to construct cell/scaffold composition,scaned it’s structure.Result:The hydrogel has good biocompatibility.The compound has network structure,aperture well-distributed,cells stuck well.Conclusion:The CHO cell line had new biological character,and successfully constructed the cell/scaffold composition,which can afford experiment base and theory foundation to study ulcer concrescence.

  5. Influence of chronic hypoxia and radiation quality on cell survival

    International Nuclear Information System (INIS)

    The purpose of this study was to investigate the influence of chronic hypoxia and anoxia on cell survival after low- and high-linear energy transfer (LET) radiation, Chinese hamster ovary K1 (CHO-K1) cells were kept for 24 h under chronic hypoxia (94.5% N2; 5% CO2; 0.5% O2) or chronic anoxia (95% N2; 5% CO2). Irradiation was performed using 250 kVp X-rays or carbon ions with a dose average LET of 100 keV/μm either directly under the chronic oxygenation states, or at different time points after reoxygenation. Moreover, the cell cycle distribution for cells irradiated under different chronic oxic states was measured over 24 h during reoxygenation. The measurements showed a fairly uniform cell cycle distribution under chronic hypoxia, similar to normoxic conditions. Chronic anoxia induced a block in G1 and a strong reduction of S-phase cells. A distribution similar to normoxic conditions was reached after 12 h of reoxygenation. CHO cells had a similar survival under both acute and chronic hypoxia. In contrast, survival after irradiation under chronic anoxia was slightly reduced compared to that under acute anoxia. We conclude that, in hamster cells, chronic anoxia is less effective than acute anoxia in inducing radioresistance for both X-rays and carbon ions, whereas in hypoxia, acute and chronic exposures have a similar impact on cell killing. (author)

  6. Communication: Photodissociation of CH{sub 3}CHO at 308 nm: Observation of H-roaming, CH{sub 3}-roaming, and transition state pathways together along the ground state surface

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen, E-mail: kclin@ntu.edu.tw [Department of Chemistry, National Taiwan University, Taipei 106, Taiwan and Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan (China)

    2015-01-28

    Following photodissociation of acetaldehyde (CH{sub 3}CHO) at 308 nm, the CO(v = 1–4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH{sub 3}CO core and CH{sub 3}-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH{sub 3}CO. By analyzing the CH{sub 4} emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH{sub 3}-roamings. A branching fraction of H-roaming/CH{sub 3}-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  7. Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

    OpenAIRE

    Pakhomov, Andrei G.; Xiao, Shu; Pakhomova, Olga N.; Semenov, Iurii; Kuipers, Marjorie A.; Ibey, Bennett L.

    2014-01-01

    Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PR...

  8. Radiation and biophysical studies on cells and viruses. Progress report, April 1, 1976--June 30, 1977

    International Nuclear Information System (INIS)

    Progress is reported on the following research projects: genetic structure of DNA, chromosomes, and nucleoproteins; particle beam studies of radiosensitive sites; division delay in CHO cells induced by partly penetrating alpha particles; location of cellular sites for mutation induction; sites for radioinduced cell transformation using partly penetrating particle beams; gamma-ray and particle irradiation of nucleoproteins and other model systems; quantitation of surface antigens on normal and neoplastic cells by x-ray fluorescence; hyperthermic effects on cell survival and DNA repair mechanisms; and studies on radioinduced cell transformation

  9. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.)

  10. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  11. Performance monitoring of a mammalian cell based bioprocess using Raman spectroscopy.

    Science.gov (United States)

    Li, Boyan; Ray, Bryan H; Leister, Kirk J; Ryder, Alan G

    2013-09-24

    Being able to predict the final product yield at all stages in long-running, industrial, mammalian cell culture processes is vital for both operational efficiency, process consistency, and the implementation of quality by design (QbD) practices. Here we used Raman spectroscopy to monitor (in terms of glycoprotein yield prediction) a fed-batch fermentation from start to finish. Raman data were collected from 12 different time points in a Chinese hamster ovary (CHO) based manufacturing process and across 37 separate production runs. The samples comprised of clarified bioprocess broths extracted from the CHO cell based process with varying amounts of fresh and spent cell culture media. Competitive adaptive reweighted sampling (CoAdReS) and ant colony optimization (ACO) variable selection methods were used to enhance the predictive ability of the chemometric models by removing unnecessary spectral information. Using CoAdReS accurate prediction models (relative error of predictions between 2.1% and 3.3%) were built for the final glycoprotein yield at every stage of the bioprocess from small scale up to the final 5000 L bioreactor. This result reinforces our previous studies which indicate that media quality is one of the most significant factors determining the efficiency of industrial CHO-cell processes. This Raman based approach could thus be used to manage production in terms of selecting which small scale batches are progressed to large-scale manufacture, thus improving process efficiency significantly. PMID:24016587

  12. Mutation and gene transfer of neutral amino acid transport System L genes in mammalian cells

    International Nuclear Information System (INIS)

    The authors are attempting to clone the genes coding for amino acid transport System L. Chinese hamster ovary (CHO) cell mutants that are temperature sensitive in their leucyl-tRNA synthetase show temperature-dependent regulation of System L. Temperature resistant mutants isolated from these cells have constitutively derepressed System L activity. Somatic cell fusion studies using these mutants have suggested that a trans-acting element controls regulation of System L. Mutants with reduced transport activity were isolated by a 3H-suicide selection. The growth of these mutant cells is limited by the transport defect. CHO mutants were transformed with a human cosmid library, followed by selection at high temperatures and low leucine concentrations. Some transformants have increased levels of System L activity, suggesting that human genes coding for leucine transport have been incorporated into the CHO genome. Human sequences were rescued by a lambda in vitro packaging system. These sequences hybridize to vector and total human DNA. Experiments are being done to confirm that these sequences indeed code for transport System L. They are also attempting to label membrane components of amino acid transporters by group-specific modifying reagents

  13. Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation.

