Sample records for hamster cho cell

  1. DNA methylation in CHO cells.

    PubMed

    Wippermann, Anna; Noll, Thomas

    2017-09-20

    Chinese hamster ovary (CHO) cells account for the production of the majority of biopharmaceutical molecules - however, the molecular basis for their versatile properties is not entirely understood yet and the underlying cellular processes need to be characterized in detail. One such process that is supposed to contribute significantly to CHO cell phenotype is methylation of DNA at cytosine residues. DNA methylation was shown to be involved in several central biological processes in humans and to contribute to diseases like cancer. Early studies of DNA methylation in CHO mostly focused on methylation of single recombinant genes and promoters and proved a correlation between DNA methylation status and recombinant gene expression or production stability. More recent publications utilized the CHO genomic and transcriptomic data available since 2011 and provided first insights into the CHO DNA methylation landscape and DNA methylation changes in response to effector molecules or culture conditions. Generally, further genome-wide studies of DNA methylation in CHO will be required to shed light on the relevance of this process regarding biopharmaceuticals production and might, e.g., address a potential link between CHO cell metabolism and DNA methylation or provide novel targets for rational cell line engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

    PubMed

    Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

    2018-05-15

    Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

  4. Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

    PubMed

    Rasmussen, B; Davis, R; Thomas, J; Reddy, P

    1998-11-01

    The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.

  5. Glycoengineering of CHO Cells to Improve Product Quality.

    PubMed

    Wang, Qiong; Yin, Bojiao; Chung, Cheng-Yu; Betenbaugh, Michael J

    2017-01-01

    Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest. Furthermore, altering the glycan composition can be used to modulate the production quality of a recombinant biotherapeutic from CHO and other mammalian hosts. This review first describes the glycosylation network in mammalian cells and compares the glycosylation patterns between CHO and human cells. Next genetic strategies used in CHO cells to modulate the sialylation patterns through overexpression of sialyltransfereases and other glycosyltransferases are summarized. In addition, other approaches to alter sialylation including manipulation of sialic acid biosynthetic pathways and inhibition of sialidases are described. Finally, this review also covers other strategies such as the glycosylation site insertion and manipulation of glycan heterogeneity to produce desired glycoforms for diverse biotechnology applications.

  6. Separation of CHO cells using hydrocyclones.

    PubMed

    Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

    2008-01-01

    Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

  7. Glycoengineering in CHO Cells: Advances in Systems Biology.

    PubMed

    Tejwani, Vijay; Andersen, Mikael R; Nam, Jong Hyun; Sharfstein, Susan T

    2018-03-01

    For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post-translational modifications, particularly glycosylation, which unlike protein synthesis, is a non-templated process. Consequently, both native and recombinant glycoprotein production generate heterogeneous mixtures containing variable amounts of different glycoforms. Stability, potency, plasma half-life, and immunogenicity of the glycoprotein biologic are directly influenced by the glycoforms. Recently, CHO cells have also been explored for production of therapeutic glycosaminoglycans (e.g., heparin), which presents similar challenges as producing glycoproteins biologics. Approaches to controlling heterogeneity in CHO cells and directing the biosynthetic process toward desired glycoforms are not well understood. A systems biology approach combining different technologies is needed for complete understanding of the molecular processes accounting for this variability and to open up new venues in cell line development. In this review, we describe several advances in genetic manipulation, modeling, and glycan and glycoprotein analysis that together will provide new strategies for glycoengineering of CHO cells with desired or enhanced glycosylation capabilities. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Inhibition of proteinase 3 (PR3) by suramin and fetal calf serum (FCS): effect of PR3 and suramin on Chinese hamster ovary cells (CHO-cells).

    PubMed

    Karam, Gholamreza Asadi; Rasaee, Mohammad Javad; Mahmoodi, Mehdi; Khaksari, Mohammad

    2005-07-01

    Proteinase 3 (PR3) is a lysosomal protease that is stored in azurophilic granules neutrophilic granulocytes and monocytes. A number of inhibitors for this proteinase are reported. Comprehensive studies on the inhibitory effect of suramin and heat treated fetal calf serum (deltaFCS) on PR3 have not been reported. It has been reported that PR3 is able to destroy the cytoskeletal integral proteins, but we have not find any reports which showed the effect of this protease on Chinese hamster ovary cells (CHO-cells) in culture medium. Suramin has proven to be useful as an antitumor drug, but there was not any report on the effect of suramin on CHO-cells. The effects of various concentrations of deltaFCS (from 0.5% up to 10%) and suramin (from 0.8 microM up to 100 microM) on PR3 and different concentrations of suramin (from 0.8 microM up to 1000 microM) on CHO-cells were investigated. Data analysis were performed by, Kolmogorov-Smirnov test, ANOVA test and Tukey HSD post tests. Results showed that deltaFCS and suramin have an inhibitory effect on PR3 and these effects increased with increasing the concentration significantly (p < 0.01). PR3 with the concentration of 2.2 Unit/ml has no effect on CHO-cells. Although suramin with the concentration of less than 125 microM cell growth retarded for only a few hours, but with the concentration of 125 to 250 microM inhibit the cell growth for a week, and after that cells gain normal growth gradually. Suramin with concentration of more than 500 microM inhibited the cell growth completely. Although suramin reversibly inhibit the PR3 activity but in concentration of less than 250 microM it had no long-term effect on CHO-cells. Therefore it can be used in the investigation of proteases. There were unknown components in deltaFCS, which cause the inhibition of PR3 activity. This finding is very important in PR3 production in culture medium. However CHO-cells are resistant to PR3 and suramin in low concentration.

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

    PubMed

    Di Virgilio, A L; Reigosa, M; Arnal, P M; Fernández Lorenzo de Mele, M

    2010-05-15

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO(2)) and aluminium oxide (Al(2)O(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 24h 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 24h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 microg/mL TiO(2) and 0.5-10 microg/mL Al(2)O(3). SCE frequencies were higher for cells treated with 1-5 microg/mL TiO(2). The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO(2). No SCE induction was achieved after treatment with 1-25 microg/mL Al(2)O(3). In conclusion, findings showed cytotoxic and genotoxic effects of TiO(2) and Al(2)O(3) NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  10. Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

    PubMed

    Kamachi, Yasuharu; Omasa, Takeshi

    2018-04-01

    Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The art of CHO cell engineering: A comprehensive retrospect and future perspectives.

    PubMed

    Fischer, Simon; Handrick, René; Otte, Kerstin

    2015-12-01

    Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out.

    PubMed

    Laux, Holger; Romand, Sandrine; Nuciforo, Sandro; Farady, Christopher J; Tapparel, Joel; Buechmann-Moeller, Stine; Sommer, Benjamin; Oakeley, Edward J; Bodendorf, Ursula

    2018-05-19

    An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using "Transcription Activator-Like Effector Nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

    PubMed

    Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

    2015-01-01

    Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

  14. Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture.

    PubMed

    Han, Seora; Rhee, Won Jong

    2018-05-01

    Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano-sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase-3 activation, meaning that the exosomes enhanced cellular activities under pro-apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial. © 2018 Wiley Periodicals, Inc.

  15. Functional heterogeneity and heritability in CHO cell populations.

    PubMed

    Davies, Sarah L; Lovelady, Clare S; Grainger, Rhian K; Racher, Andrew J; Young, Robert J; James, David C

    2013-01-01

    In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and

  16. Model-based analysis of N-glycosylation in Chinese hamster ovary cells

    PubMed Central

    Krambeck, Frederick J.; Bennun, Sandra V.; Betenbaugh, Michael J.

    2017-01-01

    The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of

  17. Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

    PubMed Central

    Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

    2017-01-01

    Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

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

    NASA Astrophysics Data System (ADS)

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

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

  19. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    PubMed

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

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

    NASA Astrophysics Data System (ADS)

    Awasthi, Kumud Kant; Awasthi, Anjali; Kumar, Narender; Roy, Partha; Awasthi, Kamlendra; John, P. J.

    2013-09-01

    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.

  1. A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level.

    PubMed

    Baik, Jong Youn; Lee, Kelvin H

    2017-05-01

    Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Computational modelling of genome-scale metabolic networks and its application to CHO cell cultures.

    PubMed

    Rejc, Živa; Magdevska, Lidija; Tršelič, Tilen; Osolin, Timotej; Vodopivec, Rok; Mraz, Jakob; Pavliha, Eva; Zimic, Nikolaj; Cvitanović, Tanja; Rozman, Damjana; Moškon, Miha; Mraz, Miha

    2017-09-01

    Genome-scale metabolic models (GEMs) have become increasingly important in recent years. Currently, GEMs are the most accurate in silico representation of the genotype-phenotype link. They allow us to study complex networks from the systems perspective. Their application may drastically reduce the amount of experimental and clinical work, improve diagnostic tools and increase our understanding of complex biological phenomena. GEMs have also demonstrated high potential for the optimisation of bio-based production of recombinant proteins. Herein, we review the basic concepts, methods, resources and software tools used for the reconstruction and application of GEMs. We overview the evolution of the modelling efforts devoted to the metabolism of Chinese Hamster Ovary (CHO) cells. We present a case study on CHO cell metabolism under different amino acid depletions. This leads us to the identification of the most influential as well as essential amino acids in selected CHO cell lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells

    PubMed Central

    Davami, Fatemeh; Baldi, Lucia; Rajendra, Yashas; M. Wurm, Florian

    2014-01-01

    The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells. PMID:25317401

  4. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  5. Aryl- and alkyl-phosphorus-containing flame retardants induced mitochondrial impairment and cell death in Chinese hamster ovary (CHO-k1) cells.

    PubMed

    Huang, Chao; Li, Na; Yuan, Shengwu; Ji, Xiaoya; Ma, Mei; Rao, Kaifeng; Wang, Zijian

    2017-11-01

    Phosphorus-containing flame retardants (PFRs) are increasingly in demand worldwide as replacements for brominated flame retardants (BFRs), but insufficient available toxicological information on PFRs makes assessing their health risks challenging. Mitochondria are important targets of various environmental pollutants, and mitochondrial dysfunction may lead to many common diseases. In the present study, mitochondria impairment-related endpoints were measured by a high content screening (HCS) assay for 11 selected non-halogen PFRs in Chinese hamster ovary (CHO-k1) cells. A cluster analysis was used to categorize these PFRs into three groups according to their structural characteristics and results from the HCS assay. Two groups, containing long-chain alkyl-PFRs and all aryl-PFRs, were found to cause mitochondrial impairment but showed different mechanisms of toxicity. Due to the high correlation between cell death and mitochondrial impairment, two PFRs with different structures, trihexyl phosphate (THP) and cresyl diphenyl phosphate (CDP), were selected and compared with chlorpyrifos (CPF) to elucidate their mechanism of inducing cell death. THP (an alkyl-PFR) was found to utilize a similar pathway as CPF to induce apoptosis. However, cell death induced by CDP (an aryl-PFR) was different from classical necrosis based on experiments to discriminate among the different modes of cell death. These results confirm that mitochondria might be important targets for some PFRs and that differently structured PFRs could function via distinct mechanisms of toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Adhesion and migration of CHO cells on micropatterned single layer graphene

    NASA Astrophysics Data System (ADS)

    Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

    2017-06-01

    Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

  7. The emerging CHO systems biology era: harnessing the 'omics revolution for biotechnology.

    PubMed

    Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan E; Betenbaugh, Michael J

    2013-12-01

    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 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 production and bioprocessing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture.

    PubMed

    Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-03-02

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

  9. miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

    PubMed

    Fischer, Simon; Paul, Albert Jesuran; Wagner, Andreas; Mathias, Sven; Geiss, Melanie; Schandock, Franziska; Domnowski, Martin; Zimmermann, Jörg; Handrick, René; Hesse, Friedemann; Otte, Kerstin

    2015-10-01

    Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells. © 2015 Wiley Periodicals, Inc.

  10. Mechanisms driving the lactate switch in Chinese hamster ovary cells.

    PubMed

    Hartley, Fiona; Walker, Tracy; Chung, Vicky; Morten, Karl

    2018-03-31

    The metabolism of Chinese Hamster Ovary (CHO) cells in a production environment has been extensively investigated. However, a key metabolic transition, the switch from lactate production to lactate consumption, remains enigmatic. Though commonly observed in CHO cultures, the mechanism(s) by which this metabolic shift is triggered is unknown. Despite this, efforts to control the switch have emerged due to the association of lactate consumption with improved cell growth and productivity. This review aims to consolidate current theories surrounding the lactate switch. The influence of pH, NAD + /NADH, pyruvate availability and mitochondrial function on lactate consumption are explored. A hypothesis based on the cellular redox state is put forward to explain the onset of lactate consumption. Various techniques implemented to control the lactate switch, including manipulation of the culture environment, genetic engineering, and cell line selection are also discussed. © 2018 Wiley Periodicals, Inc.

  11. Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

    NASA Astrophysics Data System (ADS)

    Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

    2014-03-01

    Exposure to nano-second 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. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. 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 role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to

  12. Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

    PubMed

    Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

    2015-09-18

    BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

  13. THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS

    PubMed Central

    Kedinger, Claude; Simard, Rene

    1974-01-01

    α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells. PMID:4474178

  14. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    PubMed

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

  15. Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

    NASA Astrophysics Data System (ADS)

    Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

    2010-02-01

    Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

  16. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    PubMed

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  17. Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures.

    PubMed

    Hiller, Gregory W; Ovalle, Ana Maria; Gagnon, Matthew P; Curran, Meredith L; Wang, Wenge

    2017-07-01

    A simple method originally designed to control lactate accumulation in fed-batch cultures of Chinese Hamster Ovary (CHO) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. The method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. When the short-duration cell-controlled perfusion is performed in the production bioreactor and is immediately followed by a conventional fed-batch culture using highly concentrated feeds, the overall productivity of the culture is approximately doubled when compared with a highly optimized state-of-the-art fed-batch process. The technology was applied with near uniform success to five CHO cell processes producing five different humanized monoclonal antibodies. The increases in productivity were due to the increases in sustained viable cell densities. Biotechnol. Bioeng. 2017;114: 1438-1447. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells.

    PubMed

    Chun, B H; Bang, W G; Park, Y K; Woo, S K

    2001-11-01

    The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed.

  19. Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells.

    PubMed

    Bhanumathy, Cunnigaiper D; Nakao, Steven K; Joseph, Suresh K

    2006-02-10

    myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.

  20. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

    PubMed Central

    DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

    2010-01-01

    Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of <0.0001 for the 100 individual samples. We, therefore, conclude that the improved IgG anti-PT ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

  1. A signature of 12 microRNAs is robustly associated with growth rate in a variety of CHO cell lines.

    PubMed

    Klanert, Gerald; Jadhav, Vaibhav; Shanmukam, Vinoth; Diendorfer, Andreas; Karbiener, Michael; Scheideler, Marcel; Bort, Juan Hernández; Grillari, Johannes; Hackl, Matthias; Borth, Nicole

    2016-10-10

    As Chinese Hamster Ovary (CHO) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. At the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. In this context, microRNAs (miRNAs) that exhibit a robust correlation to the growth rate of CHO cells were determined by analyzing miRNA expression profiles in a comprehensive collection of 46 samples including CHO-K1, CHO-S and CHO-DUKXB11, which were adapted to various culture conditions, and analyzed in different growth stages using microarrays. By applying Spearman or Pearson correlation coefficient criteria of>|0.6|, miRNAs with high correlation to the overall growth, or growth rates observed in exponential, serum-free, and serum-free exponential phase were identified. An overlap of twelve miRNAs common for all sample sets was revealed, with nine positively and three negatively correlating miRNAs. The here identified panel of miRNAs can help to understand growth regulation in CHO cells and contains putative engineering targets as well as biomarkers for cell lines with advantageous growth characteristics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

  3. Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Hacker, David L

    2017-01-01

    We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

  4. Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.

    PubMed

    Han, Yi; Liu, Xing-Mao; Liu, Hong; Li, Shi-Chong; Wu, Ben-Chuan; Ye, Ling-Ling; Wang, Qu-Wei; Chen, Zhao-Lie

    2006-11-01

    Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

  5. A mechanistic study on the effect of dexamethasone in moderating cell death in Chinese Hamster Ovary cell cultures.

    PubMed

    Jing, Ying; Qian, Yueming; Ghandi, Mahmoud; He, Aiqing; Borys, Michael C; Pan, Shih-Hsie; Li, Zheng Jian

    2012-01-01

    Dexamethasone (DEX) was previously shown (Jing et al., Biotechnol Bioeng. 2010;107:488-496) to play a dual role in increasing sialylation of recombinant glycoproteins produced by Chinese Hamster Ovary (CHO) cells. DEX addition increased sialic acid levels of a recombinant fusion protein through increased expression of α2,3-sialyltransferase and β1,4-galactosyltransferase, but also decreased the sialidase-mediated, extracellular degradation of sialic acid through slowing cell death at the end of the culture period. This study examines the underlying mechanism for this cytoprotective action by studying the transcriptional response of the CHO cell genome upon DEX treatment using DNA microarrays and gene ontology term analysis. Many of those genes showing a significant transcriptional response were associated with the regulation of programmed cell death. The gene with the highest change in expression level, as validated by Quantitative PCR assays with TaqMan® probes and confirmed by Western Blot analysis, was the antiapoptotic gene Tsc22d3, also referred to as GILZ (glucocorticoid-induced leucine zipper). The pathway by which DEX suppressed cell death towards the end of the culture period was also confirmed by showing involvement of glucocorticoid receptors and GILZ through studies using the glucocorticoid antagonist mifepristone (RU-486). These findings advance the understanding of the mechanism by which DEX suppresses cell death in CHO cells and provide a rationale for the application of glucocorticoids in CHO cell culture processes. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  6. Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

    PubMed Central

    Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A.; Esko, Jeffrey D.; Linhardt, Robert J.; Sharfstein, Susan T.