    Science.gov (United States)

    Shelby, J Patrick; Edgar, J Scott; Chiu, Daniel T

    2005-01-01

    This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure. PMID:15850426

  14. Uronyl 2-O sulfotransferase potentiates Fgf2-induced cell migration.

    Science.gov (United States)

    Nikolovska, Katerina; Spillmann, Dorothe; Seidler, Daniela G

    2015-02-01

    Fibroblast growth factor 2 (Fgf2) is involved in several biological functions. Fgf2 requires glycosaminoglycans, like chondroitin and dermatan sulfates (hereafter denoted CS/DS) as co-receptors. CS/DS are linear polysaccharides composed of repeating disaccharide units [-4GlcUAb1-3-GalNAc-b1-] and [-4IdoUAa1-3-GalNAc-b1-],which can be sulfated. Uronyl 2-O-sulfotransferase (Ust)introduces sulfation at the C2 of IdoUA and GlcUA resulting inover-sulfated units. Here, we investigated the role of Ust-mediated CS/DS 2-O sulfation in Fgf2-induced cell migration. We found that CHO-K1 cells overexpressing Ust contain significantly more CS/DS2-O sulfated units, whereas Ust knockdown abolished CS/DS 2-O sulfation. These structural differences in CS/DS resulted in altered Fgf2 binding and increased phosphorylation of ERK1/2 (also known as MAPK3 and MAPK1, respectively). As a functional consequence of CS/DS 2-O sulfation and altered Fgf2 binding, cell migration and paxillin activation were increased. Inhibition of sulfation, knockdown of Ust and inhibition of FgfR resulted in reduced migration. Similarly, in 3T3 cells Fgf2 treatment increased migration, which was abolished by Ust knockdown. The proteoglycan controlling the CHO migration was syndecan 1. Knockdown of Sdc1 in CHO-K1 cells overexpressing Ust abolished cell migration.We conclude that the presence of distinctly sulfated CS/DS can tune the Fgf2 effect on cell migration. PMID:25480151

  15. Method for detecting DNA strand breaks in mammalian cells using the Deinococcus radiodurans PprA protein

    International Nuclear Information System (INIS)

    In a previous study, we identified the novel protein PprA that plays a critical role in the radiation resistance of Deinococcus radiodurans. In this study, we focussed on the ability of PprA protein to recognize and bind to double-stranded DNA carrying strand breaks, and attempted to visualize radiation-induced DNA strand breaks in mammalian cultured cells by employing PprA protein using an immunofluorescence technique. Increased PprA protein binding to CHO-K1 nuclei immediately following irradiation suggests the protein is binding to DNA strand breaks. By altering the cell permeabilization conditions, PprA protein binding to CHO-K1 mitochondria, which is probably resulted from DNA strand break immediately following irradiation, was also detected. The method developed and detailed in this study will be useful in evaluating DNA damage responses in cultured cells, and could also be applicable to genotoxic tests in the environmental and pharmaceutical fields

  16. Low-dose radiation employed in diagnostic imaging causes genetic effects in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Ponzinibbio, Maria V.; Peral-Garcia, Pilar; Seoane, Analia (Inst. de Genetica Veterinaria, Univ. Nacional de La Plata CONICET, La Plata (Argentina)), e-mail: aseoane@fcv.unlp.edu.ar; Crudeli, Cintia (Agencia Nacional de Promocion Cientifica y Tecnologica, La Plata (Argentina))

    2010-11-15

    Background: Exposure to environmental, diagnostic, and occupational sources of radiation frequently involves low doses. Although these doses have no immediately noticeable impact on human health there is great interest in their long-term biological effects. Purpose: To assess immediate and time-delayed DNA damage in two cell lines exposed to low doses of ionizing radiation by using the comet assay and micronucleus test, and to compare these two techniques in the analysis of low-dose induced genotoxicity. Material and Methods: CHO and MRC-5 cells were exposed to 50 milliSievert (mSv) of ionizing radiation and assayed immediately after irradiation and at 16 or 12 passages post-irradiation, respectively. Comet assay and micronucleus test were employed. Results: The comet assay values observed in 50 mSv-treated cells were significantly higher than in the control group for both sample times and cell lines (P < 0.001). Micronuclei frequencies were higher in treated cells than in the control group (P < 0.01, CHO cells passage 16; P < 0.05, MRC-5 cells immediately after exposure; P < 0.01 MRC-5 cells passage 12). Correlation analysis between the two techniques was statistically significant (correlation coefficient 0.82, P < 0.05 and correlation coefficient 0.86, P < 0.05 for CHO and MRC-5 cells, respectively). Cells scored at passages 12 or 16 showed more damage than those scored immediately after exposure in both cell lines (no statistically significant differences). Conclusion: Cytomolecular and cytogenetic damage was observed in cells exposed to very low doses of X-rays and their progeny. A single low dose of ionizing radiation was sufficient to induce such response, indicating that mammalian cells are exquisitely sensitive to it. Comet and micronucleus assays are sensitive enough to assess this damage, although the former seems to be more efficient

  17. Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

    OpenAIRE

    Sage, E.; Lamolet, B; Brulay, E; Moustacchi, E; Chteauneuf, A; Drobetsky, E A

    1996-01-01

    To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cell...

  18. Evaluation of genotoxic risk and oxidative DNA damage in mammalian cells exposed to mycotoxins, patulin and citrinin

    International Nuclear Information System (INIS)

    Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 μM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA

  19. Fundamentals of Expression in Mammalian Cells.

    Science.gov (United States)

    Dyson, Michael R

    2016-01-01

    Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions. PMID:27165328

  20. Action of caffeine on the survival of x-irradiated cells

    International Nuclear Information System (INIS)

    Post-irradiation treatment of HeLa S3 cells with 1 mM caffeine results in a marked diminution of the surviving fraction as scored by colony formation. The decrease is dose-dependent; the effect of a 24-h post-irradiation treatment of a non-synchronous population with caffeine is to change the terminal slope of the survival curve and its intercept. Do is reduced from 130 to 60 rad; the extrapolation number is increased about twofold. The amount of post-irradiation killing is maximal if cells are exposed to caffeine at a concentration of at least 1 mM for 8 hours; less than 10% of unirradiated cells are killed under these conditions. Dose-response curves were also obtained for synchronous cells at various phases of the cell cycle. Similar results were obtained at all cell ages, but the magnitude of the effect is age-dependent. This age dependence was further explored in experiments in which mitotically collected cells were exposed to 300 or 500 rad doses at 2-hour intervals throughout the cell cycle. Treatment with caffeine for 24 hours after irradiation enhances the killing of cells late in the cycle more than in G1. The sensitivities of two other cell lines, CHO and EMT6, also were examined; both are substantially less sensitive to caffeine. The smaller cell-cycle dependence of CHO cells is qualitatively the same as that of HeLa cells

  1. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  2. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    OpenAIRE

    Tamás Juhász; Eszter Szentléleky; Csilla Szűcs Somogyi; Roland Takács; Nóra Dobrosi; Máté Engler; Andrea Tamás; Dóra Reglődi; Róza Zákány

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during cho...