    2012-01-01

    Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary. PMID:22326251

  7. Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

    PubMed

    Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

    2012-03-01

    Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

    PubMed

    Vito, Davide; Smales, C Mark

    2018-05-21

    The role of non-coding RNAs in determining growth, productivity and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non-coding RNAs have received less attention. One such class are the non-coding RNAs known collectively as long non-coding RNAs (lncRNAs). We have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. We report on those lncRNAs present in a model host CHO cell line under batch and fed-batch conditions on two different days and relate the expression of different lncRNAs to each other. We demonstrate that the mouse microarray was suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT-PCR. We then further analysed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed-batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. We discuss the implications for the publication of this rich dataset and how this may be used by the community. This article is protected by copyright. All rights reserved.

  9. Growth, metabolic activity, and productivity of immobilized and freely suspended CHO cells in perfusion culture.

    PubMed

    Hilal-Alnaqbi, Ali; Hu, Alan Y C; Zhang, Zhibing; Al-Rubeai, Mohamed

    2013-01-01

    Chinese hamster ovary (CHO) cells producing β-galactosidase (β-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10(8) cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10(7) cells/mL medium. The β-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific β-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  10. Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

    PubMed

    Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

    2013-12-16

    Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

  11. CHO microRNA engineering is growing up: Recent successes and future challenges☆

    PubMed Central

    Jadhav, Vaibhav; Hackl, Matthias; Druz, Aliaksandr; Shridhar, Smriti; Chung, Cheng-Yu; Heffner, Kelley M.; Kreil, David P.; Betenbaugh, Mike; Shiloach, Joseph; Barron, Niall; Grillari, Johannes; Borth, Nicole

    2013-01-01

    microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines. PMID:23916872

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1986-05-05

    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 (/sup 32/P)phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with (/sup 32/P)phosphatidylethanolamine, the mutant incorporated themore » 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.« less

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

    PubMed

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

    2016-09-01

    Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    PubMed

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. Mutation and repair in an ultraviolet-sensitive Chinese hamster ovary cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wood, R.D.

    1981-11-01

    An ultraviolet (UV) light-sensitive mutant of Chinese hamster ovary cells (CHO) has been isolated and characterized with respect to a number of post-irradiation responses. The UV-sensitive mutant, termed 43-3B, has the same growth rate and chromosome number as the wild-type CHO-9. 43-3B is hypersensitive to the lethal effects of UV light (D/sub 0/ of 0.3 J/m/sup 2/ as compared to 3.2 J/m/sup 2/ for the wild-type). A marked UV-hypermutability is observed in 43-3B as compared to the wild-type, as measured with markers for induced resistance to 6-thioguanine, ouabain, and diphtheria toxin. A factor of 38 to 65 more mutations aremore » induced per unit fluence in 43-3B than in CHO-9. The UV-sensitive mutant is also sensitive to killing by simulated solar light, although the D/sub 0/ ratio is not as great as for germicidal UV. 43-3B exhibits only about 17% of the wild-type level of UV-stimulated DNA repair synthesis, as measured by autoradiography of G/sub 1/ phase cells. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild-type CHO-9, but little recovery was seen in 43-3B. The present investigation demonstrates that a sensitive/wild-type pair of CHO cell lines can be used in comparative studies to determine the involvement of repair in a wide range of post-irradiation phenomena.« less

  16. A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells.

    PubMed

    Gerster, Anja; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Schulze, Andreas; Krause, Felix; Müller, Dethardt

    2016-12-01

    To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

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

    PubMed

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

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with Chinese hamster ovary (CHO) cells to provide metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fischer-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B1 (AFB1) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(A)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB1 was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating metabolic pathways important in the production and detoxification of genotoxic products in vivo.

  18. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

    PubMed

    Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

    2017-08-10

    To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  19. Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

    PubMed

    Kawabe, Yoshinori; Komatsu, Shinya; Komatsu, Shodai; Murakami, Mai; Ito, Akira; Sakuma, Tetsushi; Nakamura, Takahiro; Yamamoto, Takashi; Kamihira, Masamichi

    2018-05-01

    Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

    PubMed

    Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

    2017-08-01

    Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    PubMed Central

    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

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

    PubMed

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

    2015-10-09

    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.

  3. Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.

    PubMed

    Huang, Edwin P; Marquis, Christopher P; Gray, Peter P

    2004-11-20

    The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (c) 2004 Wiley Periodicals, Inc

  4. Analyzing Clonal Variation of Monoclonal Antibody-Producing CHO Cell Lines Using an In Silico Metabolomic Platform

    PubMed Central

    Ghorbaniaghdam, Atefeh; Chen, Jingkui; Henry, Olivier; Jolicoeur, Mario

    2014-01-01

    Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones. PMID

  5. CHOmine: an integrated data warehouse for CHO systems biology and modeling

    PubMed Central

    Hanscho, Michael; Ruckerbauer, David E.; Zanghellini, Jürgen; Borth, Nicole

    2017-01-01

    Abstract The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. Database URL: http://www.chogenome.org PMID:28605771

  6. CHOmine: an integrated data warehouse for CHO systems biology and modeling.

    PubMed

    Gerstl, Matthias P; Hanscho, Michael; Ruckerbauer, David E; Zanghellini, Jürgen; Borth, Nicole

    2017-01-01

    The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. http://www.chogenome.org. © The Author(s) 2017. Published by Oxford University Press.

  7. 13 C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures.

    PubMed

    McAtee Pereira, Allison G; Walther, Jason L; Hollenbach, Myles; Young, Jamey D

    2018-02-06

    13 C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism of industrial cell lines. In this study, 13 C MFA was used to characterize the metabolic response of Chinese hamster ovary (CHO) cells to a novel medium variant designed to reduce ammonia production. Ammonia inhibits growth and viability of CHO cell cultures, alters glycosylation of recombinant proteins, and enhances product degradation. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel 13 C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how 13 C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantitative mammalian cell genetic toxicology: study of the cytotoxicity and mutagenicity of 70 individual environmental agents related to energy technologies and 3 subfractions of a crude synthetic oil in the CHO/HGPRT system. [Hamsters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hsie, A W; ,; Neill, J P

    1978-01-01

    Conditions necessary for quantifying mutation-induction to 6-thioguanine resistance, which selects for >98% mutants deficient in the activity of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) in a near-diploid Chinese hamster ovary (CHO) cell line, referred to as CHO/HGPRT system, have been defined. Employing this mutation assay, we have determined the mutagenicity of diversified agents including 11 direct-acting alkylating agents, 16 nitrosamines, 10 heterocyclic nitrogen mustards, 15 metallic compounds, 5 quinolines, 5 aromatic amines, 27 polycyclic hydrocarbons, 13 miscellaneous chemicals, 7 ionizing and non-ionizing physical agents. The direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine is mutagenic while its noncarcinogenic analogue N-methyl-N'-nitro-N-nitroguanidine is not. Coupled with the rat livermore » S/sub 9/-activation system, procarcinogens such as nitrosopyrrolidine, benzo(a)pyrene, and 2-acetylaminofluorene are mutagenic while their analogues 2,5-dimethylnitrosopyrrolidine, pyrene and fluorene are not. The assay appears to be applicable for monitoring the genetic toxicity of crude organic mixtures in addition to diverse individual chemical and physical agents. The quantitative nature of the assay enables a study of EMS exposure dose: the mutagenic potential of EMS can be described as 310 x 10/sup -6/ mutants (cell mg ml/sup -1/ h)./sup -1/ It is also feasible to expand the CHO/HGPRT system for quantifying cytotoxicity and mutagenicity to determination of chromosomal aberrations and sister chromatid exchanges in cells treated under identical conditions which allows a simultaneous study of these four distinctive biological effects.« less

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

    NASA Astrophysics Data System (ADS)

    Nicoletti, Sarah E.

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

  10. Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells.

    PubMed

    Wan, Aini; Xu, Dongsheng; Liu, Kedong; Peng, Lin; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

    2017-08-09

    Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA-IGF-1 reached 100 mg/L. The fusion protein HSA-IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA-IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA-IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA-IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

  11. Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

    PubMed

    de Wit, C; Fautz, C; Xu, Y

    2000-09-01

    Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

  12. Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures.

    PubMed

    Kong, D; Gentz, R; Zhang, J

    1998-03-01

    Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2-6 × 10(6) cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4-10 mg/L. Specific productivity was maintained at 1.8-3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted.

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

    PubMed

    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. Sindbis virus glycoproteins are abnormally glycosylated in Chinese hamster ovary cells deprived of glucose.

    PubMed

    Davidson, S K; Hunt, L A

    1985-07-01

    We have previously demonstrated that Sindbis virus infection of Chinese hamster ovary (CHO) cells altered the protein glycosylation machinery of the cell, so that both normal, full-size (nine mannose-containing) oligosaccharides and abnormal, "truncated' (five mannose-containing) oligosaccharides are transferred from lipid-linked precursors to newly synthesized viral membrane glycoproteins. In the present studies, we have examined the precursor oligosaccharides on viral glycoproteins that were pulse-labelled with [3H]mannose in the presence or absence of glucose, since glucose starvation of uninfected CHO cells has been reported to induce synthesis of truncated precursor oligosaccharides. Pulse-labelling in the absence of glucose led to a greater than 10-fold increase in the relative amount of the truncated precursor oligosaccharides being transferred to the newly synthesized viral glycoproteins and to an apparent underglycosylation of some precursor viral polypeptides, with some asparaginyl sites not acquiring covalently linked oligosaccharides. The mature virion glycoproteins from CHO cells which were pulse-labelled in the absence of glucose and then 'chased' in the presence of glucose contained proportionately more unusual Man3GlcNAc2-size oligosaccharides. These small neutral-type oligosaccharides were apparently not as good a substrate for further processing into complex acidic-type oligosaccharides as the normal Man5GlcNAc2 intermediate that results from the full-size precursor oligosaccharides.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jones, Meredith B., E-mail: mbauman7@jhu.edu; Tomiya, Noboru, E-mail: ntomiya1@jhu.edu; Betenbaugh, Michael J., E-mail: beten@jhu.edu

    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 inmore » 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.« less

  16. Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

    PubMed

    Hu, Zhilan; Guo, Donglin; Yip, Shirley S M; Zhan, Dejin; Misaghi, Shahram; Joly, John C; Snedecor, Bradley R; Shen, Amy Y

    2013-01-01

    Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers.

  17. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1986-05-05

    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 /sup 32/Pi and L-(U-/sup 14/C)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% ofmore » 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.« less

  18. Endoplasmic Reticulum Stress Response and Mutant Protein Degradation in CHO Cells Accumulating Antithrombin (C95R) in Russell Bodies.

    PubMed

    Kimura, Koji; Inoue, Kengo; Okubo, Jun; Ueda, Yumiko; Kawaguchi, Kosuke; Sakurai, Hiroaki; Wada, Ikuo; Morita, Masashi; Imanaka, Tsuneo

    2015-01-01

    Newly synthesized secretory proteins are folded and assembled in the endoplasmic reticulum (ER), where an efficient protein quality control system performs a critically important function. When unfolded or aggregated proteins accumulate in the ER, certain signaling pathways such as the unfolded protein response (UPR) and ER-overload response (EOR) are functionally active in maintaining cell homeostasis. Recently we prepared Chinese hamster ovary (CHO) cells expressing mutant antithrombin (AT)(C95R) under control of the Tet-On system and showed that AT(C95R) accumulated in Russell bodies (RB), large distinctive structures derived from the ER. To characterize whether ER stress takes place in CHO cells, we examined characteristic UPR and EOR in ER stress responses. We found that the induction of ER chaperones such as Grp97, Grp78 and protein disulfide isomerase (PDI) was limited to a maximum of approximately two-fold. The processing of X-box-binding protein-1 (XBP1) mRNA and the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) subunit were not induced. Furthermore, the activation of nuclear factor-kappa B (NF-κB) was not observed. In contrast, CHO cells displayed UPR and EOR when the cells were treated with thapsigargin and tumor necrosis factor (TNF)-α, respectively. In addition, a portion of the mutant AT(C95R) was degraded through proteasomes and autophagy. CHO cells do respond to ER stress but the folding state of mutant AT(C95R) does not appear to activate the ER stress signal pathway.

  19. Untangling the mechanism of 3-methyladenine (3-MA) in enhancing the specific productivity: Transcriptome analysis of recombinant CHO cells treated with 3-MA.

    PubMed

    Baek, Eric; Lee, Jae Seong; Lee, Gyun Min

    2018-06-25

    3-Methyladenine (3-MA) is a chemical additive that enhances the specific productivity (q p ) in recombinant Chinese hamster ovary (rCHO) cell lines. Different from its widely known function of inhibiting autophagy, 3-MA has instead shown to increase autophagic flux in various rCHO cell lines. Thus, the mechanism by which 3-MA enhances the q p requires investigation. To evaluate the effect of 3-MA on transcriptome dynamics in rCHO cells, RNA-seq was performed with Fc-fusion protein-producing rCHO cells treated with 3-MA. By analyzing genes that were differentially expressed following the addition of 3-MA during culture, the role of 3-MA in the biological processes of rCHO cells was identified. One pathway markedly influenced by the addition of 3-MA was the unfolded protein response (UPR). Having a close relationship with autophagy, the UPR reestablishes protein folding homeostasis under endoplasmic reticulum (ER) stress. The addition of 3-MA increased the expression of key regulators of the UPR, such as Atf4, Ddit3, and Creb3l3, further supporting the idea that the enhancement of ER capacity acts as a key in increasing the q p . Consequently, the downstream effectors of UPR, which include autophagy-promoting genes, were upregulated as well. Hence, the role of 3-MA in increasing UPR pathway could have made a salient contribution to the increased autophagic flux in rCHO cells. Taken together, transcriptome analysis improved the understanding of the role of 3-MA in gene expression dynamics in rCHO cells and its mechanism in enhancing the q p . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

    PubMed

    Coppen, S R; Newsam, R; Bull, A T; Baines, A J

    1995-04-20

    The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

  1. Cytotoxicity Evaluation of Anatase and Rutile TiO₂ Thin Films on CHO-K1 Cells in Vitro.

    PubMed

    Cervantes, Blanca; López-Huerta, Francisco; Vega, Rosario; Hernández-Torres, Julián; García-González, Leandro; Salceda, Emilio; Herrera-May, Agustín L; Soto, Enrique

    2016-07-26

    Cytotoxicity of titanium dioxide (TiO₂) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO₂ thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO₂ films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO₂ films' thickness values fell within the nanometer range (290-310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO₂ thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO₂ thin films, the number of CHO-K1 cells on the control substrate and on all TiO₂ thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO₂ films annealed at 800 °C. These results indicate that TiO₂ thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO₂ thin films in biomedical science.

  2. CHOPPI: A Web Tool for the Analysis of Immunogenicity Risk from Host Cell Proteins in CHO-Based Protein Production

    PubMed Central

    Bailey-Kellogg, Chris; Gutiérrez, Andres H; Moise, Leonard; Terry, Frances; Martin, William D; De Groot, Anne S

    2014-01-01

    Despite high quality standards and continual process improvements in manufacturing, host cell protein (HCP) process impurities remain a substantial risk for biological products. Even at low levels, residual HCPs can induce a detrimental immune response compromising the safety and efficacy of a biologic. Consequently, advanced-stage clinical trials have been cancelled due to the identification of antibodies against HCPs. To enable earlier and rapid assessment of the risks in Chinese Hamster Ovary (CHO)-based protein production of residual CHO protein impurities (CHOPs), we have developed a web tool called CHOPPI, for CHO Protein Predicted Immunogenicity. CHOPPI integrates information regarding the possible presence of CHOPs (expression and secretion) with characterizations of their immunogenicity (T cell epitope count and density, and relative conservation with human counterparts). CHOPPI can generate a report for a specified CHO protein (e.g., identified from proteomics or immunoassays) or characterize an entire specified subset of the CHO genome (e.g., filtered based on confidence in transcription and similarity to human proteins). The ability to analyze potential CHOPs at a genomic scale provides a baseline to evaluate relative risk. We show here that CHOPPI can identify clear differences in immunogenicity risk among previously validated CHOPs, as well as identify additional “risky” CHO proteins that may be expressed during production and induce a detrimental immune response upon delivery. We conclude that CHOPPI is a powerful tool that provides a valuable computational complement to existing experimental approaches for CHOP risk assessment and can focus experimental efforts in the most important directions. Biotechnol. Bioeng. 2014;111: 2170–2182. PMID:24888712

  3. Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

    PubMed

    Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2018-07-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

  4. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    PubMed

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-07-01

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

    PubMed

    Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

    2017-02-01

    Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

  6. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    PubMed Central

    Thoring, Lena; Wüstenhagen, Doreen A.; Borowiak, Maria; Stech, Marlitt; Sonnabend, Andrei; Kubick, Stefan

    2016-01-01

    Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA. PMID:27684475

  7. Anti-cell death engineering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition, Beclin-1 for autophagy induction.

    PubMed

    Lee, Jae Seong; Ha, Tae Kwang; Park, Jin Hyoung; Lee, Gyun Min

    2013-08-01

    Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti-apoptosis engineering. Recently, autophagy has received attention as a new anti-cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti-apoptosis and pro-autophagy in CHO cells (DG44) was attempted by co-overexpressing an anti-apoptotic protein, Bcl-2, and a key regulator of autophagy pathway, Beclin-1, respectively. Co-overexpression of Bcl-2 and Beclin-1 exhibited a longer culture period as well as higher viability during serum-free suspension culture, compared with the control (without co-overexpression of Bcl-2 and Beclin-1) and Bcl-2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl-2 overexpression, Beclin-1 overexpression successfully induced the increase in the autophagic marker protein, LC3-II, and autophagosome formation with the decrease in mTOR activity. Co-immunoprecipitation and qRT-PCR experiments revealed that the enforced expression of Beclin-1 increased Ulk1 expression and level of free-Beclin-1 that did not bind to the Bcl-2 despite the Bcl-2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co-overexpression of Bcl-2 and Beclin-1 also protected the cells from cell death more efficiently than Bcl-2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro-autophagy engineering together with anti-apoptosis engineering yields a synergistic effect and successfully enhances the anti-cell death engineering of CHO cells. Copyright © 2013 Wiley Periodicals, Inc.

  8. Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

    PubMed

    Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2017-01-01

    Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

  9. Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

    PubMed Central

    Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh

    2017-01-01

    Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios. PMID:28662065

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

    PubMed

    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. Copyright © 2016. Published by Elsevier B.V.

  11. The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition.

    PubMed

    Purdy, P H; Fox, M H; Graham, J K

    2005-08-01

    Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.

  12. Laboratory production of human prolactin from CHO cells adapted to serum-free suspension culture.

    PubMed

    Arthuso, Fernanda Santos; Bartolini, Paolo; Soares, Carlos Roberto Jorge

    2012-08-01

    Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 10(6) cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5-7 μg hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.

  13. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hasegawa, K.; Kuge, O.; Nishijima, M.

    1989-11-25

    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 themore » 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.« less

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

    PubMed

    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. © 2015 International Federation for Cell Biology.

  15. Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

    PubMed

    Avgerinos, G C; Drapeau, D; Socolow, J S; Mao, J I; Hsiao, K; Broeze, R J

    1990-01-01

    We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

  16. Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

    PubMed Central

    Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

    2014-01-01

    Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins. PMID:24390926

  17. Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vita, N.; Magazin, M.; Marchese, E.

    We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with (35S)-methionine, or with (3H)-glucosamine and (3H)-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the (35S)-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and themore » structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.« less

  18. Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

    PubMed Central

    Moffett, J; Englesberg, E

    1984-01-01

    Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results. PMID:6538929

  19. Mechanisms underlying radiosensitivity : iIvestigations in xrs-5, an X-ray sensitive hamster cell line

    NASA Astrophysics Data System (ADS)

    Johnston, Peter James

    The damage caused to cells by ionising radiation is believed to center on damage to the DNA. In particular, the induction of DNA double strand breaks (DSB) have been implicated in biological end-points such as cell killing and the formation of chromosomal aberrations. The xrs-5 cell line is a mutant Chinese hamster ovary fibroblast (CHO-K1) mutant which exhibits sensitivity to ionising radiation and a number of other DNA damaging agents. This mutation, postulated to involve the hamster homologue of the human XRCC5 gene, is believed to be involved in the repair of the DSB. In addition, there are constitutive differences between the wild type and xrs cells involving the structure and function of the nucleus and higher order chromatin structures. The aims of this thesis were to study further the xrs-5 cell line and its response to DNA damage and to investigate the possible link between chromatin structure and DSB repair. By the examination of the response of xrs-5 cells to a number of DNA damaging agents and potential modulators of this response using the cytokinesis block micronucleus assay [Fenech and Morley, 1985] a possible cell cycle defect was identified in addition to elevated levels of chromosomal damage. Xrs-5 cells appeared to be partially defective in the cell cycle checkpoints involving the passage from G2 phase to mitosis. By the use of a modified neutral filter elution procedure variations in the repair of DSB were observed between xrs-5 and CHO. Conventional neutral filter elution requires harsh lysis conditions to remove higher order chromatin structures which interfere with the elution of DNA containing DSB. By lysing cells with non-ionic detergent in the presence of 2 M NaC1, histone depleted structures which retain the higher order nuclear matrix organisation, including chromatin loops, can be produced. Elution from these structures will only occur if two or more DSB lie within a single looped domain delineated by points of attachment to the nuclear

  20. Evaluation of differential representative values between Chinese hamster cells and human lymphocytes in mitomycin C-induced cytogenetic assays and caspase-3 activity.

    PubMed

    Liao, Pei-Hu; Lin, Ruey-Hseng; Yang, Ming-Ling; Li, Yi-Ching; Kuan, Yu-Hsiang

    2012-03-01

    Chinese hamster ovary (CHO) cells, its lung fibroblasts (V79), and human lymphocytes are routinely used in in vitro cytogenetic assays, which include micronuclei (MN), sister chromatid exchange (SCE), and chromosome aberration (CA) assays. Mitomycin C (MMC), a DNA cross-link alkylating agent, is both an anticancer medicine and a carcinogen. To study the differential representative values of cell types in MMC-treated cytogenetic assays and its upstream factor, cysteine aspartic acid-specific protease (caspase)-3. Among the chosen cell types, lymphocytes expressed the highest sensitivity in all three MMC-induced assays, whereas CHO and V79 showed varied sensitivity in different assays. In MN assay, the sensitivity of CHO is higher than or equal to V79; in SCE assay, the sensitivity of CHO is the same as V79; and in CA assay, the sensitivity of CHO is higher than V79. In-depth analysis of CA revealed that in chromatid breaks and dicentrics formation, lymphocyte was the most sensitive of all and CHO was more sensitive than V79; and in acentrics and interchanges formation, lymphocyte was much more sensitive than the others. Furthermore, we found caspase-3 activity plays an important role in MMC-induced cytogenetic assays, with MMC-induced caspase-3 activity resulting in more sensitivity in lymphocytes than in CHO and V79. Based on these findings, lymphocyte will make a suitable predictive or representative control reference in cytogenetic assays and caspase-3 activity with its high specificity, positive predictive value, and sensitivity.

  1. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    PubMed Central

    Cervantes, Blanca; López-Huerta, Francisco; Vega, Rosario; Hernández-Torres, Julián; García-González, Leandro; Salceda, Emilio; Herrera-May, Agustín L.; Soto, Enrique

    2016-01-01

    Cytotoxicity of titanium dioxide (TiO2) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science. PMID:28773740

  2. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    PubMed

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  3. Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

    PubMed

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

    2015-01-01

    Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection. © 2015 American Institute of Chemical Engineers.

  4. Genetic engineering of CHO cells for viral resistance to minute virus of mice.

    PubMed

    Mascarenhas, Joaquina X; Korokhov, Nikolay; Burger, Lisa; Kassim, Ademola; Tuter, Jason; Miller, Daniel; Borgschulte, Trissa; George, Henry J; Chang, Audrey; Pintel, David J; Onions, David; Kayser, Kevin J

    2017-03-01

    Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN ® GS -/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to

  5. Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

    PubMed

    Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

    1999-03-01

    Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

  6. Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

    PubMed

    Gácsi, Mariann; Antal, Otilia; Vasas, Gábor; Máthé, Csaba; Borbély, György; Saker, Martin L; Gyori, János; Farkas, Anna; Vehovszky, Agnes; Bánfalvi, Gáspár

    2009-06-01

    In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

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

    PubMed

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    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.

  8. Application of a cell-once-through perfusion strategy for production of recombinant antibody from rCHO cells in a Centritech Lab II centrifuge system.

    PubMed

    Kim, Byoung Jin; Chang, Ho Nam; Oh, Duk Jae

    2007-01-01

    Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

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

    PubMed

    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.

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

    PubMed

    Lee, Suk Kyoo; Lee, Gyun Min

    2003-06-30

    Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.

  11. Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells.

    PubMed

    Westwood, J T; Steinhardt, R A

    1989-04-01

    Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.

  12. Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

    PubMed

    Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

    2018-04-15

    Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Effect of diisopropanolamine upon choline uptake and phospholipid synthesis in Chinese hamster ovary cells.

    PubMed

    Stott, W T; Kleinert, K M

    2008-02-01

    Aminoalcohols differ in mammalian toxicity at least in part based upon their ability to alter the metabolism of phospholipids and to cause depletion of the essential nutrient choline in animals. This study examined the incorporation of diisopropanolamine (DIPA) into phospholipids (PLs) and effects of DIPA upon choline uptake and phospholipid synthesis in Chinese hamster ovary (CHO) cells. Results were compared to those of a related secondary alcohol amine, diethanolamine (DEA), whose systemic toxicity is closely associated with its metabolic incorporation into PLs and depletion of choline pools. DIPA caused a dose-related inhibition of (3)H-choline uptake by CHO cells that was approximately 3-4 fold less potent, based upon an IC50, than that reported for DEA. DIPA, in contrast to DEA, did not cause changes in the synthesis rates of (33)P-phosphatidylethanolamine, (33)P-phosphatidylcholine or (33)P-sphingomyelin at either non-toxic or moderately toxic concentrations. Only approximately 0.004%, of administered (14)C-DIPA was metabolically incorporated into PLs, over 30-fold less than the incorporation of (14)C-DEA under similar conditions. Overall, these data and previous pharmacokinetic and toxicity data obtained in vivo suggests that DIPA is distinct from DEA and lacks significant choline and PL metabolism related toxicity in animals.

  14. Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-gamma production in suspension and inside microcarriers in protein-free media.

    PubMed

    Ballez, J S; Mols, J; Burteau, C; Agathos, S N; Schneider, Y J

    2004-03-01

    We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-gamma) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-gamma secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-gamma secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-gamma to a level of 6.6 mug h(-1) g(-1) of microcarriers after 160 h when a cellular density of about 4 x 10(8) cell g(-1) of carriers was reached.

  15. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

    PubMed

    Zhang, Ye; Stobbe, Per; Silvander, Christian Orrego; Chotteau, Véronique

    2015-11-10

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period. Copyright © 2015 The Authors. Published by Elsevier B.V. All

  16. Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

    PubMed Central

    Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

    1999-01-01

    This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

  17. Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes.

    PubMed

    Ogawa, T; Dobrinski, I; Avarbock, M R; Brinster, R L

    1999-02-01

    It was recently demonstrated that rat spermatogenesis can occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat. In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis would result. The hamster diverged at least 16 million years ago from the mouse and produces spermatozoa that are larger than, and have a shape distinctly different from, those of the mouse. In four separate experiments with a total of 13 recipient mice, hamster spermatogenesis was identified in the testes of each mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved hamster testis cells generated spermatogenesis in recipients. However, abnormalities were noted in hamster spermatids and acrosomes in seminiferous tubules of recipient mice. Hamster spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and heads and tails were separated. Thus, defects in spermiogenesis occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell. The generation of spermatogenesis from frozen hamster cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding has immediate application to valuable animals of many species, because the cells could be stored until suitable recipients are identified or culture techniques devised to expand the stem cell population.

  18. In vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal.

    PubMed

    Mutkus, Lysette; Aschner, Judy L; Syversen, Tore; Shanker, Gouri; Sonnewald, Ursula; Aschner, Michael

    2005-01-01

    Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/ aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [3H]-d-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST- transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.

  19. [The expression of interferon-lambda1 in CHO cell].

    PubMed

    Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

    2013-06-01

    To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

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

    PubMed

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

    2015-12-01

    Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-β (Aβ) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate-conjugated Aβ(1-42) was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aβ oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13).

    PubMed

    Li, Shengwei; Gao, Xiaoping; Peng, Rui; Zhang, Sheng; Fu, Wei; Zou, Fangdong

    A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells.

  2. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

    PubMed

    Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca

    2012-09-18

    In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. The protective effect of niacinamide on CHO AA8 cell line against ultraviolet radiation in the context of main cytoskeletal proteins.

    PubMed

    Izdebska, Magdalena; Hałas-Wiśniewska, Marta; Adamczyk, Iwona; Lewandowska, Ismena; Kwiatkowska, Iga; Gagat, Maciej; Grzanka, Alina

    2018-03-13

    Niacinamide is a stable and water-soluble form of vitamin B3, a valuable and versatile cosmetic ingredient, which is well absorbed and tolerated by the skin. A large body of literature has reported on the antioxidant and cell repair properties of niacinamide. Therefore, it has been shown to be useful in the protection of the skin against ultraviolet B (UVB) radiation and free radicals. Despite numerous hypotheses on the mechanism of vitamin B3, its protective effects have not yet been fully elucidated. The aim of the study was to determine the protective effects of niacinamide on CHO AA8 cell line against UVB radiation. We assessed the following factors: cell death, cell cycle phase distributions, reorganization of main cytoskeletal proteins, such as F-actin, vimentin and β-tubulin, and also alterations at the ultrastructural level. The material used for our research was Chinese hamster ovary cell line (CHO AA8). We used 4 research groups: 1) control cells; 2) cells treated with niacinamide; 3) cells exposed to UV radiation; and 4) cells co-incubated with niacinamide and next exposed to ultraviolet. The cell death and cell cycle were evaluated by a Tali® based-image cytometer. A fluorescence microscope was used to assess the reorganization of cytoskeletal proteins, whereas a transmission electron microscope enabled the evaluation of the alterations at the ultrastructural level of cells. We showed that UV-induced apoptosis and cell cycle distributions during treatment with niacinamide resulted in a non-statistical significance in cell survival and no significant changes in the morphology and cytoskeleton in comparison to the control group. In turn, a combination of both factors led to an increase in the population of live cells and a decreased level of apoptotic cells in comparison to UV-exposed cells. Our results confirmed the harmful effects of UV radiation on CHO AA8 cell line. Furthermore, niacinamide can protect cells against these factors, and the mechanism

  4. Surface antibody and cytokine response to recombinant Chinese hamster ovary cell (CHO) hepatitis B vaccine.

    PubMed

    Zhang, Wei; Han, Lili; Lin, Changying; Wang, Huai; Pang, Xinghuo; Li, Liqiu; Gao, Pei; Lin, Hui; Gong, Xiaohong; Tang, Yaqing; Ma, Jianxin; Zhang, Haiyan; Wang, Chen; Yang, Peng; Li, Hui; Sun, Meiping; He, Xiong

    2011-08-26

    To compare the immune responses of the 10 μg and 20 μg doses of CHO hepatitis B vaccine on adults. Adults aged 18-45 years who gave a history of never having received hepatitis B vaccine and lacked serologic evidence of infection to hepatitis B virus (HBV) infection or previous vaccination were enrolled into the study. A total of 642 eligible participants were randomized to receive 3 doses of either the 10 μg or the 20 μg formulation of CHO hepatitis B vaccine in a 0-1-6 month schedule. Each study subject had a serologic specimen collected one month following the third vaccine dose that was tested for markers of HBV infection and anti-HBs by Abbott I2000. Persons who tested negative for anti-HBs negative persons were tested for HBV DNA. Logistic regression was used to identify factors associated with antibody response. Among the participants, 153 subjects had their lymphocytes cultivated and tested for cytokine production. Enzyme-linked immunospot (ELISPOT) was used to test spot numbers of IL-4, IFN-γ which produced by lymphocyte. The anti-HBs seroconversion rate was 88.8% (95% CI: 85.4-92.2%) and 95.3% (95% CI: 93.0-97.6%), respectively in 10 μg and 20 μg group. Geometric mean titers (GMT) were 173.42 mIU/ml and 585.51 mIU/ml, respectively in 10 μg and 20 μg groups. Multivariate analysis demonstrated that diabetes, spouse is hepatitis B virus infector, older age and receipt of the 10 μg dose were all negatively associated with antibody response (P<.05). Cellular immunity results showed: IL-4 immunity spot numbers in 20 μg group was higher than 10 μg group. With anti-HBs increased, the IL-4 immunity spot numbers increased significantly which had significant positive correlation (Spearman coefficient=0.538, P<0.0001). IFN-γ spot numbers had no statistical significant between the two groups. The humoral immunity and cytokines response among the group that received the 20 μg CHO hepatitis B vaccine dose was superior compared to the group that received

  5. Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

    PubMed

    Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

    1998-11-06

    The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

  6. Effects of YM471, a nonpeptide AVP V1A and V2 receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells

    PubMed Central

    Tsukada, Junko; Tahara, Atsuo; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Taniguchi, Nobuaki; Tanaka, Akihiro

    2001-01-01

    YM471, (Z)-4′-{4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V1A, V1B and V2) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [3H]-AVP binding to V1A and V2 receptors with Ki values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V1B and oxytocin receptors with Ki values of 16.4 μM and 31.6 nM, respectively. In CHO cells expressing V1A receptors, YM471 potently inhibited AVP-induced intracellular Ca2+ concentration ([Ca2+]i) increase, exhibiting an IC50 value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca2+]i increase (IC50=193 nM), and did not affect AVP-induced [Ca2+]i increase in CHO cells expressing V1B receptors. Furthermore, in CHO cells expressing V2 receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC50 value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V1A and V2 receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP. PMID:11429400

  7. Effects of YM471, a nonpeptide AVP V(1A) and V(2) receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells.

    PubMed

    Tsukada, J; Tahara, A; Tomura, Y; Wada Ki; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Taniguchi, N; Tanaka, A

    2001-07-01

    YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.

  8. Sequencing, Annotation and Analysis of the Syrian Hamster (Mesocricetus auratus) Transcriptome

    PubMed Central

    Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L.; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F.; Feldmann, Heinz

    2014-01-01

    Background The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. Results A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. Conclusions This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional

  9. The C. elegans VIG-1 and FRM-1 modulate carbachol-stimulated ERK1/2 activation in chinese hamster ovary cells expressing the muscarinic acetylcholine receptor GAR-3.

    PubMed

    Shin, Youngmi; Cho, Nam Jeong

    2014-04-01

    Many neurotransmitter receptors are known to interact with a variety of intracellular proteins that modulate signaling processes. In an effort to understand the molecular mechanism by which acetylcholine (ACh) signaling is modulated, we searched for proteins that interact with GAR-3, the Caenorhabditis elegans homolog of muscarinic ACh receptors. We isolated two proteins, VIG-1 and FRM-1, in a yeast two-hybrid screen of a C. elegans cDNA library using the third intracellular (i3) loop of GAR-3 as bait. To test whether these proteins regulate ACh signaling, we utilized Chinese hamster ovary (CHO) cells stably expressing GAR-3 (GAR-3/CHO cells). Previously we have shown that the cholinergic agonist carbachol stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in an atropine-sensitive manner in this cell line. When VIG-1 was transiently expressed in GAR-3/CHO cells, carbachol-stimulated ERK1/2 activation was substantially reduced. In contrast, transient expression of FRM-1 significantly enhanced carbachol-stimulated ERK1/2 activation. Neither VIG-1 nor FRM-1 expression appeared to alter the affinity between GAR-3 and carbachol. In support of this notion, expression of these proteins did not affect GAR-3-mediated phospholipase C activation. To verify the modulation of ERK1/2 activity by VIG-1 and FRM-1, we used an i3 loop deletion mutant of GAR-3 (termed GAR-3Δi3). Carbachol treatment evoked robust ERK1/2 activation in CHO cells stably expressing the deletion mutant (GAR-3Δi3/CHO cells). However, transient expression of either VIG-1 or FRM-1 had little effect on carbachol-stimulated ERK1/2 activation in GAR-3Δi3/CHO cells. Taken together, these results indicate that VIG-1 and FRM-1 regulate GAR-3-mediated ERK1/2 activation by interacting with the i3 loop of GAR-3.