  3. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    International Nuclear Information System (INIS)

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well

  4. Multi-omics data driven analysis establishes reference codon biases for synthetic gene design in microbial and mammalian cells.

    Science.gov (United States)

    Ang, Kok Siong; Kyriakopoulos, Sarantos; Li, Wei; Lee, Dong-Yup

    2016-06-01

    In this study, we analyzed multi-omics data and subsets thereof to establish reference codon usage biases for codon optimization in synthetic gene design. Specifically, publicly available genomic, transcriptomic, proteomic and translatomic data for microbial and mammalian expression hosts, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Chinese hamster ovary (CHO) cells, were compiled to derive their individual codon and codon pair frequencies. Then, host dependent and -omics specific codon biases were generated and compared by principal component analysis and hierarchical clustering. Interestingly, our results indicated the similar codon bias patterns of the highly expressed transcripts, highly abundant proteins, and efficiently translated mRNA in microbial cells, despite the general lack of correlation between mRNA and protein expression levels. However, for CHO cells, the codon bias patterns among various -omics subsets are not distinguishable, forming one cluster. Thus, we further investigated the effect of different input codon biases on codon optimized sequences using the codon context (CC) and individual codon usage (ICU) design parameters, via in silico case study on the expression of human IFNγ sequence in CHO cells. The results supported that CC is more robust design parameter than ICU for improved heterologous gene design. PMID:26850284

  5. On the Formation and Isomer Specific Detection of Propenal (C2H3CHO) and Cyclopropanone (c-C3H4O) in Interstellar Model Ices - A Combined FTIR and Reflectron Time-of-Flight Mass Spectroscopic Study

    Science.gov (United States)

    Abplanalp, Matthew J.; Borsuk, Aleca; Jones, Brant M.; Kaiser, Ralf I.

    2015-11-01

    The formation routes of two structural isomers—propenal (C2H3CHO) and cyclopropanone (c-C3H4O)—were investigated experimentally by exposing ices of astrophysical interest to energetic electrons at 5.5 K thus mimicking the interaction of ionizing radiation with interstellar ices in cold molecular clouds. The radiation-induced processing of these ices was monitored online and in situ via Fourier Transform Infrared spectroscopy and via temperature programmed desorption exploiting highly sensitive reflectron time-of-flight mass spectrometry coupled with single photon ionization in the post irradiation phase. To selectively probe which isomer(s) is/are formed, the photoionization experiments were conducted with 10.49 and 9.60 eV photons. Our studies provided compelling evidence on the formation of both isomers—propenal (C2H3CHO) and cyclopropanone (c-C3H4O)—in ethylene (C2H4)—carbon monoxide (CO) ices forming propenal and cyclopropanone at a ratio of (4.5 ± 0.9):1. Based on the extracted reaction pathways, the cyclopropanone molecule can be classified as a tracer of a low temperature non-equilibrium chemistry within interstellar ices involving most likely excited triplet states, whereas propenal can be formed at ultralow temperatures, but also during the annealing phase via non-equilibrium as well as thermal chemistry (radical recombination). Since propenal has been detected in the interstellar medium and our laboratory experiments demonstrate that both isomers originated from identical precursor molecules our study predicts that the hitherto elusive second isomer—cyclopropanone—should also be observable toward those astronomical sources such as Sgr B2(N) in which propenal has been detected.

  6. Influence of acute hypoxia and radiation quality on cell survival

    International Nuclear Information System (INIS)

    The purpose of this study was to measure the effect of acute oxygen depletion on cell survival for different types of radiation, experiments have been performed using Chinese hamster ovary (CHO) cells and R-3327-AT1 (RAT-1) rat prostate cancer cells. A special chamber has been developed to perform irradiations under different levels of oxygenation. The oxygen concentrations used were normoxia (air), hypoxia (94.5% N2, 5% CO2, 0.5% O2) and anoxia (95% N2, 5% CO2). Cells were exposed to X-rays and to C-, N- or O-ions with linear energy transfer (LET) values ranging from 100-160 keV/μm. The oxygen enhancement ratio (OER) and relative biological effectiveness (RBE) values have been calculated from the measured clonogenic survival curves. For both cell lines, the X-ray OER depended on the survival level. For particle irradiation, OER was not dependent on the survival level but decreased with increasing LET. The RBE of CHO cells under oxic conditions reached a plateau for LET values above 100 keV/μm, while it was still increasing under anoxia. In conclusion, the results demonstrated that our chamber could be used to measure radiosensitivity under intermediate hypoxia. Measurements suggest that ions heavier than carbon could be of additional advantage in the irradiation, especially of radioresistant hypoxic tumor regions. (author)

  7. The effect of caffeine and adenine on radiation induced suppression of DNA synthesis, and cell survival

    International Nuclear Information System (INIS)

    Exposure of cultured mammalian cells to ionizing radiation or UV light results in a transient decrease in the rate of DNA synthesis. This depression in synthetic rate may be attenuated or deferred via a post-irradiation treatment with caffeine or adenine. It has been suggested that this attenuation may increase the fixation of damage and, therefore, increase radiation sensitivity. However, it has been previously reported that, for V79 cells treated with caffeine or adenine, no correlation exists between the extent of depression and cell survival. The present investigation expands upon these findings by examining the effect of caffeine or adenine post-irradiation treatment on two cell lines with normal UV sensitivity, mouse 3T3 and CHO AA8 cells, and one UV sensitive cell line, CHO UV5 cells. Both caffeine and adenine have been found to reduce, or delay, the suppression in DNA synthesis in all three cell lines. Surprisingly, caffeine appeared to induced even the UV5 cells to recover DNA synthetic ability. The amount of reduction in suppression of DNA synthesis, however, varies between the different cell lines and no consistent relationship with cell survival has emerged