  10. Intracellular trehalose via transporter TRET1 as a method to cryoprotect CHO-K1 cells.

    PubMed

    Uchida, Tsutomu; Furukawa, Maho; Kikawada, Takahiro; Yamazaki, Kenji; Gohara, Kazutoshi

    2017-08-01

    Trehalose is a promising natural cryoprotectant, but its cryoprotective effect is limited due to difficulties in transmembrane transport. Thus, expressing the trehalose transporter TRET1 on various mammalian cells may yield more trehalose applications. In this study, we ran comparative cryopreservation experiments between the TRET1-expressing CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells transfected with an empty vector (CHO-vector). The experiments involve freezing under various trehalose concentrations in an extracellular medium. The freeze-thawing viabilities of CHO-TRET1 cells are higher than those of CHO-vector cells for most freezing conditions. This result differs from control experiments with a transmembrane type cryoprotectant, dimethyl sulfoxide (Me 2 SO), which had similar viabilities in each condition for both cell types. We conclude that the trehalose loaded into the cells with TRET1 significantly improves the cryoprotective effect. The higher viabilities occurred when the extracellular trehalose concentration exceeded 200 mM, with 250-500 mM being optimal, and a cooling rate below 30 K/min, with 5-20 K/min being optimal. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system.

    PubMed

    Fields, Wanda; Fowler, Kathy; Hargreaves, Victoria; Reeve, Lesley; Bombick, Betsy

    2017-04-01

    Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses. 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily "heat-not-burn" cigarette (Eclipse). The VITROCELL® VC10® provided consistent generation and delivery of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC 50 values; comparative analysis showed that the heat-not-burn cigarette was significantly (P<0.001) less cytotoxic than the 3R4F combustible cigarette, consistent with the lower levels of chemical constituents liberated by primarily-heating the cigarette versus burning. Copyright © 2017. Published by Elsevier Ltd.

  12. CHO-cell mutant with a defect in cytokinesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, L.H.; Lindl, P.A.

    1976-01-01

    In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie, 34 percent) of polyploid cells at the permissive temperature of 34/sup 0/C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5/sup 0/ and 39.5/sup 0/. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completionmore » of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivity.« less

  13. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    PubMed

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  14. Southern Analysis of Genomic Alterations in Gamma-Ray-Induced Aprt- Hamster Cell Mutants

    PubMed Central

    Grosovsky, Andrew J.; Drobetsky, Elliot A.; deJong, Pieter J.; Glickman, Barry W.

    1986-01-01

    The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus. PMID:3013724

  15. Manipulation of the sodium-potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures.

    PubMed

    Wang, Samantha B; Lee-Goldman, Alexandria; Ravikrishnan, Janani; Zheng, Lili; Lin, Henry

    2018-04-01

    Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed. © 2017 Wiley Periodicals, Inc.

  16. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

    PubMed

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-06-01

    Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn 56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

  17. Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

    PubMed

    Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

    2018-01-11

    A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

  18. Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

    PubMed

    Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

    2010-06-01

    Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

  19. Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

    PubMed

    Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

    2013-01-01

    Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation

  20. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles.

    PubMed

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

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

    PubMed

    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

  2. A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization.

    PubMed

    Mozley, Olivia L; Thompson, Ben C; Fernandez-Martell, Alejandro; James, David C

    2014-01-01

    In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex

  3. [Continuously perfused cultivation of genetically-engineered CHO cells producing prothrombin in a modified Super-Spinner].

    PubMed

    Chen, Z L; Iding, K; Lütkemeyer, D; Lehmann, J

    2001-01-01

    A Super-Spinner was Modified by mounting a stainless steel filter(pore size 75 microns) to the impeller shaft to retain cells while fresh nutrient is perfused. Using Macroporous microcarrier Cytopore 1, continuously perfused cultivation of a recombinant CHO cell line, CHO2DS producing prothrombin was performed with the perfusion of a protein-free medium DF6S. The cell retention rate was more than 90% during the 24 days continuously perfused cultivation. The viable cell density of CHO2DS and prothrombin concentration reached 4.62 x 10(6)(cells.m/L) and 11.3(mg/L) respectively after 9 days culture.

  4. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

    PubMed

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

    2014-02-01

    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  5. 2-Aminoanthracene, 5-fluorouracil, colchicine, benzo[a]pyrene, cadmium chloride and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster ovary (CHO) cells at Covance Laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

    PubMed

    Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, Debbie; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

    2010-10-29

    The reference genotoxic agents 2-aminoanthracene (a metabolism dependent weak clastogen), 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation), cadmium chloride (an inorganic carcinogen), and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster ovary (CHO) cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked positive increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants.

    PubMed

    Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

    2015-06-01

    Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L(-1) Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.

  7. Mutagenicity of silver nanoparticles in CHO cells dependent on particle surface functionalization and metabolic activation

    NASA Astrophysics Data System (ADS)

    Guigas, Claudia; Walz, Elke; Gräf, Volker; Heller, Knut J.; Greiner, Ralf

    2017-06-01

    The potential of engineered nanomaterials to induce genotoxic effects is an important aspect of hazard identification. In this study, cytotoxicity and mutagenicity as a function of metabolic activation of three silver nanoparticle (AgNP) preparations differing in surface coating were determined in Chinese hamster ovary (CHO) subclone K1 cells. Three silver nanoparticle preparations ( x 90,0 <30 nm) stabilized with polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate (AgPure™), citrate (Citrate-Ag), and polyvinylpyrrolidone (PVP-Ag) were used for the experiments. The cytotoxic effect of AgNPs was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) test using different concentrations of nanoparticles, while the mutagenicity was evaluated using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. The cytotoxicity of all three AgNPs was lower in a cell culture medium containing 10% fetal calf serum (FCS) than in medium without FCS. The HPRT test without metabolic activation system S9 revealed that compared to the other AgNP formulations, citrate-coated Ag showed a lower genotoxic effect. However, addition of S9 increased the mutation frequency of all AgNPs and especially influenced the genotoxicity of Citrate-Ag. The results showed that exogenous metabolic activation of nanosilver is crucial even if interactions of the metabolic activation system, nanosilver, and cells are not really understood up to now.

  8. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells.

    PubMed

    Minshall, R D; Tan, F; Nakamura, F; Rabito, S F; Becker, R P; Marcic, B; Erdös, E G

    1997-11-01

    Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

  9. Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

    PubMed Central

    Bäckström, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C

    2003-01-01

    We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer. PMID:12950230

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

    PubMed

    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. Copyright © 2015. Published by Elsevier Inc.

  11. Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

    PubMed Central

    Leurs, R.; Smit, M. J.; Menge, W. M.; Timmerman, H.

    1994-01-01

    1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7921611

  12. Protective activity of Cynara scolymus L. leaf extract against chemically induced complex genomic alterations in CHO cells.

    PubMed

    Jacociunas, Laura Vicedo; de Andrade, Heloisa Helena Rodrigues; Lehmann, Mauricio; Pedersini, Larissa Wölfle; Ferraz, Alexandre de Barros Falcão; da Silva, Juliana; Dihl, Rafael Rodrigues

    2013-09-15

    Cynara scolymus L., popularly known as artichoke, has been widely used in traditional medicine as an herbal medicament for therapeutic purposes. The study aimed at assessing the protective activity of Cynara scolymus leaf extract (LE) against DNA lesions induced by the alkylating agent ethylmethnesulphonate (EMS) in Chinese hamster ovary cells (CHO). The ability of C. scolymus L. LE to modulate the mutagenicity of EMS was examined using the cytokinesis block micronucleus (CBMN) cytome assay in three antigenotoxic protocols, pre- post- and simultaneous treatments. In the pre-treatment, C. scolymus L. LE reduced the frequencies of MNi and NBUDs induced by EMS in the lower concentration. In contrast, at the highest concentration (5 mg/ml) artichoke enhanced the frequency of MNi, potentiating EMS genotoxicity. In the simultaneous treatment only the induction of MNi was repressed by the exposure of cells to C. scolymus L. LE. No modification in genotoxicity was observed in LE post-treatment. The results obtained in this study suggest that lower concentrations of artichoke prevent chemically induced genomic damage in mammalian cells. In this context, the protective activity of C. scolymus L. could be associated to its constitutive antioxidants compounds. Copyright © 2013 Elsevier GmbH. All rights reserved.

  13. Laser light prevents apoptosis in Cho K-1 cell line.

    PubMed

    Carnevalli, Célia M M; Soares, Cristina Pacheco; Zângaro, Renato Amaro; Pinheiro, Antonio L B; Silva, Newton Soares

    2003-08-01

    The present study investigated the effects of low-level laser therapy (LLLT) on the mitochondria, nucleus, and cytoskeleton of CHO K-1 cells by the use of specific fluorescent probes. The use of LLLT has been recommended by several authors for acceleration of the healing process. The literature on the effects of LLLT in this process is highly contradictory because of difficulties in identifying its effects on cells. CHO K-1 cells were cultivated using MEM containing 5% FBS and were irradiated or not with a semiconductor laser (lambda = 830 nm; phi approximately 0.8 mm; 10 mW; 2 J/cm2). The cells were incubated with specific fluorescent probes--0.1 microM for 30 min with 5,5', 6,6'-tetrachloro-1, 1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) for the mitochondria; 5 mM for 5 min of 4',6'-diamidino, 2'-phenylindole (DAPI)for the nucleus, and 0.1 M of 1:100 PHEM of rhodamine-phalloidin during 1 h for the cytoskeleton--and were analyzed by epifluorescence. Positive biomodulatory effects were observed on irradiated cells compared to their controls as seen on JC-1, DAPI, and rhodamine-phalloidin labeling. Irradiated cells showed an increased level of cellular division, as evidenced by analyzing the intermediary filaments of the cytoskeleton and the chromosomes. Another important observation was that cells maintained under the condition of nutritional deficiency had both membrane and genetic material that was more preserved in comparison to the controls, in which the presence of an apoptotic nucleus could be observed in some cells. The results of the present study demonstrate that LLLT, in addition to providing positive biomodulation, acts in the re-establishment of cellular homeostasis when the cells are maintained under the condition of nutritional stress; it also prevents apoptosis in CHO K-1 cells.

  14. Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

    PubMed

    Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

    2007-02-01

    There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

  15. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

    PubMed Central

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    Background: The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. Methods: In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. Results: In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. Conclusion: The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells. PMID:26232332

  16. Mesothelial cell proliferation induced by intrapleural instillation of man-made fibers in rats and hamsters.

    PubMed

    Rutten, A A; Bermudez, E; Mangum, J B; Wong, B A; Moss, O R; Everitt, J I

    1994-07-01

    Long-term inhalation exposure to a biopersistent man-made ceramic fiber (RCF 1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed rats. To understand better the mechanisms involved in the intraspecies pathobiology of fiber-exposed mesothelium, the ability of the two different man-made fibers to induce cell proliferation in hamster and rat pleural mesothelial cells was investigated. Three dose levels of either glass fibers (MMVF 10) or ceramic fibers (RCF 1) were instilled intrapleurally into male Fischer 344 rats and male Syrian Golden hamsters. Rats and hamsters were exposed to approximately equal numbers of long thin fibers per kilogram of body weight using a single intrapleural instillation. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump, and mesothelial cell proliferation was assessed at 7 and 28 days postinstillation (PI) using immunocytochemical visualization of labeled S-phase cells. Both rats and hamsters exhibited dose-dependent increases in proliferation of pleural mesothelial cells following exposure to both fiber types. Interspecies differences in mesothelial cell proliferation were noted for fiber type and pleural site. At 28 days PI, RCF-induced mesothelial cell proliferation was found to be more pronounced in hamsters than in rats in the caudal visceral pleural. Comparing both fibers either by equal mass or by equal fiber numbers, mesothelial cell proliferation in RCF 1-treated animals was higher than in animals exposed to MMVF 10, especially in hamsters, and may be a factor in the difference in mesothelioma induced by the two fibers. The higher sustained (28 day) mesothelial cell proliferation in the visceral pleural of hamsters exposed to RCF may contribute to the species-specific differences in mesothelioma incidence found in long-term rodent inhalation studies.

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

    PubMed

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

    2016-01-05

    Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (<10%) and good accuracy (100±10%) were achieved for nearly all the 30 compounds. Applying this method to CHO cell extracts, statistically significant differences in the metabolite levels were measured between two cell lines originated from the same host, indicating differences in genetic makeup or metabolic activities and nutrient supply levels in the culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells.

    PubMed

    Hinz, J M; Meuth, M

    1999-02-01

    In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.

  19. CELLULAR TOXICITY IN CHINESE HAMSTER OVARY CELL CULTURES. 2. A STATISTICAL APPRAISAL OF SENSITIVITY WITH THE RABBIT ALVEOLAR MACROPHAGE, SYRIAN HAMSTER EMBRYO, BALB 3T3 MOUSE, AND HUMAN NEONATAL FIBROBLAST CELL SYSTEMS

    EPA Science Inventory

    Chinese hamster ovary, rabbit alveolar macrophage, Syrian Hamster embryo, mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances. The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimet...

  20. Cell-Matrix Interactions in Breast Carcinoma Invasion.

    DTIC Science & Technology

    1998-01-01

    concentrated in hemidesmosomes, adhesive junctions which connect the basement membrane to the intracellular keratin cytoskeleton. In virtually all...fibronectin receptor contribute to the adhesive abnormalities of transformed fibroblasts by overexpressing this integrin in Chinese hamster ovary (CHO) cells...normal breast epithelium , the integrins expressed in breast carcinoma cells are diffusely distributed over the cell surface (Zutter et al., 1990

  1. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

    PubMed

    Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

    2018-03-29

    To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

  2. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    PubMed

    Gonzalez, Gaëlle; Vituret, Cyrielle; Di Pietro, Attilio; Chanson, Marc; Boulanger, Pierre; Hong, Saw-See

    2012-01-01

    Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  3. Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells.

    PubMed

    Hu, Suwen; Deng, Lei; Wang, Huamao; Zhuang, Yingping; Chu, Ju; Zhang, Siliang; Li, Zhonghai; Guo, Meijin

    2011-05-01

    The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 10(5) cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID

  5. EMODIN DOWNREGULATES CELL PROLIFERATION MARKERS DURING DMBA INDUCED ORAL CARCINOGENESIS IN GOLDEN SYRIAN HAMSTERS.

    PubMed

    Manimaran, Asokan; Buddhan, Rajamanickam; Manoharan, Shanmugam

    2017-01-01

    Cell-cycle disruption is the major characteristic features of neoplastic transformation and the status of cell-cycle regulators can thus be utilized to assess the prognostic significance in patients with cancer. The PCNA, cyclin D1, CDK4, CDK6 and survivin expression in the buccal mucosa was utilized to evaluate the Emodin efficacy on abnormal cell proliferation during 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis in golden Syrian hamsters. Topical application of DMBA, three times a week for 14 weeks, on the hamsters' buccal pouches developed well differentiated squamous cell carcinoma. Cyclin D1 and PCNA over-expression and up-regulation of CDK4, CDK6 and survivin were noticed in the buccal mucosa of hamsters treated with DMBA alone. Emodin administration (50mg/kg b.w) orally to hamsters treated with DMBA down-regulated the expression of cell proliferation markers in the buccal mucosa. The anti-cell proliferative role of Emodin is owing to its modulating efficacy on cell-cycle markers towards the tumor suppression during DMBA induced oral carcinogenesis.

  6. Utilization of a quantitative mammalian cell mutation system, CHO/HGPRT, in experimental mutagenesis and genetic toxicology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hsie, A. W.; Couch, D. B.; O'Neill, J. P.

    1977-01-01

    Development of the CHO/HGPRT system is described and a host-mediated CHO/HGPRT assay is discussed. The following topics are discussed: evidence for the genetic origin of mutation induction in the CHO/HGPRT system; dose-response relationship for EMS-mediated mutation induction and cell lethality; apparent dosimetry of EMS-induced mutagenesis; structure-activity relationship of alkylating agents and ICR compounds; mutagenicity and cytotoxicity of congeners of two classes of nitrosi compounds; and preliminary validation of the CHO/HGPRT assay in predicting chemical carcinogenicity. (HLW)

  7. High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

    PubMed

    Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

    2017-07-01

    Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

  8. Tetramer-organizing polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.

    PubMed

    Schopfer, Lawrence M; Lockridge, Oksana

    2016-06-01

    Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode polyproline-rich peptides. The function of the polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. CHOgenome.org 2.0: Genome resources and website updates.

    PubMed

    Kremkow, Benjamin G; Baik, Jong Youn; MacDonald, Madolyn L; Lee, Kelvin H

    2015-07-01

    Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

    PubMed

    Mols, J; Peeters-Joris, C; Wattiez, R; Agathos, S N; Schneider, Y-J

    2005-01-01

    Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

  11. Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

    PubMed

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2017-11-01

    The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C 6 glucose containing feed media during constant operation at 20 × 10 6 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C 6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C 6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017. © 2017 American Institute of Chemical Engineers.

  12. Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC.

    PubMed

    Kochanowski, N; Blanchard, F; Cacan, R; Chirat, F; Guedon, E; Marc, A; Goergen, J-L

    2006-01-15

    Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

  13. T cells are not required for pathogenesis in the Syrian hamster model of hantavirus pulmonary syndrome.

    PubMed

    Hammerbeck, Christopher D; Hooper, Jay W

    2011-10-01

    Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology.

  14. Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor.

    PubMed

    Wang, M-D; Yang, M; Huzel, N; Butler, M

    2002-01-20

    A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days. The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27. The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate. The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture. The EPO accumulated to a total production of 28,000 kUnits over the whole culture period. The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up. The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask. The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period. The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor. These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks. Copyright 2002 John Wiley & Sons, Inc.

  15. A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

    PubMed

    Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

    2015-05-01

    Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers

  16. A new CHO (Chinese hamster ovary)-derived cell line expressing anti-TNFα monoclonal antibody with biosimilar potential.

    PubMed

    Luchese, Mateus Dalcin; Lopes Dos Santos, Mariana; Garbuio, Angelica; Targino, Roselaine Campos; Mansueli, Carla Ploeger; Tsuruta, Lilian Rumi; Quintilio, Wagner; Moro, Ana Maria

    2018-05-31

    Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.

  17. Atypical fibrosarcomas derived from cutaneous ganglion cell-like cells in 2 domestic Djungarian hamsters (Phodopus sungorus).

    PubMed

    Kondo, Hirotaka; Onuma, Mamoru; Shibuya, Hisashi; Sato, Tsuneo; Abbott, Jeffrey R

    2011-07-01

    Androgen-dependent atypical fibromas are benign tumors derived from ganglion-cell-like cells that are particular to Djungarian hamsters (Phodopus sungorus). Masses excised from 2 hamsters were composed of pleomorphic ganglion cell-like cells supported by small to moderate amounts of collagenous matrix. Intracytoplasmic fibrils were present in silver-stained sections, and immunohistochemistry showed that the cells expressed vimentin, androgen receptor, and, in one case, estrogen receptor α. In contrast to previously reported atypical fibromas, these tumors had features of anaplasia and were locally invasive. We diagnosed the tumors as atypical fibrosarcomas and consider them an unusual malignant counterpart of atypical fibroma. Copyright 2011 by the American Association for Laboratory Animal Science

  18. Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique

    USDA-ARS?s Scientific Manuscript database

    A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been ...

  19. Serotonergic modulation of hippocampal pyramidal cells in euthermic, cold-acclimated, and hibernating hamsters

    NASA Technical Reports Server (NTRS)

    Horrigan, D. J.; Horwitz, B. A.; Horowitz, J. M.

    1997-01-01

    Serotonergic fibers project to the hippocampus, a brain area previously shown to have distinctive changes in electroencephalograph (EEG) activity during entrance into and arousal from hibernation. The EEG activity is generated by pyramidal cells in both hibernating and nonhibernating species. Using the brain slice preparation, we characterized serotonergic responses of these CA1 pyramidal cells in euthermic, cold-acclimated, and hibernating Syrian hamsters. Stimulation of Shaffer-collateral/commissural fibers evoked fast synaptic excitation of CA1 pyramidal cells, a response monitored by recording population spikes (the synchronous generation of action potentials). Neuromodulation by serotonin (5-HT) decreased population spike amplitude by 54% in cold-acclimated animals, 80% in hibernating hamsters, and 63% in euthermic animals. The depression was significantly greater in slices from hibernators than from cold-acclimated animals. In slices from euthermic animals, changes in extracellular K+ concentration between 2.5 and 5.0 mM did not significantly alter serotonergic responses. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin mimicked serotonergic inhibition in euthermic hamsters. Results show that 5-HT is a robust neuromodulator not only in euthermic animals but also in cold-acclimated and hibernating hamsters.

  20. Stimulation of islet cell proliferation enhances pancreatic ductal carcinogenesis in the hamster model.

    PubMed Central

    Pour, P. M.; Kazakoff, K.

    1996-01-01

    Previous studies have shown that some N-nitrosobis (2-oxopropyl)amine (BOP)-induced ductal/ductular pancreatic cancers in the hamster model develop within islets and that streptozotocin (SZ) pretreatment that caused islet degeneration and atrophy inhibits pancreatic cancer induction. Hence, it appears that in this model islets play a significant role in exocrine pancreatic carcinogenesis. To examine whether stimulation of islet cell proliferation (nesidioblastosis) enhances pancreatic exocrine cancer development, we tested the effect of the pancreatic carcinogen BOP in hamsters after induction of nesidioblastosis by cellophane wrapping. Before wrapping, hamsters were treated with SZ to inhibit pancreatic tumor induction in the unwrapped pancreatic tissues. Control groups with a wrapped pancreas did not receive SZ. Six weeks after SZ treatment, all hamsters were treated with BOP (10 mg/kg body weight) weekly for 10 weeks and the experiment was terminated 38 weeks after the last BOP treatment. Many animals recovered from their diabetes at the time when BOP was injected and many more after BOP treatment. Only nine hamsters remained diabetic until the end of the experiment. Both SZ-treated and control groups developed proliferative and malignant pancreatic ductal-type lesions primarily in the wrapped area (47%) but less frequently in the larger segments of the pancreas, including the splenic lobe (34%), gastric lobe (13%), and duodenal lobe (6%). Only a few lesions developed in the unwrapped pancreatic region of nine diabetic hamsters with atrophic islets, whereas seven of these hamsters had tumors in the wrapped area. Histologically, most tumors appeared to originate from islets, many invasive carcinomas had foci of islets, and some tumor cells showed reactivity with anti-insulin. The results show that, in the BOP hamster model, islets are the site of formation of the major fraction of exocrine pancreatic cancer and that induction of nesidioblastosis enhances

  1. T Cells Are Not Required for Pathogenesis in the Syrian Hamster Model of Hantavirus Pulmonary Syndrome ▿

    PubMed Central

    Hammerbeck, Christopher D.; Hooper, Jay W.

    2011-01-01

    Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology. PMID:21775442

  2. [Dependence of ion transport across the plasma membrane on the density of the cell culture. I. Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)].

    PubMed

    Marakhova, I I; Pospelova, T V; Vinogradova, T A; Vereninov, A A; Ignatova, T N

    1985-09-01

    Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth. With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively. The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed. The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes. The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.

  3. Survival of Chinese Hamster Ovary Cells Following Ultrahigh Dose Rate Electron and Bremsstrahlung Radiation

    DTIC Science & Technology

    1990-04-01

    and a stepped lead flattening filter. The electron energy used for these studies was 13 MeV. Dosimetry was performed by the Health Physics Division...VolI LJSAFSAPA-TR-90-4 AD-A222 722 SURVIVAL OF CHINESE HAMSTER OVARY CELLS FOLLOWING ULTRAHIGH DOSE RATE ELECTRON AND BREMISSTRAHLUNG RADIATION...Include Security ;a!. iatcn) Survival of Chinese Hamster Ovary Cells Following Ultrahigh Dose Rate Electron and Bremsstrahlung Radiation 12 PERSONAL

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

    PubMed

    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. © 2016 Wiley Periodicals, Inc.

  5. Partial apamin sensitivity of human small conductance Ca2+-activated K+ channels stably expressed in Chinese hamster ovary cells.

    PubMed

    Dale, T J; Cryan, J E; Chen, M X; Trezise, D J

    2002-11-01

    The bee venom toxin apamin is an important drug tool for characterising small conductance Ca(2+)-activated K(+) channels (SK channels). In recombinant expression systems both rSK2 and rSK3 channels are potently blocked by apamin, whilst the sensitivity of SK1 channels is somewhat less clear. In the present study we have conducted a detailed analysis by patch clamp electrophysiology of the effects of apamin on human SK channels (SK1, SK2 and SK3) stably expressed in Chinese hamster ovary (CHO-K1) cells. CHO-K1 cell lines expressing either hSK1, 2 or 3 channels were first validated using specific antibodies and Western blotting. Specific protein bands of a size corresponding to the predicted channel tetramer (approximately 250-290 kDa) were detected. In each cell line, but not wild-type untransfected cells, large, time-independent inwardly rectifying Ca(2+)-dependent K(+) currents were observed under voltage-clamp. In CHO-hSK1, this current was markedly reduced by apamin (IC(50) value 8 nM), however, a significant fraction of the current remained unblocked (39+/-5%), even at saturating concentrations (1 microM apamin). The apamin-sensitive and -insensitive currents possess very similar biophysical and pharmacological properties. Each are Ca(2+)-dependent, inwardly rectify and have relative ionic permeabilities of K(+)>Cs(+)>Li(+)=Na(+). Both components were resistant to block by charybdotoxin and iberiotoxin, known IK and BK channel blockers, but were attenuated by the tricyclic antidepressant cyproheptadine (>95% block at 1 mM). The SK channel opener 1-EBIO could still produce channel activation in the presence of apamin. Importantly, hSK2 and hSK3 channels also exhibit partial apamin sensitivity in our experimental paradigm (IC(50) values of 0.14 nM and 1.1 nM, respectively, and maximal percentage inhibition values of 47+/-7% and 58+/-9%, respectively). Our data indicate that, at least in a recombinant expression system, all three SK channels can be partially

  6. Cytotoxic effects of Cochlospermum regium (Mart & Schrank) Pilger aqueous root extract on mammalian cells.

    PubMed

    Ceschini, Livônios; Campos, Elida Geralda

    2006-01-16

    We investigated the effect of Cochlospermum regium (Mart & Schrank) Pilger aqueous root extract on Chinese hamster ovarian (CHO)-K1 cells. The extract significantly decreased proliferation of CHO-K1 cells (EC(50)=1.5mg/mL). Apoptosis induction was analysed by fluorescent microscopy. Cell cultures treated with Cochlospermum regium extract for 4h contained 13.6% apoptotic cells after 24h (investigated by fluorescent DNA-microscopy with acridine orange/ethidium bromide staining). Characteristic chromatin condensation and fragmentation, verified by 4',6-diamidino-2-phenylindole (DAPI) staining, was observed in the cells after treatment with Cochlospermum regium extract. The results confirm the toxicity of Cochlospermum regium root extract to immortal, non-tumorigenic mammalian cells in vitro.

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

    PubMed Central

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

    2015-01-01

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

  8. A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

    PubMed

    Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

    2015-09-01

    Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays

    NASA Technical Reports Server (NTRS)

    Kraemer, S. M.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    1997-01-01

    Small mutations, megabase deletions, and aneuploidy are involved in carcinogenesis and genetic defects, so it is important to be able to quantify these mutations and understand mechanisms of their creation. We have previously quantified a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in a hamster-human hybrid cell line AL. S1- mutants have lost expression of a human cell surface antigen, S1, which is encoded by the M1C1 gene at 11p13 so that mutants can be detected via a complement-mediated cytotoxicity assay in which S1+ cells are killed and S1- cells survive. But loss of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that mutants that have lost the entire chromosome 11 die and escape detection. To circumvent this, we fused AL with Chinese hamster ovary (CHO) cells to produce a new hybrid, ALC, in which the requirement for maintaining 11p15.5 is relieved, allowing us to detect mutations events involving loss of 11p15.5. We evaluated the usefulness of this hybrid by conducting mutagenesis studies with colcemid, 137Cs gamma-radiation and UV 254 nm light. Colcemid induced 1000 more S1- mutants per unit dose in ALC than in AL; the increase for UV 254 nm light was only two-fold; and the increase for 137Cs gamma-rays was 12-fold. The increase in S1- mutant fraction in ALC cells treated with colcemid and 137Cs gamma-rays were largely due to chromosome loss and 11p deletions often containing a breakpoint within the centromeric region.

  10. Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

    PubMed

    Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

    2012-12-18

    The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

  11. Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells.

    PubMed

    Long, Yan; Wang, Wei-ping; Yuan, Hui; Ma, Shi-ping; Feng, Nan; Wang, Ling; Wang, Xiao-liang

    2013-05-01

    To investigate the reverse mode function of Na(+)/Ca(2+) exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA. CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca(2+) level ([Ca(2+)]i) was measured using Ca(2+) imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na(+)-free medium. Ca(2+) paradox was induced by Ca(2+)-free EBSS medium, followed by Ca(2+)-containing solution (1.8 or 3.8 mmol/L CaCl2). The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca(2+)]i, which was followed by a Ca(2+) level plateau at higher external Ca(2+) concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca(2+)]i at higher external Ca(2+) concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L). Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca(2+)]i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.

  12. Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

    PubMed

    Popp, Oliver; Larraillet, Vincent; Kettenberger, Hubert; Gorr, Ingo H; Hilger, Maximiliane; Lipsmeier, Florian; Zeck, Anne; Beaucamp, Nicola

    2015-06-01

    In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention. © 2014 Wiley Periodicals, Inc.

  13. A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.

    PubMed Central

    Piechaczek, C; Fetzer, C; Baiker, A; Bode, J; Lipps, H J

    1999-01-01

    We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure. PMID:9862961

  14. Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

    PubMed

    Juárez, Mariana; González-De la Rosa, Claudia H; Memún, Elisa; Sigala, Juan-Carlos; Lara, Alvaro R

    2017-03-01

    Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Microarray platform affords improved product analysis in mammalian cell growth studies

    PubMed Central

    Li, Lingyun; Migliore, Nicole; Schaefer, Eugene; Sharfstein, Susan T.; Dordick, Jonathan S.; Linhardt, Robert J.

    2014-01-01

    High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity. PMID:24227746

  16. The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

    NASA Technical Reports Server (NTRS)

    Ponnaiya, B.; Limoli, C. L.; Corcoran, J.; Kaplan, M. I.; Hartmann, A.; Morgan, W. F.

    1998-01-01

    PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

  17. Modified multiple drug resistance phenotype of Chinese hamster ovary cells selected with X-rays and vincristine versus X-rays only.

    PubMed Central

    McClean, S.; Hill, B. T.

    1994-01-01

    Exposure of Chinese hamster ovary (CHO) cells to fractionated X-irradiation [ten fractions of 9 Gray (Gy)] resulted in the expression of a multiple drug resistance phenotype which was distinct from that of drug-selected cells in two features: (i) resistance to vinca alkaloids and epipodophyllotoxins but sensitivity to anthracyclines was retained; (ii) overexpression of P-glycoprotein (Pgp) but regulated by post-translational stability rather than by any elevation in Pgp mRNA (Hill et al., 1990). It was also reported that when these cells (designated DXR-10) were subsequently exposed to another ten fractions of 9 Gy (20 x 9 Gy in total), no further increases in drug resistance or in the extent of Pgp expression were observed. To examine this apparent plateauing of the drug resistance phenotype following X-ray pretreatment, DXR-10 cells were instead treated with ten pulsed vincristine exposures. The resultant cell line, designated DXR-10/VCR-10, proved to be more resistant to vincristine, implying that the effect of further drug selection was additive to that of X-ray pretreatment. In addition, these cells showed resistance to doxorubicin and increased Pgp expression which was matched by a concomitant elevation in Pgp mRNA. These findings appear to confirm that Pgp expression is differentially regulated in tumour cells showing drug resistance after drug as opposed to X-ray selection. Images Figure 2 Figure 3 Figure 5 PMID:7908216

  18. Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores

    PubMed Central

    Sinha, Rakesh K.; Pospíšil, Pavel; Maheshwari, Priti; Eudes, François

    2016-01-01

    Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity. PMID:28082995

  19. Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

    PubMed

    Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

    2011-10-01

    Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    PubMed

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  1. A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

    DTIC Science & Technology

    2006-11-01

    fibers that served as the junction for the replacement of the OAdV7 tail domain, as well as other common sequences, is highlighted. The final Ad5Luc1-OvF...Fold increase in luciferase activity vs. Ad5 b Reference CHO Hamster ovary L/N 22 Soudais et al., 2000 RD Rhabdomyosarcoma L/N 1.5 Dmitriev et al., 1998...of OV-3 cells (human ovarian cancer, 23-fold) and CAR-deficient CHO cells (Chinese hamster ovary, 22-fold), suggesting that RD cells do not express

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

    PubMed

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

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA.

    PubMed

    Takagi, M; Ilias, M; Yoshida, T

    2000-01-01

    The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1982-01-01

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

  5. Comparative study of the effects of phosphatidylcholine rich in DHA and EPA on Alzheimer's disease and the possible mechanisms in CHO-APP/PS1 cells and SAMP8 mice.

    PubMed

    Che, Hongxia; Zhou, Miaomiao; Zhang, Tiantian; Zhang, Lingyu; Ding, Lin; Yanagita, Teruyoshi; Xu, Jie; Xue, Changhu; Wang, Yuming

    2018-01-24

    Metabolic stress induced by a high-fat (HF) diet leads to cognitive dysfunction and aging. In the present study, Chinese hamster ovary cells stably transfected with amyloid precursor protein (APP) and presenilin 1 (PS1) (CHO-APP/PS1 cells) and SAMP8 mice fed with an HF diet were used to study the effects of docosahexaenoic acid (DHA)-enriched phosphatidylcholine (DHA-PC) and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (EPA-PC) on Alzheimer's disease (AD) and the possible mechanisms involved in these effects. Behavior test results indicated that DHA-PC exerted better effects than EPA-PC on improving memory and cognitive deficiency. Further analysis showed that DHA-PC and EPA-PC could significantly decrease β-amyloid (Aβ) concentrations in CHO-APP/PS1 cells and SAMP8 mice by inhibiting APP, PS1, and BACE1 expression. Moreover, both DHA-PC and EPA-PC can increase the activities of the antioxidant index, including SOD, T-AOC, GSH, and GSH-PX, and inhibit levels of MDA, NO, and NOS. In addition, the expressions of inflammatory factors (TNF-α, IL-1β) and apoptosis were significantly suppressed via improving the ratio of Bcl-2/Bax and decreasing the expression of pro-apoptosis factors. Interestingly, only DHA-PC could improve the expression of neurotrophic factors, including BDNF, synaptophysin, and growth associated protein 43. DHA-PC and EPA-PC could ameliorate memory and cognitive function of HF diet-fed SAMP8 mice via inhibiting Aβ generation, suppressing oxidative stress and apoptosis, down-regulating inflammatory response, and improving neurotrophic activity. Therefore, DHA-PC and EPA-PC may be applied as food supplements and/or functional ingredients to relieve neurodegenerative disease.

  6. Optimization of a pH-shift control strategy for producing monoclonal antibodies in Chinese hamster ovary cell cultures using a pH-dependent dynamic model.