  8. Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

    International Nuclear Information System (INIS)

    Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal

  9. Effect of taxol on chromosome aberrations induced by gamma radiation or by doxorubicin in Chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Francisca da Luz Dias

    1997-09-01

    Full Text Available Combined therapy with radiation and chemotherapy has being increasingly used in cancer treatment. The effect of combinations of taxol (0.08 mug/ml with doxorubicin (DXR, 0.5 or 1.0 mug/ml or gamma radiation (20 or 40 cGy was examined in two different treatment schedules (pretreatment or simultaneous treatment using Chinese hamster ovary (CHO cells treated at the G2 phase of the cell cycle. The results showed that taxol did not have a radiosensitizing effect on the chromosomal aberrations induced by gamma radiation nor did it have a potentiating effect on the chromosomal aberrations induced by DXR in CHO cells treated in the G2 phase of the cell cycleA terapia combinada com radiação e agentes químicos tem aumentado nos regimes de tratamento do câncer. Nesse estudo foi investigado o efeito das combinações de taxol (0,08 mig/ml com a doxorubicina (DXR 0,5 ou 1,0 mig/ml ou com as radiações gama (20 ou 40 cGy em dois diferentes protocolos de tratamento (pré-tratamento e simultâneo em células CHO tratadas na fase G2 do ciclo celular. Os resultados mostraram que o taxol não teve efeito sensibilizador sobre as aberrações cromossômicas induzidas pelas radiações gama, nem teve efeito potenciador sobre as aberrações cromossômicas induzidas pela DXR em células CHO tratadas na fase G2 do ciclo celular

  10. Thresholds for Phosphatidylserine Externalization in Chinese Hamster Ovarian Cells following Exposure to Nanosecond Pulsed Electrical Fields (nsPEF)

    OpenAIRE

    Vincelette, Rebecca L.; Roth, Caleb C.; McConnell, Maureen P.; Payne, Jason A.; Beier, Hope T.; Ibey, Bennett L.

    2013-01-01

    High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to ...

  11. Dose-dependent thresholds of 10-ns electric pulse induced plasma membrane disruption and cytotoxicity in multiple cell lines.

    Directory of Open Access Journals (Sweden)

    Bennett L Ibey

    Full Text Available In this study, we determined the LD(50 (50% lethal dose for cell death, and the ED(50 (50% of cell population staining positive for propidium (Pr iodide uptake, and phosphatidylserine (PS externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3 exposed to 10-ns electric pulses (EP. We found that the LD(50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD(50 lower than the ED(50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

  12. Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji, E-mail: kzsuzuki@nagasaki-u.ac.jp [Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Kodama, Seiji [Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-machi, Sakai 599-8570 (Japan); Watanabe, Masami [Kyoto University Research Reactor Institute, Kumatori-cho Sennan-gun, Osaka 590-0494 (Japan)

    2010-01-05

    Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.

  13. Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line

    Institute of Scientific and Technical Information of China (English)

    Bushra Tabassum; Idrees Ahmad Nasir; Tayyab Husnain

    2011-01-01

    RNA interference(RNAi)is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the eapsid protein gene of potato virus Y(CP-PVY)was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

  14. Rejoining of prematurely condensed chromosomes in radiosensitive xrs-5 cells

    International Nuclear Information System (INIS)

    Xrs-5 cells, a radiosensitive, DNA double strand break repair deficient mutant of CHO cells have been studied with the premature chromosome condensation (PCC) technique. This mutant displayed a higher number of initial chromosome breaks with x-ray treatment as well as partial deficiency in the rejoining of interphase chromosome breaks with the standard PCC protocol. Moreover, hypertonic treatment during the incubation period which allowed for PCC did not change the yield of PCC breaks in x-irradiated xrs-5 cells. Notably the number of PCC breaks after treatment with hypertonic media is similar in CHO and xrs-5 cells. Recently, a gene product responsible for the xrs phenotype was identified as a Ku-like DNA end binding protein. The present paper summarizes completed information regarding the induction and repair of the α- and β-forms of PCC breaks in xrs-5 cells and demonstrates that this gene product predominantly affects the fast form (β-form) of interphase chromosome breaks

  15. Development and blind clinical validation of a microRNA based predictor of response to treatment with R-CHO(E)P in DLBCL

    DEFF Research Database (Denmark)

    Knudsen, Steen; Hother, Christoffer; Grønbæk, Kirsten;

    2015-01-01

    MicroRNAs (miRNA) are a group of short noncoding RNAs that regulate gene expression at the posttranscriptional level. It has been shown that microRNAs are independent predictors of outcome in patients with diffuse large B-cell lymphoma (DLBCL) treated with the drug combination R-CHOP. Based on the...

  16. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC12, one being increased in amount while the other was reduced to an insignificant level. In ZnC12-treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC12-treated cells. While ZnC12 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC12-treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC12-treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  17. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    Energy Technology Data Exchange (ETDEWEB)

    Einstein, R.; Gabel, C.A. (Columbia Univ. College of Physicians and Surgeons, NY (USA))

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  18. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    International Nuclear Information System (INIS)

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated

  19. Numerical model for the deformation of nucleated cells by optical stretchers

    KAUST Repository

    Sraj, Ihab

    2015-07-01

    In this paper, we seek to numerically study the deformation of nucleated cells by single diode-laser bar optical stretchers. We employ a recently developed computational model, the dynamic ray-tracing method, to determine the force distribution induced by optical stretchers on a cell encapsulating a nucleus of different optical properties. These optical forces are shape dependent and can deform real non-rigid objects; thus resulting in dynamically changing distributions with cell and nucleus deformation. A Chinese hamster ovary (CHO) cell is a common biological cell that is of interest to the biomedical community because of its use in recombinant protein therapeutics and is an example of a nucleated cell. To this end, we model CHO cells as two concentric three-dimensional elastic capsules immersed in a fluid where the hydrodynamic forces are calculated using the immersed boundary method. We vary the inner capsule size to simulate different nucleus sizes. Our results show that the presence of a nucleus has a major effect on the force distribution on the cell surface and consequently on its net deformation. Scattering and gradient forces are reported for different nucleus sizes and the effect of nucleus size on the cell deformation is discussed quantitatively. © 2015 IOP Publishing Ltd.