    PubMed

    Hogiri, Tomoharu; Tamashima, Hiroshi; Nishizawa, Akitoshi; Okamoto, Masahiro

    2018-02-01

    To optimize monoclonal antibody (mAb) production in Chinese hamster ovary cell cultures, culture pH should be temporally controlled with high resolution. In this study, we propose a new pH-dependent dynamic model represented by simultaneous differential equations including a minimum of six system component, depending on pH value. All kinetic parameters in the dynamic model were estimated using an evolutionary numerical optimization (real-coded genetic algorithm) method based on experimental time-course data obtained at different pH values ranging from 6.6 to 7.2. We determined an optimal pH-shift schedule theoretically. We validated this optimal pH-shift schedule experimentally and mAb production increased by approximately 40% with this schedule. Throughout this study, it was suggested that the culture pH-shift optimization strategy using a pH-dependent dynamic model is suitable to optimize any pH-shift schedule for CHO cell lines used in mAb production projects. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production.

    PubMed

    Takuma, Shinya; Hirashima, Chikashi; Piret, James M

    2007-08-15

    The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions. (c) 2007 Wiley Periodicals, Inc.

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

    PubMed Central

    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.

    2016-01-01

    ABSTRACT 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

  9. Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein.

    PubMed

    Gillard, Michel; Chatelain, Pierre; Fuks, Bruno

    2006-04-24

    A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53+/-7, 55+/-9, 70

  10. Prevention of Simian Virus 40 Tumors by Hamster Fetal Tissue: Influence of Parity Status of Donor Females on Immunogenicity of Fetal Tissue and on Immune Cell Cytotoxicity

    PubMed Central

    Girardi, Anthony J.; Reppucci, Phyllis; Dierlam, Peggy; Rutala, William; Coggin, Joseph H.

    1973-01-01

    Fetal tissue from primiparous hamsters prevented simian virus 40 (SV40) tumorigenesis in male hamsters, whereas fetal tissue from multiparous hamsters did not. The parity status of normal (uninoculated) hamsters also influenced the cytotoxicity of their lymphoid cells against tumor cells. Lymph node cells from nonpregnant primiparous and multiparous animals were cytotoxic in microcytotoxicity tests against SV40, polyoma, and adenovirus 7 tumor cells, but were not active against control BHK cells. Lymph node cells from virgin female donors were inactive. Peritoneal exudate cells from these donors reacted in similar fashion against SV40 tumor cells in vitro and in adoptive transfer tests in vivo. However, the cytotoxicity of peritoneal exudate cells from multiparous hamsters was greatly reduced during pregnancy, a time when noncytotoxic humoral antibody reactive with surface antigen of SV40 tumor cells is present. This humoral antibody is not detected during first pregnancy, and peritoneal exudate cells obtained from pregnant primiparous hamsters demonstrated a high degree of cytotoxicity. PMID:4346032

  11. Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

    PubMed

    Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

    2000-07-03

    In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta Me

  12. Characteristics of the uridine uptake system in normal and polyoma transformed hamster embryo cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lemkin, J.A.

    1973-01-01

    The lability of the uridine uptake system in the normal and polyoma transformed hamster embryo fibroblast was studied. The major areas investigated were: the kinetic parameters of uridine transport, a comparison of changes in cellular ATP content by factors which modulate uridine uptake, and a comparison of the qualitative and quantitative effects of the same modulating agent on uridine transport, cell growth, and cellular ATP content. Uridine uptake into cells in vitro was examined using tritiated uridine as a tracer to measure the amount of uridine incorporated into the acid soluble and acid-insoluble fractions of the cells studied. The ATPmore » content of the cells was determined by the firefly bioluminescence method. It was found that the K/sub t/ for uridine uptake into the normal hamster embryo cell and two polyoma transformed hamster embryo cell lines was identical. However, the V/sub max/ for uridine transport was higher in both polyoma transformed cell lines. Furthermore, the K/sub t/ in both the normal and transformed cell cultured in serum-less or serum-containing media was identical, although the V/sub max/ was higher in the serum-stimulated cell in both the normal and transformed cell. Stimulation of the normal cell with adenosine produced a different K/sub t/ for uridine transport. Preliminary investigations have demonstrated that treatment of the polyoma transformed with adenosine also induces a different K/sub t/ (not shown). The K/sub i/ for phloretin inhibition in serum-less and serum-stimulated normal and polyoma transformed cells was found to be identical in each case.« less

  13. Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

    PubMed

    Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

    2015-05-01

    CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Thyroid cell lines in research on goitrogenesis.

    PubMed

    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.

  15. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    NASA Technical Reports Server (NTRS)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  16. Transformation of Primary Hamster Brain Cells with JC Virus and Its DNA

    PubMed Central

    Frisque, R. J.; Rifkin, D. B.; Walker, D. L.

    1980-01-01

    We transformed primary hamster brain cells with four isolates of JC virus and JC virus DNA. Several properties of these transformants were characterized and compared to those of simian virus 40 transformants isolated under identical conditions. Images PMID:6251275

  17. Elucidating the role of copper in CHO cell energy metabolism using (13)C metabolic flux analysis.

    PubMed

    Nargund, Shilpa; Qiu, Jinshu; Goudar, Chetan T

    2015-01-01

    (13)C-metabolic flux analysis was used to understand copper deficiency-related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein-producing CHO cells. Stationary-phase labeling experiments with U-(13)C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed-batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC-MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%-79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%-23% and 74%, respectively) compared with the Cu-containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper-deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation. © 2015 American Institute of Chemical Engineers.

  18. Notch as a Diagnostic Marker and Therapeutic Target in Human Breast Cancer

    DTIC Science & Technology

    2008-05-01

    JAG1. The soluble JAG1-ECD-FLAG was expressed in Chinese Hamster ovary K1 (CHO-K1) cells and then CHO clones were screened for their ability to... medium was collected from CHO-K1- hJAG1-ECD-Flag (clone14) grown in culture. The purification strategy to obtain hJAG1-ECD-Flag is as follows: 1) pre...expressed in Chinese hampster ovary K1 (CHO-K1) cells and then CHO clones were screened for their ability to express high levels of secreted JAG1-Flag

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  1. CHO cell enlargement oscillates with a temperature-compensated period of 24 min

    NASA Technical Reports Server (NTRS)

    Pogue, R.; Morre, D. M.; Morre, D. J.

    2000-01-01

    The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14 degrees C, 24 degrees C or 34 degrees C) while the rate of cell enlargement increased with temperature over this same range of temperatures.

  2. Crocidolite asbestos and SV40 are cocarcinogens in human mesothelial cells and in causing mesothelioma in hamsters

    PubMed Central

    Kroczynska, Barbara; Cutrone, Rochelle; Bocchetta, Maurizio; Yang, Haining; Elmishad, Amira G.; Vacek, Pamela; Ramos-Nino, Maria; Mossman, Brooke T.; Pass, Harvey I.; Carbone, Michele

    2006-01-01

    Only a fraction of subjects exposed to asbestos develop malignant mesothelioma (MM), suggesting that additional factors may render some individuals more susceptible. We tested the hypothesis that asbestos and Simian virus (SV40) are cocarcinogens. Asbestos and SV40 in combination had a costimulatory effect in inducing ERK1/2 phosphorylation and activator protein-1 (AP-1) activity in both primary Syrian hamster mesothelial cells (SHM) and primary human mesothelial cells (HM). Ap-1 activity caused the expression and activation of matrix metalloprotease (MMP)-1 and MMP-9, which in turn led to cell invasion. Experiments using siRNA and chemical inhibitors confirmed the specificity of these results. The same effects were observed in HM and SHM. Experiments in hamsters showed strong cocarcinogenesis between asbestos and SV40: SV40 did not cause MM, asbestos caused MM in 20% of hamsters, and asbestos and SV40 together caused MM in 90% of hamsters. Significantly lower amounts of asbestos were sufficient to cause MM in animals infected with SV40. Our results indicate that mineral fibers and viruses can be cocarcinogens and suggest that lower amounts of asbestos may be sufficient to cause MM in individuals infected with SV40. PMID:16966607

  3. Role of various DNA repair pathways in chromosomal inversion formation in CHO mutants.

    PubMed

    Cartwright, Ian M; Kato, Takamitsu A

    2015-01-01

    In an effort to better understand the formation of chromosomal inversions, we investigated the role of various DNA repair pathways, including the non-homologous end joining (NHEJ), homologous recombination (HR), and Fanconi Anemia (FA) repair pathways for the formation of radiation induced chromosomal inversions. CHO10B2 wild type, CHO DNA repair-deficient, and CHO DNA repair-deficient corrected mutant cells were synchronized into G1 phase and exposed to gamma-rays. First post-irradiation metaphase cells were analyzed for chromosomal inversions by a differential chromatid staining technique involving a single cycle pre-irradiation ethynyl-uridine treatment and statistic calculations. It was observed that inhibition of the NHEJ pathway resulted in an overall decrease in the number of radiation-induced inversions, roughly a 50% decrease when compared to the CHO wild type. Interestingly, inhibition of the FA pathway resulted in an increase in both the number of spontaneous inversions and the number of radiation-induced inversions observed after exposure to 2 Gy of ionizing radiation. It was observed that FA-deficient cells contained roughly 330% (1.24 inversions per cell) more spontaneous inversions and 20% (0.4 inversions per cell) more radiation-induced inversions than the wild-type CHO cell lines. The HR mutants, defective in Rad51 foci, showed similar number of spontaneous and radiation-induced inversion as the wild-type cells. Gene complementation resulted in both spontaneous and radiation-induced inversions resembling the CHO wild-type cells. We have concluded that the NHEJ repair pathway contributes to the formation of radiation-induced inversions. Additionally, through an unknown molecular mechanism it appears that the FA signal pathway prevents the formation of both spontaneous and radiation induced inversions.

  4. Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

    PubMed

    Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

    2018-05-01

    The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

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

    PubMed

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

    2015-03-01

    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 process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

  6. A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

    PubMed

    Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

    2018-01-01

    Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid

  7. High-throughput synchronization of mammalian cell cultures by spiral microfluidics.

    PubMed

    Lee, Wong Cheng; Bhagat, Ali Asgar S; Lim, Chwee Teck

    2014-01-01

    The development of mammalian cell cycle synchronization techniques has greatly advanced our understanding of many cellular regulatory events and mechanisms specific to different phases of the cell cycle. In this chapter, we describe a high-throughput microfluidic-based approach for cell cycle synchronization. By exploiting the relationship between cell size and its phase in the cell cycle, large numbers of synchronized cells can be obtained by size fractionation in a spiral microfluidic channel. Protocols for the synchronization of primary cells such as mesenchymal stem cells, and immortal cell lines such as Chinese hamster ovarian cells (CHO-CD36) and HeLa cells are provided as examples.

  8. Synergetic cholesterol-lowering effects of main alkaloids from Rhizoma Coptidis in HepG2 cells and hypercholesterolemia hamsters.

    PubMed

    Kou, Shuming; Han, Bing; Wang, Yue; Huang, Tao; He, Kai; Han, Yulong; Zhou, Xia; Ye, Xiaoli; Li, Xuegang

    2016-04-15

    Hyperlipidemia contributes to the progression of cardiovascular diseases. Main alkaloids from Rhizoma Coptidis including berberine (BBR), coptisine (COP), palmatine (PAL), epiberberine (EPI) and jatrorrhizine (JAT), improved dyslipidemia in hypercholesterolemic hamsters to a different degree. In this study, HepG2 cells and hypercholesterolemic hamsters were used to investigate the synergetic cholesterol-lowering efficacy of these five main alkaloids. The cellular lipid and cholesterol accumulation and in HepG2 cells were evaluated by Oil Red O staining and HPLC analysis. LDL receptor, 3-Hydroxy-3-methylglutaryl CoA reductase (HMGCR) and cholesterol 7-alpha-hydroxylase (CYP7A1) that involving cholesterol metabolism in HepG2 cells were measured by qRT-PCR, western blot and immunofluorescence analysis. The serum profiles including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c), as well as TC and total bile acids (TBA) of feces in hypercholesterolemic hamsters were also measured. As compared to single alkaloids, the combination of five main alkaloids (COM) reduced the lipid and cholesterol accumulation in HepG2 cells more effectively and performed an advantageous effect on controlling TC, TG, LDL-c and HDL-c in hypercholesterolemic hamsters. More effective reduction of TBA and TC levels in feces of hamsters were achieved after the administration of COM. These effects were derived from the up-regulation of LDL receptor and CYP7A1, as well as HMGCR downregulation. Our results demonstrated that COM showed a synergetic cholesterol-lowering efficacy, which was better than single alkaloids and it might be considered as a potential therapy for hypercholesterolemia. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.

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

  10. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line.

    PubMed

    Toyohara, Yukiyo; Hashitani, Susumu; Kishimoto, Hiromitsu; Noguchi, Kazuma; Yamamoto, Nobuto; Urade, Masahiro

    2011-07-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model.

  11. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line

    PubMed Central

    TOYOHARA, YUKIYO; HASHITANI, SUSUMU; KISHIMOTO, HIROMITSU; NOGUCHI, KAZUMA; YAMAMOTO, NOBUTO; URADE, MASAHIRO

    2011-01-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model. PMID:22848250

  12. alpha-Adrenergic-mediated activation of human reconstituted fibrinogen receptor (integrin alphaIIbbeta3) in Chinese hamster ovary cells.

    PubMed

    Butta, Nora; Larrucea, Susana; Gonzalez-Manchon, Consuelo; Alonso, Sonia; Parrilla, Roberto

    2004-12-01

    This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.

  13. Detection of osteoclastic cell-cell fusion through retroviral vector packaging.

    PubMed

    Kondo, Takako; Ikeda, Kyoji; Matsuo, Koichi

    2004-11-01

    Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.

  14. Hematologic Assessment in Pet Rats, Mice, Hamsters, and Gerbils: Blood Sample Collection and Blood Cell Identification.

    PubMed

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-09-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

    PubMed Central

    Wilson, Paul F.; Hinz, John M.; Urbin, Salustra S.; Nham, Peter B.; Thompson, Larry H.

    2010-01-01

    The repair of DNA double-strand breaks (DSB) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and nonhomologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after γ-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300 cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction (~20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had ~2-fold higher chromatid-type CAs and a remarkable ~25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al. DNA Repair 4, 782–792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2. PMID:20434408

  16. Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

    PubMed

    Isbrucker, R; Daas, A; Wagner, L; Costanzo, A

    2016-01-01

    Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

  17. Evaluating the toxicity of bDtBPP on CHO-K1 cells for testing of single-use bioprocessing systems considering media selection, cell culture volume, mixing, and exposure duration.

    PubMed

    Shah, Rhythm R; Linville, Taylor W; Whynot, Andrew D; Brazel, Christopher S

    2016-09-01

    Single-use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale-up. Bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4-di-tert-butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single-use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO-K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO-K1 cells. While CHO-K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry-standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO-K1 cells. It was found that the sensitivity of cell culture to leachates from single-use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318-1323, 2016. © 2016 American Institute of Chemical Engineers.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer 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 delaymore » 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 G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.« less

  19. Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.

    PubMed

    Florin, Lore; Lipske, Carolin; Becker, Eric; Kaufmann, Hitto

    2011-04-10

    DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Development of thermotolerance in CHO cells: modification by procaine.

    PubMed

    Rastogi, D; Henle, K J; Nagle, W A; Moss, A J; Neilan, B A; Rastogi, S P

    1987-01-01

    We have tested the reported ability of procaine to inhibit the induction and the development of thermotolerance in Chinese hamster ovary cells. Thermotolerance was induced either by hyperthermia alone (10 min, 45 degrees C) or by combining hyperthermia and procaine (5 min, 45 degrees C + 10 mM procaine) with heating times adjusted to yield similar cell survival after the conditioning treatments. Both the kinetics of thermotolerance development in fresh medium without procaine and the magnitude of thermotolerance 6 h after heat conditioning were similar for the two treatment groups. Development of thermotolerance in the presence of procaine was tested by adding the drug at 5 or 10 mM to culture medium between, but not during two fractionated heat treatments. Thermotolerance development was observed even in the presence of 10 mM procaine, but only if cell survival was corrected for the 37 degrees C-procaine toxicity. Complete survival curves of cells incubated for 6 h at 37 degrees C in 7.5 mM procaine between heat conditioning and test heating showed a D0 that was only 35 per cent lower than that of thermotolerant controls. The data are consistent with the reported sensitization to heat killing by procaine, but show that thermotolerance induction and development were only minimally perturbed by procaine.

  1. In vitro evaluation of low-intensity light radiation on murine melanoma (B16F10) cells.

    PubMed

    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.

  2. Generic Raman-based calibration models enabling real-time monitoring of cell culture bioreactors.

    PubMed

    Mehdizadeh, Hamidreza; Lauri, David; Karry, Krizia M; Moshgbar, Mojgan; Procopio-Melino, Renee; Drapeau, Denis

    2015-01-01

    Raman-based multivariate calibration models have been developed for real-time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO-based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers.

  3. Metabolic design of macroscopic bioreaction models: application to Chinese hamster ovary cells.

    PubMed

    Provost, A; Bastin, G; Agathos, S N; Schneider, Y-J

    2006-12-01

    The aim of this paper is to present a systematic methodology to design macroscopic bioreaction models for cell cultures based upon metabolic networks. The cell culture is seen as a succession of phases. During each phase, a metabolic network represents the set of reactions occurring in the cell. Then, through the use of the elementary flux modes, these metabolic networks are used to derive macroscopic bioreactions linking the extracellular substrates and products. On this basis, as many separate models are obtained as there are phases. Then, a complete model is obtained by smoothly switching from model to model. This is illustrated with batch cultures of Chinese hamster ovary cells.

  4. Origin and evolution of binucleated cells and binucleated cells with micronuclei in cisplatin-treated CHO cultures.

    PubMed

    Rodilla, V

    1993-08-01

    It has recently been described that cisplatin is an agent able to induce binucleated cells (BC) in cultured CHO cells. Both the origin and the significance of those cells within a population are unknown although several hypothesis have been suggested such as blocking of cytokinesis or cell fusion. Using interval photography we have found that at least two mechanisms are involved in the production of BC. These cells can arise in a culture as a result of an incomplete process of cell division, i.e. karyokinesis with incomplete cytokinesis or as a result of the mitotic division of a pre-existent BC. The mitotic division of a BC can give rise to different types of daughter cells. These BC sometimes enter mitosis but fail to divide and as a consequence they remain BC. When the process of division is successful (in the vast majority of cases), the results that have been found are either two mononucleated cells or one mononucleated and one binucleated cell. The possible implications and significance of BC and BC with micronuclei in a given population are discussed.