  20. Effects of UV light on DNA chain growth and replicon initiation in human cells

    International Nuclear Information System (INIS)

    Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. Using DNA fiber autoradiography the authors report here similar findings for human cells. Wild-type human cells did not exhibit signigicant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. The authors have previously shown that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. The authors report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation. (Author). 24 refs.; 4 figs.; 2 tabs

  1. Cloning and identification of measles virus receptor gene from marmoset cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The measles virus (MV) strains with mutated hemagglutinin gene (ha) lost the capacity to infect its sensitive host cells (Vero cells), but it may infect the marmoset B-lymphoblastoid cell line B95a. From above, we can presume that there is a novel cellular receptor for those measles virus strains on B95a cell s. Using the yeast two-hybrid system, we screened and cloned a novel gene--bip (B-lympho- blastoid interaction protein of marmoset) from B95a cell cDNA library, which encoded a protein interacting with measles virus hemagglutinin protein (Ha). The bip cDNA was 1540 base pairs in length and contained a unique open rea ding frame (ORF) of 1011 base pairs encoding a transmembrane protein of 337 amino acid residues. The primary structure of amino acids residue is predicted that the Bip comprised a hydrophobic transmembrane domain and a hydrophobic leader region. The researches about the deletion mutants showed that the deletion of tran smembrane domain in Bip did not affect the interaction between Bip and Ha protei ns. Expression of bip in measles virus non-permissive cell line--CHO (Chinese hamster ovary) cells was performed to prove that CHO/Bip can be infected by meas les virus and then turned to the MV permissive cells. We concluded that the bip gene is a novel measles virus receptor gene in marmoset B-lymphoblastoid cells.

  2. Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions

    OpenAIRE

    Del Piccolo, Nuala; Placone, Jesse; He, Lijuan; Agudelo, Sandra Carolina; Hristova, Kalina

    2012-01-01

    Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation...

  3. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  4. The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface

    OpenAIRE

    van Bemmelen, Miguel Xavier; Huser, Delphine; Gautschi, Ivan; Schild, Laurent

    2015-01-01

    The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathi...

  5. Molecular suicide studies of 125I and 3H disintegration in the DNA of Chinese hamster cells

    International Nuclear Information System (INIS)

    Several recent experiments are discussed which yield some new data to help further understand the dramatic sensitivity of mammalian cells to 125I induced reproductive death. The authors discuss the effects of halogenated pyrimidines on removing the shoulder of the survival curve after tritium thymidine suicide; the effect of oxygen on 125I decay effects as a functions of the localization of the decays within the nucleus; and recent data on the induction of chromosome aberrations by 125I DNA decays in CHO cells stored in the G1-stage of the cell cycle. (B.R.H.)

  6. Differential Dynamics of α5 Integrin, Paxillin, and α-Actinin during Formation and Disassembly of Adhesions in Migrating Cells

    OpenAIRE

    Laukaitis, Christina M; Webb, Donna J.; Donais, Karen; Horwitz, Alan F.

    2001-01-01

    To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed α5 integrin, α-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. α5-GFP also rescued the adhesive defects in CHO B2 cells, which are α5 integrin deficient. In ruffling cells, α5-GFP and α-actinin–GFP localized prominently at the leading edge in membrane protrusions. ...

  7. Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields

    International Nuclear Information System (INIS)

    Potassium ion influx was measured by monitoring 42KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+

  8. Achievements and Challenges of CdS/CdTe Solar Cells

    OpenAIRE

    Zhou Fang; Xiao Chen Wang; Hong Cai Wu; Ce Zhou Zhao

    2011-01-01

    Thin film CdS/CdTe has long been regarded as one promising choice for the development of cost-effective and reliable solar cells. Efficiency as high as 16.5% has been achieved in CdS/CdTe heterojunction structure in laboratory in 2001, and current techniques for CdS/CdTe solar cells gradually step toward commercialization. This paper reviews some novel techniques mainly within two years to solve this problem from aspects of promotion of fabrication technology, structural modification, and cho...

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

    International Nuclear Information System (INIS)

    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

  10. Detailed analysis of the response of different cell lines to carbon irradiation

    CERN Document Server

    Hromcikova, H; Lokajícek, M

    2005-01-01

    Published survival data for Chinese hamster ovarian cells CHO-K1 and their radiosensitive mutant xrs5 after irradiation by carbon ions of energies from 2.4 to 266.4 MeV/u have been analyzed using the probabilistic two-stage radiobiological model, which enables to represent the interplay of damage induction and repair processes. The results give support for the hypothesis that the differences in radiation sensitivity of diverse cell lines are given primarily by their different repair capabilities, and indicate the need for explicitly representing the outcome of repair processes in radiobiological models and treatment planning approaches in radiotherapy.

  11. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells

    Directory of Open Access Journals (Sweden)

    Irvine Alistair

    2005-06-01

    Full Text Available Abstract Background The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing. Results In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression. Conclusion Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Juliana Branco Novo

    2012-01-01

    Full Text Available 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.