  5. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    PubMed

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates. © 2015 American Institute of Chemical Engineers.

  6. A statistical approach to determining criticality of residual host cell DNA.

    PubMed

    Yang, Harry; Wei, Ziping; Schenerman, Mark

    2015-01-01

    We propose a method for determining the criticality of residual host cell DNA, which is characterized through two attributes, namely the size and amount of residual DNA in biopharmaceutical product. By applying a mechanistic modeling approach to the problem, we establish the linkage between residual DNA and product safety measured in terms of immunogenicity, oncogenicity, and infectivity. Such a link makes it possible to establish acceptable ranges of residual DNA size and amount. Application of the method is illustrated through two real-life examples related to a vaccine manufactured in Madin Darby Canine Kidney cell line and a monoclonal antibody using Chinese hamster ovary (CHO) cell line as host cells.

  7. Growth factor withdrawal in combination with sodium butyrate addition extends culture longevity and enhances antibody production in CHO cells.

    PubMed

    Hong, Jong Kwang; Lee, Gyun Min; Yoon, Sung Kwan

    2011-09-10

    The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, q(Ab). Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced q(Ab) and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, q(Glc), q(Lac) and Y(Lac/Glc), and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance q(Ab) and extend culture longevity. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Reduced cytotoxicity in PCB-exposed Chinese Hamster Ovary (CHO) cells pretreated with vitamin E.

    PubMed

    Murati, Teuta; Šimić, Branimir; Pleadin, Jelka; Vukmirović, Maja; Miletić, Marina; Durgo, Ksenija; Kniewald, Jasna; Kmetič, Ivana

    2017-01-01

    The aim of this study was to evaluate protective effects of vitamin E (50 -150 μM) in ovary cells upon cytotoxic effects induced by two structurally distinct PCB congeners - planar "dioxin-like" PCB 77 and non-planar di-ortho-substituted PCB 153 with an emphasis on identifying differences in the mechanism of vitamin E action depending on the structure of congeners. Application of three bioassays confirmed that PCBs decrease ovarian cell proliferation with slightly profound effects of PCB 77. PCB - induced ROS production and lipid peroxidation were significant for both congeners with also more noticeable effect for PCB 77. Vitamin E pre-incubation has improved viability of cells, reduced ROS formation and lipid peroxidation induced by PCBs' treatment. Preincubation with vitamin E was more effective when cells where treated with non-planar PCB 153. Altogether, vitamin E action was protective, congener specific and more effective when ovary cells were exposed to ortho-substituted PCB congener. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Etiology of Tetracycline-Associated Pseudomembranous Colitis in Hamsters

    PubMed Central

    Toshniwal, Renu; Fekety, Robert; Silva, Joseph

    1979-01-01

    Tetracyclines were implicated in the 1950s in induction of protracted diarrhea and pseudomembranous colitis. Because the pathogenetic mechanism of these illnesses has been questioned recently, we studied tetracycline in hamster models of antibiotic-associated colitis. Orogastric administration of tetracycline caused diarrhea and death, with evidence of hemorrhagic typhlitis. Filtrates of cecal contents were toxic when inoculated into normal hamsters and cell culture monolayers, and toxicity was neutralized with Clostridium sordellii antitoxin. Tetracycline-resistant C. difficile was cultured from stools of these hamsters, but Staphylococcus aureus was not isolated. The value of tetracycline for treatment or prevention of clindamycin-induced colitis in hamsters was also studied, and it was found that daily orogastric administration of tetracycline was poorly protective against clindamycin-induced colitis. PMID:485127

  10. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    PubMed

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  11. [Application of dhfr gene negative Chinese hamster ovary cell line to express hepatitis B virus surface antigen].

    PubMed

    Yi, Y; Zhang, M; Liu, C

    2001-06-01

    To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line. HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin. Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml. In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

  12. Regulation of murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in Chinese hamster ovary cells

    PubMed Central

    Lansdell, K A; Kidd, J F; Delaney, S J; Wainwright, B J; Sheppard, D N

    1998-01-01

    We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique.The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl− channels that had previously been activated by protein kinase A.Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl− channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux.The alkaline phosphatase inhibitor, (−)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR. PMID:9769419

  13. Dynamized Preparations in Cell Culture

    PubMed Central

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

    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 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. PMID:18955237

  14. Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors

    PubMed Central

    Scharfstein, Julio; Schmitz, Veronica; Morandi, Veronica; Capella, Marcia M. A.; Lima, Ana Paula C. A.; Morrot, Alexandre; Juliano, Luiz; Müller-Esterl, Werner

    2000-01-01

    The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease. PMID:11067878

  15. Multimodal biophotonic workstation for live cell analysis.

    PubMed

    Esseling, Michael; Kemper, Björn; Antkowiak, Maciej; Stevenson, David J; Chaudet, Lionel; Neil, Mark A A; French, Paul W; von Bally, Gert; Dholakia, Kishan; Denz, Cornelia

    2012-01-01

    A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

    PubMed

    Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

    2012-03-01

    N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

  17. Effect of ambient temperature on the proliferation of brown adipocyte progenitors and endothelial cells during postnatal BAT development in Syrian hamsters.

    PubMed

    Nagaya, Kazuki; Okamatsu-Ogura, Yuko; Nio-Kobayashi, Junko; Nakagiri, Shohei; Tsubota, Ayumi; Kimura, Kazuhiro

    2018-04-02

    In Syrian hamsters, brown adipose tissue (BAT) develops postnatally through the proliferation and differentiation of brown adipocyte progenitors. In the study reported here, we investigated how ambient temperature influenced BAT formation in neonatal hamsters. In both hamsters raised at 23 or 30 °C, the interscapular fat changed from white to brown coloration in an age-dependent manner and acquired the typical morphological features of BAT by day 16. However, the expression of uncoupling protein 1, a brown adipocyte marker, and of vascular endothelial growth factor α were lower in the group raised at 30 °C than in that raised at 23 °C. Immunofluorescent staining revealed that the proportion of Ki67-expressing progenitors and endothelial cells was lower in the 30 °C group than in the 23 °C group. These results indicate that warm ambient temperature suppresses the proliferation of brown adipocyte progenitors and endothelial cells and negatively affects the postnatal development of BAT in Syrian hamsters.

  18. ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014.

    PubMed

    Fernández de Las Heras, Laura; Mascaraque, Victoria; García Fernández, Esther; Navarro-Llorens, Juana María; Perera, Julián; Drzyzga, Oliver

    2011-07-20

    Cholesterol catabolism has been reported in different bacteria and particularly in several Rhodococcus species, but the genetic of this complex pathway is not yet very well defined. In this work we report the isolation and sequencing of a 9.8 kb DNA fragment of Rhodococcus sp. strain CECT3014, a bacterial strain that we here identify as a Rhodococcus erythropolis strain. In this DNA fragment we found several ORF that are probably involved in steroid catabolism, and choG, a gene encoding a putative cholesterol oxidase whose functional characterization we here report. ChoG protein is a class II cholesterol oxidase with all the structural features of the enzymes of this group. The disruption of the choG gene does not alter the ability of strain CECT3014 cells to grow on cholesterol, but it abolishes the production of extracellular cholesterol oxidase. This later effect is reverted when the mutant cells are transformed with a plasmid expressing choG. We conclude that choG is the gene responsible for the inducible extracellular cholesterol oxidase activity of strain CECT3014. This activity distributes between the cellular membrane and the culture supernatant in a way that suggests it is produced by the same ChoG protein that occurs in two different locations. RT-PCR transcript analysis showed a dual scheme of choG expression: a low constitutive independent transcription, plus a cholesterol induced transcription of choG into a polycistronic kstD-hsd4B-choG mRNA. Copyright © 2010 Elsevier GmbH. All rights reserved.

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

    EPA Science Inventory

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

  20. Neoplastic transformation of hamster embryo cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Han, Z.; Suzuki, H.; Suzuki, F.; Suzuki, M.; Furusawa, Y.; Kato, T.; Ikenaga, M.

    1998-11-01

    We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/μm. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/μm, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.

  1. Neoplastic transformation of hamster embyro cells by heavy ions.

    PubMed

    Han, Z; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

    1998-01-01

    We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/micrometer. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/micrometer, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.

  2. Localization of azithromycin in Toxoplasma gondii-infected cells.

    PubMed Central

    Schwab, J C; Cao, Y; Slowik, M R; Joiner, K A

    1994-01-01

    Agents effective against intracellular pathogens must enter infected cells, crossing vacuolar membranes surrounding the organisms and then penetrating into the microbe and localizing to the microbial target site. We have characterized these parameters for azithromycin entry into Toxoplasma gondii-infected Chinese hamster ovary cells and murine macrophage-like J774 cells. Azithromycin uptake into infected host cells was concentrative and was dependent upon proton gradients. Subcellular fractionation of azithromycin-loaded infected CHO cells demonstrated > 95% intracellular drug in host cell lysosomes and cytosol, with < 5% associated with the parasite. Uptake of azithromycin into the T. gondii vacuole increased if parasites were coated with antibody prior to internalization by murine J774 cells, conditions which result in the formation of acidified phagolysosomes. No redistribution or retention of azithromycin in the parasite was observed when drug efflux from antibiotic-loaded infected CHO cells was monitored. Azithromycin entry into extracellular T. gondii was concentrative, was temperature and pH dependent, and was not different when azithromycin-sensitive and -resistant parasites were compared. These results demonstrate that azithromycin concentrates primarily in acidified compartments in parasites and host cells. The high concentration of azithromycin within these compartments may not be biologically relevant to inhibition of intracellular parasite growth by this agent. PMID:7979295

  3. Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling.

    PubMed

    Takekura, H; Takeshima, H; Nishimura, S; Takahashi, M; Tanabe, T; Flockerzi, V; Hofmann, F; Franzini-Armstrong, C

    1995-10-01

    Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of

  4. Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia.

    PubMed

    Cole, A; Armour, E P

    1988-09-01

    A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.

  5. Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone.

    PubMed

    Aghili, Zahra Sadat; Zarkesh-Esfahani, Sayyed Hamid

    2018-02-01

    Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO 4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations. © Georg Thieme Verlag KG Stuttgart · New York.

  6. Histological study of cell migration in the dermis of hamsters after immunisation with two different vaccines against visceral leishmaniasis.

    PubMed

    Moreira, Nádia das Dores; Giunchetti, Rodolfo Cordeiro; Carneiro, Cláudia Martins; Vitoriano-Souza, Juliana; Roatt, Bruno Mendes; Malaquias, Luiz Cosme Cotta; Corrêa-Oliveira, Rodrigo; Reis, Alexandre Barbosa

    2009-04-15

    Vaccine candidates, including live and/or killed parasites, Leishmania-purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti-Leishmania. More recently, the hamsters have been used to pre-selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1-24h) and late (48-168h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations.

  7. A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

    PubMed

    Roy, Gargi; Martin, Tom; Barnes, Arnita; Wang, Jihong; Jimenez, Rod Brian; Rice, Megan; Li, Lina; Feng, Hui; Zhang, Shu; Chaerkady, Raghothama; Wu, Herren; Marelli, Marcello; Hatton, Diane; Zhu, Jie; Bowen, Michael A

    2018-04-01

    The conserved glycosylation site Asn 297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn 297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

  8. Differential response of two cell lines sequentially irradiated with low X-ray doses.

    PubMed

    Güerci, A M; Dulout, F N; Grillo, C A; Seoane, A I

    2005-05-01

    An experiment was designed to compare the effect of repeated low doses of X-rays in two different cell lines: one transformed, epithelial like and aneuploid Chinese hamster ovary K-1 (CHO-K1); the other originated from a human primary culture, fibroblast, diploid and non-transformed, MRC-5. CHO and MRC-5 cells were cultured for 14 or eight passages, respectively. Irradiation was performed once per passage when cells were in the quiescent state (90 - 95% in G1/G0). Cells were exposed to 10.0 mSv X-ray doses. Ionizing radiation did not induce apoptosis or necrosis in the exposed CHO cell population. Significant increases of low-level damaged cells (degrees 1 and 2) were found for the 14 cycles of radiation when compared with controls, except for the first irradiation cycle. No significant increases in the frequency of cells with severe damage were observed. The frequency of MRC-5 cells with low-level damage increased significantly when compared with controls for radiation cycles seven and eight. Significant increases of apoptosis, necrosis and severe damage were found only for the highest dose. Transformed and non-transformed cell types responded differently to direct and indirect damage using low-dose repeat exposures to ionizing radiation. Though more investigation is needed to understand the mechanisms of radiation effects in chronic low-dose-exposed cell populations, cellular type should be taken into account in the design of in vitro experiments for understanding low-dose-irradiation effects.

  9. Nanosecond pulsed electric fields and the cell cycle

    NASA Astrophysics Data System (ADS)

    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

  10. Cholesterol-lowering effect of non-viscous soluble dietary fiber Nutriose6 in moderately hypercholesterolemic hamsters.

    PubMed

    Juhel, Christine; Tosini, Fredéric; Steib, Marlène; Wils, Daniel; Guerin-Deremaux, Laetitia; Lairon, Denis; Cara, Louis

    2011-03-01

    NUTRIOSE6 is a new wheat starch-based low-digestible carbohydrate. This study investigated the effect of this soluble non-viscous fiber on cholesterol metabolism. Hamsters fed with 0.25% cholesterol-enriched diet (CHO) were given graded amounts of NUTRIOSE6, i.e., 0% (cellulose, CHO), 3% (N3), 6% (N6) or 9% (N9) (w:w). As compared to CHO diet, 9% NUTRIOSE6 significantly lowered plasma and LDL cholesterol by 14.5 and 23.8%, respectively. The LDL-cholesterol lowering effect was also significant with the 6% dose (-21.4%). NUTRIOSE6 diets prevented hepatic cholesterol accumulation (-10 to -20%) and significantly decreased bile cholesterol (-47 to -68%) and phospholipids (-30 to -45%) concentrations. The 9% NUTRIOSE6 diet significantly decreased the rate of dietary cholesterol absorption (-25%) and markedly stimulated faecal neutral sterol (+81%) and bile salts (+220%) excretion. No significant change in cholesterol 7-alpha-hydroxylase or LDL-receptor activities was observed whereas 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was reduced by 29%. Reduced cholesterol and bile salt absorptions and lowered cholesterol synthesis are likely mechanisms underlying the cholesterol lowering effect of NUTRIOSE6. Results suggest the use of NUTRIOSE6 as a new dietary cholesterol-lowering agent that should be tested in humans as treatment and evenly prevention of mild hypercholesterolemia.

  11. Collaborative study for the standardisation of the histamine sensitizing test in mice and the CHO cell-based assay for the residual toxicity testing of acellular pertussis vaccines.

    PubMed

    Xing, D; Maes, A; Behr-Gross, M-E; Costanzo, A; Daas, A; Buchheit, K-H

    2010-04-01

    The European Pharmacopoeia (Ph. Eur.) and the World Health Organisation (WHO) require the performance of extensive quality and safety control testing before the release on the market of vaccine products for human use. Safety testing with regard to residual pertussis toxin (PT) in acellular pertussis combination vaccines is performed through assessment of fatal sensitisation of mice to histamine challenge by the vaccine product under test. Currently, use of different in-house procedures and no requirement for the inclusion of a standard reference in each assay render comparisons of results obtained for identical vaccine batches between different control laboratories very difficult. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM), an international collaborative study was organised for the standardization of the Histamine Sensitizing Test (HIST) in mice and the Chinese Hamster Ovary (CHO)-cell-based assay (performed at the bulk product level) for the residual toxicity testing of acellular pertussis vaccines or acellular pertussis-based combination vaccines. The study was run under the aegis of the Biological Standardisation Programme, jointly supported by the Council of Europe and the European Commission under the project code BSP076. Ten (10) laboratories participated in the study and were requested to perform 3 independent Histamine Sensitizing Tests in mice and to report results of the lethal end-point measurement as prescribed by the Ph. Eur. monographs. Some of them also reported data from an in-house validated CHO-cell-based assay. In addition, some of the laboratories reported concomitantly data obtained by measurement of the drop in temperature induced after the histamine challenge, a method currently under investigation to be added as an alternative end-point for the HIST in the Ph. Eur. monographs for acellular pertussis-based combination vaccines in order to alleviate animal suffering (in application of the 3

  12. Engineering CHO cells with an oncogenic KIT improves cells growth, resilience to stress, and productivity.

    PubMed

    Mahameed, Mohamed; Tirosh, Boaz

    2017-11-01

    An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  14. Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

    PubMed

    Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

    2016-04-01

    Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

  15. Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

    PubMed Central

    Giannasca, P J; Boden, J A; Monath, T P

    1997-01-01

    The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal

  16. Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

    PubMed Central

    Summers, E. F.; Letts, V. A.; McGraw, P.; Henry, S. A.

    1988-01-01

    Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME. PMID:3066687

  17. Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

    PubMed Central

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh

    2016-01-01

    Abstract Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies. PMID:27214658

  18. Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

    PubMed

    Du, Yuncheng; Budman, Hector M; Duever, Thomas A

    2016-06-01

    Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

  19. Agonist and antagonist modulation of [35S]-GTPγS binding in transfected CHO cells expressing the neurotensin receptor

    PubMed Central

    Hermans, Emmanuel; Geurts, Muriel; Maloteaux, Jean-Marie

    1997-01-01

    The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The agonist-induced binding of [35S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM). The stimulation of [35S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM). The NT-induced [35S]-GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTPγS. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor. PMID:9283723

  20. Insulin-Responsive Compartments Containing GLUT4 in 3T3-L1 and CHO Cells: Regulation by Amino Acid Concentrations

    PubMed Central

    Bogan, Jonathan S.; McKee, Adrienne E.; Lodish, Harvey F.