  14. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  15. Transient transfection of mammalian cells using a violet diode laser

    Science.gov (United States)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  16. Differential biological effects of iodoacetate in mammalian cell lines; radio sensitization and radio protection

    International Nuclear Information System (INIS)

    There are several studies where it has been shown that Iodoacetate (IA) possesses in vivo anti-tumor activity. The fact that it is a model glycolytic inhibitor makes it more interesting. As seen in recent trends, glycolytic inhibitors are emerging as new strategy for cancer therapeutic research taking advantage of glycolytic phenotype of cancerous tissues. IA has been reported to have radioprotective effects in yeast cells and human lymphocytes. Biological effects of IA in response to radiation in mammalian cell lines are not well documented. We screened IA for cytotoxicity using clonogenic assay at different concentrations ranging from 0.1 to 2.5 μg/ml using three different mammalian cell lines; A-549 (human lung carcinoma cell line), MCF-7 (human mammary cancer cell line) and a noncancerous CHO (Chinese hamster ovary cell line). For studying radioprotective/radio sensitizing efficacy, cells were exposed to 4 Gy of 60Co-γ radiation using a teletherapy source at a dose rate of 1 Gy/min, following which IA post-treatment was carried out. Clonogenic and micronucleus assay were performed to assess radioprotection/sensitization. The results indicated that IA was highly cytotoxic in cancerous cell lines A-549 (IC50=1.25 μg/ml) and MCF-7 (IC50 = 1.9 μg/ml). In contrast, it was totally non-toxic in non-cancerous cell line, viz. CHO, in the same concentration range. In addition, IA exhibited radio protective effect in CHO cell line, whereas in other two cancer cell lines, viz. A-549 and MCF-7, radio sensitizing effect was seen as judged by induction of cell killing and micronuclei. In conclusion, lA, a model glycolytic inhibitor, was found to be selectively cytotoxic in cancer cells as compared to normal cells. Further, it reduced radiation induced damage (micronuclei and cell killing) in normal cells but increased it in cancer cells indicating its potential use in cancer therapy. (author)

  17. Mapping analysis of scaffold/matrix attachment regions (s/MARs) from two different mammalian cell lines

    International Nuclear Information System (INIS)

    Scaffold/matrix attachment regions (S/MARs) are potential element that can be integrated into expression vector to increase expression of recombinant protein. Many studies on S/MAR have been done but none has revealed the distribution of S/MAR in a genome. In this study, we have isolated S/MAR sequences from HEK293 and Chinese hamster ovary cell lines (CHO DG44) using two different methods utilizing 2 M NaCl and lithium-3,5-diiodosalicylate (LIS). The isolated S/MARs were sequenced using Next Generation Sequencing (NGS) platform. Based on reference mapping analysis against human genome database, a total of 8,994,856 and 8,412,672 contigs of S/MAR sequences were retrieved from 2M NaCl and LIS extraction of HEK293 respectively. On the other hand, reference mapping analysis of S/MAR derived from CHO DG44 against our own CHO DG44 database have generated a total of 7,204,348 and 4,672,913 contigs from 2 M NaCl and LIS extraction method respectively

  18. Mapping analysis of scaffold/matrix attachment regions (s/MARs) from two different mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pilus, Nur Shazwani Mohd; Ahmad, Azrin; Yusof, Nurul Yuziana Mohd [School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Johari, Norazfa [Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2014-09-03

    Scaffold/matrix attachment regions (S/MARs) are potential element that can be integrated into expression vector to increase expression of recombinant protein. Many studies on S/MAR have been done but none has revealed the distribution of S/MAR in a genome. In this study, we have isolated S/MAR sequences from HEK293 and Chinese hamster ovary cell lines (CHO DG44) using two different methods utilizing 2 M NaCl and lithium-3,5-diiodosalicylate (LIS). The isolated S/MARs were sequenced using Next Generation Sequencing (NGS) platform. Based on reference mapping analysis against human genome database, a total of 8,994,856 and 8,412,672 contigs of S/MAR sequences were retrieved from 2M NaCl and LIS extraction of HEK293 respectively. On the other hand, reference mapping analysis of S/MAR derived from CHO DG44 against our own CHO DG44 database have generated a total of 7,204,348 and 4,672,913 contigs from 2 M NaCl and LIS extraction method respectively.

  19. Effect of 3-aminobenzamide on the cell cycle in x-irradiated Chinese hamster cells replicating in medium containing 5-bromodeoxyuridine

    International Nuclear Information System (INIS)

    A specific inhibitor of poly(ADP-ribose)polymerase- 3 -aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-Kl cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU-cells), and increase their radiosensitivity; 2) induce G2-delay in BDU-cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2-delay in X-irradiated BDU-cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure

  20. Large scale isolation of DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    A semiautomated procedure has been designed and used successfully to permit the isolation of a large number of uv (ultraviolet light) sensitive mutant colonies of Chinese hamster ovary (CHO) cells. 149 uv sensitive mutants of CHO cells have been isolated to date by this procedure. This represents the largest in vitro isolation to date of uv sensitive mutants of mammalian cells The isolation of uv sensitive clones of EM9-1 represents the first stepwise isolation of double mutants of mammalian cells with sensitivity to an increased range of carcinogenic agents. Using a rapid screening test for complementation group assignment, 70 of these mutants have been assigned to a total of 5 complementation classes. Representatives of 4 of these classes have been studied for competence in repair replication following uv irradiation and have demonstrated a defective phenotype in uv repair. Representatives of 2 of these repair-deficient classes have been studies for uv mutagenic response for three selective markers, and have demonstrated an approximately ninefold enhancement in mutant yield per unit uv exposure relative to the parental cels. The sensitivity, repair phenotype, and mutational response ofthe isolates studied resemble that of human mutant xeroderma pigmentosum cells

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

    Science.gov (United States)

    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

  2. Caspase-dependent retinal ganglion cell apoptosis in the rat model of acute diabetes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Neural apoptosis is generally believed to be mediated by two distinct pathways, caspase-dependant and caspase-independent pathways. This study investigated the apoptotic pathways involved in retinal ganglion ceils in acute diabetes in rats. Methods Diabetes was induced in male Wistar rats by a peritoneal injection of streptozotocin (STZ). Expression and localization of caspase-3 and apoptosis-inducing factor (AIF) proteins in the retina of diabetic rats was examined by Western blotting and immunohistochemistry analyses. Terminal transferase dUTP nick end labeling (TUNEL) assay and immunofluorescent staining specific for caspase-3 and AIF were applied to analyze for apoptosis of retinal ganglion cells. In addition, a caspase-3 inhibitor DEVD-CHO was injected intravitreally to further determine the apoptotic pathways of retinal ganglion cells triggered in acute diabetes. Results Two weeks after induction of diabetes, a significant increase in caspase-3 protein expression and localization occurred in the nerve fiber layer, ganglion cell layer, and inner plexiform layer of the retina. Four weeks after the onset of diabetes, the increase in caspase-3 expression was profound eight weeks postinduction of diabetes (P<0.05). Meanwhile, no AIF protein expression was detected in this study. In addition, intravitreal administration of the caspase-3 inhibitor DEVD-CHO reduced apoptosis of retinal ganglion cells by its direct inhibitory action on caspase-3. Conclusion Caspase-dependent apoptotic pathways may be the main stimulant of STZ-induced retinal ganglion cell apoptosis in acute diabetes.

  3. Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

    OpenAIRE

    Chung, K N; Saikawa, Y; Paik, T H; Dixon, K H; Mulligan, T.; Cowan, K. H.; Elwood, P. C.

    1993-01-01

    A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable hum...

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

    Science.gov (United States)

    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

  5. Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Carrano, A.V.; Salazar, E.; Felton, J.S.; Hatch, F.T.

    1983-01-01

    As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido(4,3-b)indole) and IQ (2-amino-3-methylimidazo(4,5-f)quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr/sup +/ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 ..mu..g/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 ..mu..g/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. 38 references, 3 figures, 1 table.

  6. Promoter and 11-kilobase upstream enhancer elements responsible for hepatoma cell-specific expression of the rat ornithine transcarbamylase gene.

    OpenAIRE

    Murakami, T.; Nishiyori, A; Takiguchi, M.; Mori, M

    1990-01-01

    The gene for ornithine transcarbamylase (OTC; EC 2.1.3.3), a urea cycle enzyme, is expressed almost exclusively in the liver and small intestine. To identify DNA elements regulating transcription of the OTC gene in the liver, transient expression analysis was carried out by using hepatoma (HepG2) and nonhepatic (CHO) cell lines. The 1.3-kilobase 5'-flanking region of the rat OTC gene directed expression of the fused chloramphenicol acetyltransferase gene in HepG2 cells much more efficiently t...

  7. Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans.

    Science.gov (United States)

    Iqbal, M; Flick-Smith, H; McCauley, J W

    2000-02-01

    Recombinant E(rns) glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble E(rns) to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged E(rns) to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. E(rns) failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. E(rns) also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of E(rns) binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-L-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of E(rns) to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction. PMID:10644844

  8. Evaluation of Cellular Toxicity for Cisplatin, Arsenic And Acetaminophen in the Cancer and Normal Cell Line

    Directory of Open Access Journals (Sweden)

    S Saeedi Saravi

    2007-12-01

    Full Text Available Introduction: Cell culture is a process in which the cells ware isolated from original tissue, dispersed in liquid media and then placed in culture plate where the cells adhere together and propagate. Today, this method is used for assessment of cell toxicity, its mechanisms and effect of different compounds on intracellular components. Methods: Clonogenic assay was used for assessment of cell toxicity and amount of cell death after a specific time during which cells were exposed to different compounds. Thus, IC50 in caner cell lines (HePG2, SKOV3 and A549 and normal cell (LLCPK1, CHO and HGF1 was assessed after exposure to cisplatin, acetaminophen and arsenic. Results: Results showed that acetaminophen has maximum resistance and minimum sensitivity in CHO line with IC50=16.7±1.06 HePG2 with IC50=18.6±1.29. On the other hand, cisplatin showed minimum resistance and maximum sensitivity in HePG2 with IC50 = 0.87±0.07 and HGF1 with IC50 = 1.6±0.21 and lastly, arsenic showed minimum resistance and maximum sensitivity in A549 with IC50 = 4.59±0.29 and LLCPK1 with IC50= 1±0.37. Discussion: According to the evaluated IC50, there were differences between results of sensitivity of cell lines exposed to the three drugs (P<0.05. Entirely, resistance in cancer cell lines was lower than normal cells. The results showed the importance of cell defensive mechanisms encountering different substances like glutathione.

  9. Recombinant DNA repair genes

    International Nuclear Information System (INIS)

    We have developed a gene transfer system with Chinese hamster ovary (CHO) cells to identify, characterize, and potentially isolate functionally homologous human or CHO genes regulating repair initiation

  10. The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane. - Highlights: →DDT is a pesticide with a severe environmental impact →Epidemiologic correlation exists between exposition to DDT and thyroid dysfunction →DDT is a lipophilic molecule that has been shown to inhibit TSH receptor

  11. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

    Science.gov (United States)

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E

    2016-01-01

    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. PMID:26587808

  12. The Application of Dielectric Spectroscopy and Biocalorimetry for the Monitoring of Biomass in Immobilized Mammalian Cell Cultures

    Directory of Open Access Journals (Sweden)

    Harriet E. Cole

    2015-05-01

    Full Text Available The purpose of this study was to introduce dielectric spectroscopy and biocalorimetry as monitoring methods to follow immobilised Chinese Hamster Ovary (CHO cell culture development. The theory behind both monitoring techniques is explained and perfusion cultures are performed in a Reaction Calorimeter (eRC1 from Mettler Toledo as an application example. The findings of this work show that dielectric spectroscopy gives highly reliable information upon the viable cell density throughout the entire culture. On the other hand, the RC1 could only provide accurate data from day 5, when the cell density exceeded 4 × 106 vcells∙mL−1 (viable cell per mL working volume (WV. The method validation showed the limit of detection (LOD for 1.4 L cultures to be 8.86 × 106 vcells∙mL−1, a viable cell density commonly achieved in fed-batch and the early stages of a perfusion culture. This work suggests that biocalorimetry should be possible to implement at industrial scale to monitor CHO cell cultures.

  13. Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspas-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

  14. Molecular Characteristic of MAR that Increases Transgene Expression in Recombinant CHO Cells%MAR提高重组CHO细胞中转基因表达的分子特性分析

    Institute of Scientific and Technical Information of China (English)

    孙秋丽; 张俊河; 赵春澎; 王小引; 王天云

    2016-01-01

    分析核基质结合区(matrix attachment region,MAR)调控转基因表达的分子序列特征,鉴定能有效提高CHO细胞转基因表达的MAR特征性元件.将人β-珠蛋白MAR片段从5'到3'端分为6个分段(1 ~ 540,421 ~1 020,901 ~1 500,1 381~1 980,1 861 ~2 460,2 341~2 999位置),分别采用PCR进行克隆,经测序证实正确后,分别连接到含有氯霉素乙酰转移酶(chloramphenicol acetyltransferase,CAT)报告基因的表达载体SV40启动子及上游,构建β-珠蛋白MAR渐次片段介 导的表达载体,转染CHO细胞,G418筛选出稳定表达细胞株,ELISA分析CAT报告基因的表达水平,生物信息学分析MAR序列特征.结果表明,β-珠蛋白MAR全长能显著提高转基因的表达,6个渐次片段相比较,421~1 020位的第2个分段和901~l 500位的第3个分段提高转基因表达作用显著.生物信息学分析结果显示,MAR-like motif有助于转基因表达提高.

  15. Applications of Crispr/Cas9 in Gene Disruption of CHO Cells%Crispr/Cas9技术在CHO细胞中基因敲除的应用

    Institute of Scientific and Technical Information of China (English)

    孙涛; 王佳贤; 李朝东; 朱建伟

    2015-01-01

    Crispr/Cas9技术是近年发展起来的基因编辑技术,已经在人、动物、植物等各类细胞中得到广泛应用.本文基于文献和本实验室研究成果,介绍了Crispr/Cas9技术在CHO细胞中运用的基本操作方法,以及与重组蛋白表达相关的基因敲除实例,对可能遇到的问题做出了技术解释.笔者认为可利用Crispr/Cas9技术对CHO细胞进行改造以创造出新的工程细胞株,为生物药物开发及生产服务.

  16. A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery

    OpenAIRE

    Yi Huan; Linyi Li; Quan Liu; Shuainan Liu; Zhufang Shen

    2013-01-01

    The sodium/glucose cotransporter 2 (SGLT2) is responsible for the majority of glucose reabsorption in the kidney, and currently, SGLT2 inhibitors are considered as promising hypoglycemic agents for the treatment of type 2 diabetes mellitus. By constructing CHO cell lines that stably express the human SGLT2 transmembrane protein, along with a fluorescent glucose transporter assay that uses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]2-deoxyglucose (2-NBDG) as a glucose analog, we have develo...

  17. Ataxia telangiectasia mutated and Rad3 related (ATR protein kinase inhibition is synthetically lethal in XRCC1 deficient ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Rebeka Sultana

    Full Text Available INTRODUCTION: Ataxia telangiectasia mutated and Rad3 Related (ATR protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response. METHODS: In the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO and human ovarian cancer cells using ATR inhibitors (NU6027. In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed. RESULTS: ATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells. CONCLUSIONS: Our data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.

  18. Effects of turmeric and its active principle, curcumin, on bleomycin-induced chromosome aberrations in Chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Maria Cristina P. Araújo

    1999-09-01

    Full Text Available Naturally occurring antioxidants have been extensively studied for their capacity to protect organisms and cells from oxidative damage. Many plant constituents including turmeric and curcumin appear to be potent antimutagens and antioxidants. The effects of turmeric and curcumin on chromosomal aberration frequencies induced by the radiomimetic agent bleomycin (BLM were investigated in Chinese hamster ovary (CHO cells. Three concentrations of each drug, turmeric (100, 250 and 500 mg/ml and curcumin (2.5, 5 and 10 mg/ml, were combined with BLM (10 mg/ml in CHO cells treated during the G1/S, S or G2/S phases of the cell cycle. Neither turmeric nor curcumin prevented BLM-induced chromosomal damage in any phases of the cell cycle. Conversely, a potentiation of the clastogenicity of BLM by curcumin was clearly observed in cells treated during the S and G2/S phases. Curcumin was also clastogenic by itself at 10 µg/ml in two protocols used. However, the exact mechanism by which curcumin produced clastogenic and potentiating effects remains unknown.Antioxidantes de ocorrência natural têm sido exaustivamente estudados quanto a sua capacidade de proteger organimos e células contra danos oxidativos. Muitos constituintes das plantas, incluindo cúrcuma e curcumina, parecem ser potentes antimutágenos e antioxidantes. Os efeitos de cúrcuma e curcumina na freqüência de aberrações cromossômicas induzidas pelo agente radiomimético bleomicina (BLM foram investigados em células do ovário de hamster chinês (CHO. Três concentrações de cada droga, cúrcuma (100, 250 e 500 mg/ml e curcumina (2,5, 5,0 e 10 mg/ml, foram combinadas com BLM (10 mg/ml em células CHO tratadas durante as fases G1/S, S ou G2/S do ciclo celular. Nem cúrcuma nem curcumina evitaram o dano cromossômico induzido pela BLM em fase alguma do ciclo celular. Ao contrário, a potenciação da clastogenicidade da BLM pelo curcumina foi nitidamente observada em células tratadas

  19. Detailed analysis of the cell-inactivation mechanism by accelerated protons and light ions

    CERN Document Server

    Kundrát, P

    2006-01-01

    Published survival data for V79 cells irradiated by monoenergetic protons, helium-3, carbon, and oxygen ions and for CHO cells irradiated by carbon ions have been analyzed using the probabilistic two-stage model of cell inactivation. Three different classes of DNA damages formed by traversing particles have been distinguished, namely severe single-track damages which might lead to cell inactivation directly, less severe damages where cell inactivation is caused by their combinations, and damages of negligible severity that can be repaired easily. Probabilities of single ions to form these damages have been assessed in dependence on their linear energy transfer (LET) values. Damage induction probabilities increase with atomic number and LET. While combined damages play crucial role at lower LET values, single-track damages dominate in high-LET regions. The yields of single-track lethal damages for protons have been compared with the Monte Carlo estimates of complex DNA lesions, indicating that lethal events co...

  20. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    International Nuclear Information System (INIS)

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-13C-glucose and 15N-glutamate as labeled precursors. This study suggests that uniformly 15N,13C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.