    2001-01-01

    In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism. PMID:11416153

  1. Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus.

    PubMed

    Kabithe, Esther W; Place, Ned J

    2008-03-01

    Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

  2. Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules.

    PubMed

    Rees, Jennifer; Haig, David; Mack, Victoria; Davis, William C

    2017-01-01

    Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. White matter NAA/Cho and Cho/Cr ratios at MR spectroscopy are predictive of motor outcome in preterm infants.

    PubMed

    Kendall, Giles S; Melbourne, Andrew; Johnson, Samantha; Price, David; Bainbridge, Alan; Gunny, Roxanna; Huertas-Ceballos, Angela; Cady, Ernest B; Ourselin, Sebastian; Marlow, Neil; Robertson, Nicola J

    2014-04-01

    To determine (a) whether diffuse white matter injury of prematurity is associated with an increased choline (Cho)-to-creatine (Cr) ratio and a reduced N-acetylaspartate (NAA)-to-Cho ratio and whether these measures can be used as biomarkers of outcome and (b) if changes in peak area metabolite ratios at magnetic resonance (MR) spectroscopy are associated with changes in T2 and fractional anisotropy (FA) at MR imaging. The local ethics committee approved this study, and informed parental consent was obtained for each infant. At term-equivalent age, 43 infants born at less than 32 weeks gestation underwent conventional and quantitative diffusion-tensor and T2-weighted MR imaging. Single-voxel point-resolved proton (hydrogen 1) MR spectroscopy was performed from a 2-cm(3) voxel centered in the posterior periventricular white matter. Outcome was evaluated by using Bayley scales at a corrected age of 1 year. Associations were investigated with Pearson product moment or Spearman rank order correlation. Differences in ratios in infants with and infants without impairment were tested by using t tests. NAA/Cho and Cho/Cr ratios correlated with the scaled gross motor score and the composite motor score, independent of gestational age (P < .05). FA at diffusion-tensor MR imaging and T2 at MR imaging correlated with the NAA/Cho ratio (P < .05 for both) but not with the Cho/Cr ratio. Infants with motor scores of less than 85 (impaired) had an increased Cho/Cr ratio (P < .03) and a reduced NAA/Cho ratio (P < .01) compared to those without impairment. A combination of increased Cho/Cr ratio and decreased NAA/Cho ratio predicted impaired motor outcome at a corrected age of 1 year with a sensitivity of 0.80 (95% confidence interval [CI]: 0.57, 0.94) and a specificity of 0.80 (95% CI: 0.66, 0.88). The combination of Cho/Cr and NAA/Cho ratios measured in the posterior periventricular white matter at term-equivalent age is predictive of motor outcome at 1 year in infants born at less

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

    NASA Astrophysics Data System (ADS)

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

    2007-10-01

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

  5. Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

    PubMed

    Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

    2003-07-20

    Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

  6. Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors.

    PubMed

    De Jesus, Maria; Wurm, Florian M

    2011-06-01

    Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today's technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market [21]. The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l. We are focusing in this article on the upstream part of protein manufacturing. Over the past 25 years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. The relationship of metabolic burden to productivity levels in CHO cell lines.

    PubMed

    Zou, Wu; Edros, Raihana; Al-Rubeai, Mohamed

    2018-03-01

    The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  8. Comparing the clastogenic potential of atrazine with caffeine using Chinese hamster ovary (CHO) cells.

    PubMed

    Rayburn, A L; Bouma, J; Northcott, C A

    2001-04-08

    The agronomically important herbicide atrazine has been reported to cause damage to animal chromosomes at levels of atrazine found contaminating drinking water supplies. While documenting potential chromosome damage is important it is equally important to compare the damage with the potential consequences of compounds readily found in our food and water supply. In this study atrazine and caffeine, a ubiquitous food additive, were compared at equal levels and at real exposure levels for their ability to damage animals chromosomes in cell culture. Nuclei and chromosomes from treated and control cells were analyzed by flow cytometry. At extremely low levels, atrazine was found to be a more potent clastogen. Caffeine had no effect on the chromosomes at the lower levels. Both chemicals were genotoxic at the potential exposure levels with caffeine being more disruptive than atrazine. Atrazine appears to be a more potent damaging agent than caffeine at similar levels of exposure; however, the levels of caffeine one is exposed to during everyday life appears to be more damaging on the endpoints analyzed in this study than the levels of atrazine found contaminating water supplies. The advantages and limitations of whole cell clasotgenicity are also presented in light of these results.

  9. The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

    PubMed

    Amann, Thomas; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

    2018-06-03

    In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, we aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2 and-T3 with and without a disrupted B4Gal-T4 sequence resulted in only ∼1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. We revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ∼6% and ∼3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, we present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, we provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins. This article is protected by copyright. All rights reserved.

  10. Single Unit Recordings of Cells Responsive to Visual, Somatic, Acoustic, and Noxious Stimuli in the Superior Colliculus of the Golden Hamster.

    DTIC Science & Technology

    1978-08-01

    Acoustic, and Noxious Stimuli Thesis in the Superior Colliculus of the Golden 6. PERFORMING OG. REPORT NUMBER Hamster -. _ // 7. AUTHOR( a ) S. CONTRACT...OR GRANT NUMBER(s) James P. Dixon I - "JV 9. PERF 7 MING ORGANIZATION NAME A D10. PROGRAM ELEMENT, PROJECT, TASK AFIT Student at: Virginia...studied in the superior colliculus of the golden hamster. A laminar organiza- tion was observed with cells in the superficial layers responding exclusively

  11. Evaluation of genotoxic and cytotoxic properties of pesticides employed in Italian agricultural practices.

    PubMed

    De Marco, A; De Salvia, R; Polani, S; Ricordy, R; Sorrenti, F; Perticone, P; Cozzi, R; D'Ambrosio, C; De Simone, C; Guidotti, M; Albanesi, T; Duranti, G; Festa, F; Gensabella, G; Owczarek, M

    2000-07-01

    In a program coordinated by the Italian Ministry of Works, we tested in vitro four pesticides widely employed in a developed agricultural region of central Italy. The four commercial agents were chosen on the basis of their diffusion in agricultural practice, knowledge of their active principle(s), and scant availability of data concerning their toxic and genotoxic activity. The agents were Cirtoxin, Decis, Tramat Combi (TC), and Lasso Micromix (LM). All substances were tested in three in vitro systems: Chinese hamster ovary (CHO) cells, a metabolically competent hamster cell line (Chinese hamster epithelial liver; CHEL), and root tips of Vicia faba (VF). The cytotoxic and genotoxic end points challenged were micronuclei and root tip length (RTL) in VF and mitotic index (MI), proliferation index (PI), cell survival (CS), cell growth (CG), cell cycle length (CCL), sister chromatid exchanges, chromosomal aberrations, and single-cell gel electrophoresis, or comet assay, in CHEL and CHO cells. Tested doses ranged from the field dose up to 200x the field dose to take into account accumulation effects. On the whole, tested agents appear to induce genotoxic damage only at subtoxic or toxic doses, indicating a low clastogenic risk. MI, PI, CS, CG, RTL, and CCL appear to be the less sensitive end points, showing no effects in the presence of a clear positive response in some or all of the other tests. Using cytogenetic tests, we obtained positive results for TC and LM treatments in CHO but not in CHEL cells. These data could be accounted for by postulating a detoxifying activity exerted by this cell line. However, cytogenetic end points appear to be more sensitive than those referring to cytotoxicity.

  12. Interactions of the plasma needle with cells in culture

    NASA Astrophysics Data System (ADS)

    Stoffels, E.; Broers, J. L. V.; Kunts, S.; Cornelis, R. A. A.; Caubet, V.; Ramaekers, F. C. S.

    2002-10-01

    A non-thermal atmospheric plasma source (plasma needle) has been developed. This plasma operates at room temperature, low voltages and power levels, so it can be applied for fine treatment of organic material. In this work the impact of the plasma needle on living cells is explored. For this purpose CHO-K1 (Chinese hamster ovary) cells in culture have been plasma-treated and their responses have been recorded by means of propidium iodide staining. Plasma treatment at low to intermediate power levels leads to damage of the DNA in the cell nucleus, which causes cell death. Characteristic features are high precision of plasma action (influenced cells are strictly localized) and induction of cell death without destroying the cell integrity. Possibilities of using plasma treatment for removal of unwanted cells (e.g. cancer cells) will be investigated.

  13. OH radicals from the indirect actions of X-rays induce cell lethality and mediate the majority of the oxygen enhancement effect.

    PubMed

    Hirayama, Ryoichi; Ito, Atsushi; Noguchi, Miho; Matsumoto, Yoshitaka; Uzawa, Akiko; Kobashi, Gen; Okayasu, Ryuichi; Furusawa, Yoshiya

    2013-11-01

    We examined OH radical-mediated indirect actions from X irradiation on cell killing in wild-type Chinese hamster ovary cell lines (CHO and AA8) under oxic and hypoxic conditions, and compared the contribution of direct and indirect actions under both conditions. The contribution of indirect action on cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide, which suppresses indirect action by quenching OH radicals without affecting the direct action of X rays on cell killing. The contributions of indirect action on cell killing of CHO cells were 76% and 50% under oxic and hypoxic conditions, respectively, and those for AA8 cells were 85% and 47%, respectively. Therefore, the indirect action on cell killing was enhanced by oxygen during X irradiation in both cell lines tested. Oxygen enhancement ratios (OERs) at the 10% survival level (D10 or LD90) for CHO and AA8 cells were 2.68 ± 0.15 and 2.76 ± 0.08, respectively. OERs were evaluated separately for indirect and direct actions, which gave the values of 3.75 and 2.01 for CHO, and 4.11 and 1.32 for AA8 cells, respectively. Thus the generally accepted OER value of ∼3 is best understood as the average of the OER values for both indirect and direct actions. These results imply that both indirect and direct actions on cell killing require oxygen for the majority of lethal DNA damage, however, oxygen plays a larger role in indirect than for direct effects. Conversely, the lethal damage induced by the direct action of X rays are less affected by oxygen concentration.

  14. Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

    PubMed

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

    2016-09-01

    Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Morphometric and histological analysis of the lungs of Syrian golden hamsters.

    PubMed Central

    Kennedy, A R; Desrosiers, A; Terzaghi, M; Little, J B

    1978-01-01

    Hamster lung morphometry and histology have been studied in an attempt to determine differences between hamster and human lungs which may have relevance for lung carcinogenesis studies. Morphometric measurements were made on fresh lungs, lung casts, and histological sections. Cell type and frequency measurements were determined from frozen, paraffin, 1 micron plastic (glycol methacrylate) and electron microscopic sections. A standard terminology for hamster lung histology is established, and differences between hamster and human lung morphometry and histology are discussed. Images Fig. 2 Fig. 3 Fig. 4 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 Fig. 17 Fig. 18 Fig. 19 Fig. 20 Fig. 21 Fig. 22 PMID:640957

  16. 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.

    PubMed Central

    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. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol

  17. Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor

    PubMed Central

    Burton, Liza J.; Dougan, Jodi; Jones, Jasmine; Smith, Bethany N.; Randle, Diandra; Henderson, Veronica

    2016-01-01

    ABSTRACT The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression. PMID:27956696

  18. Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor.

    PubMed

    Burton, Liza J; Dougan, Jodi; Jones, Jasmine; Smith, Bethany N; Randle, Diandra; Henderson, Veronica; Odero-Marah, Valerie A

    2017-03-01

    The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression. Copyright © 2017 American Society for Microbiology.

  19. Comparing effects of carbohydrate (CHO) blockers and trivalent chromium on CHO-induced insulin resistance and elevated blood pressure in rats.

    PubMed

    Preuss, Harry G; Echard, Bobby; Bagchi, Debasis; Perricone, Nicholas V

    2013-01-01

    In Sprague-Dawley rats (SD), we compared two categories of natural dietary supplements that influence carbohydrate (CHO) metabolism via different basic mechanisms to ameliorate insulin resistance (IR) and elevated blood pressure (BP) associated with heavy sugar/starch consumption. Two dietary supplements (bean extract and l-arabinose) are often referred to as carb blockers (CBs), because they slow the gastrointestinal absorption of CHO. Trivalent chromium (CR) falls into a group of so-called insulin sensitizers, because its major effect is to enhance peripheral insulin sensitivity. We divided 48 mature male SD into 4 groups of 12. The first group received powdered baseline diet alone (Con). The remaining 3 SD groups (groups 2-4) ingested regular rat chow containing 20% w/w sucrose and 20% w/w rice starch. The second group received only this CHO-enriched chow. To the high-CHO diets of the remaining two groups, either CB to slow CHO absorption (CHO + CB) (group 3) or an insulin sensitizer, trivalent CR (CHO + CR; group 4), was added. Compared to Con group 1, adding high CHO content to the diet of group 2 significantly increased circulating glucose levels and systolic BP (SBP). Addition of CB or CR to the feed of groups 3 and 4 overcame the perturbations that occurred with high CHO challenge in group 2; that is, they lowered circulating glucose concentrations to Con levels, enhanced response to exogenous insulin, and overcame the gradual elevation of SBP. Compared to group 2, the two treatment groups (3 and 4) also showed decreased renin-angiotensin system activity, decreased serum angiotensin-converting enzyme activity, and enhanced nitric oxide activity. Our data indicate that high doses of CB and CR, despite their different mechanisms of action, can completely overcome CHO-induced IR and BP elevations. The data further suggest that CB and CR affect only the changes brought on by heavy CHO ingestion, because IR and SBP in groups 3 and 4 mirrored Con values (group 1

  20. Alpha 1-protease inhibitor moderates human neutrophil elastase-induced emphysema and secretory cell metaplasia in hamsters.

    PubMed

    Stone, P J; Lucey, E C; Virca, G D; Christensen, T G; Breuer, R; Snider, G L

    1990-06-01

    A study was undertaken to determine whether emphysema and airway secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be moderated by pretreatment with human alpha 1-protease inhibitor (API). API (4.9 mg) was given intratracheally to hamsters 1 h before 0.3 mg HNE. Eight weeks later, lung volumes and pressure-volume relationships were measured in the anaesthetized animals. Mean linear intercepts and secretory cell indices were measured in lung sections. API given 1 h before HNE moderated the development of bronchial secretory cell metaplasia. The severity of emphysema was reduced by 75%. Clearance studies indicated that 80% of the functional activity of instilled API could be lavaged from the lungs after 1 h, indicating a 4 h half-life in the lavageable compartment of the lungs. We calculate that for 50% protection from emphysema the molar ratio of lavageable API to HNE at the time of HNE instillation was 4.8 as compared with 0.78 for 50% inhibition of elastolytic activity in vitro, indicating that API is only 16% as efficient in vivo as compared with its in vitro HNE inhibitory effectiveness. Nevertheless, we conclude that human API given intratracheally is efficacious against HNE-induced emphysema and secretory cell metaplasia.

  1. HALOACETONITRILES VS. REGULATED HALOACETIC ACIDS: ARE NITROGEN CONTAINING DBPS MORE TOXIC?

    EPA Science Inventory

    Haloacetonitriles (HANs) are toxic nitrogenous drinking water disinfection by-products (N-DBPs) and are observed with chlorine, chloramine, or chlorine dioxide disinfection. Using microplate-based Chinese hamster ovary (CHO) cell assays for chronic cytotoxicity and acute genotoxi...

  2. Noninvasive measurement of three-dimensional morphology of adhered animal cells employing phase-shifting laser microscope.

    PubMed

    Takagi, Mutsumi; Kitabayashi, Takayuki; Ito, Syunsuke; Fujiwara, Masashi; Tokuda, Akio

    2007-01-01

    Noninvasive measurement of 3-D morphology of adhered animal cells employing a phase-shifting laser microscope (PLM) is investigated, in which the phase shift for each pixel in the view field caused by cell height and the difference in refractive indices between the cells and the medium is determined. By employing saline with different refractive indices instead of a culture medium, the refractive index of the cells, which is necessary for the determination of cell height, is determined under PLM. The observed height of Chinese hamster ovary (CHO) cells cultivated under higher osmolarity is lower than that of the cells cultivated under physiological osmolarity, which is in agreement with previous data observed under an atomic force microscope (AFM). Maximum heights of human bone marrow mesenchymal stem cells and human umbilical cord vein endothelial cells measured under PLM and AFM agree well with each other. The maximum height of nonadherent spherical CHO cells observed under PLM is comparable to the cell diameter measured under a phase contrast inverted microscope. Laser irradiation, which is necessary for the observation under PLM, did not affect 3-D cell morphology. In conclusion, 3-D morphology of adhered animal cells can be noninvasively measured under PLM.

  3. Early effect of boron neutron capture therapy mediated by boronophenylalanine (BPA-BNCT) on mast cells in premalignant tissue and tumors of the hamster cheek pouch.

    PubMed

    Aromando, Romina F; Trivillin, Verónica A; Heber, Elisa M; Pozzi, Emiliano; Schwint, Amanda E; Itoiz, María E

    2010-05-01

    Mast cell (MC) activation in the hamster cheek pouch cancerization model is associated with the increase in tumor cell proliferation, mediated in turn by tryptase, a protease released from mast cell granules after activation. Tryptase induces tumor cell proliferation through the activation of PAR-2 (protease activated receptor-2) on the plasma membrane of carcinoma cells. The therapeutic success of boron neutron capture therapy mediated by boronophenylalanine (BPA-BNCT) in tumor control in the hamster cheek pouch oral cancer model has been previously reported by our laboratory. Early effects of BPA-BNCT on tumors of the hamster cheek pouch include a reduction in DNA-synthesis with the concomitant decrease in the proliferation of malignant cells. The aim of the present study was to investigate the early histological changes in mast cells after BPA-BNCT in tumors and premalignant tissue of the hamster cheek pouch. Tumor-bearing pouches were treated with BPA-BNCT or beam only (neutron irradiation without prior administration of the boron compound) and sacrificed 1day after treatment. The samples were fixed in Carnoy fixative and stained with alcian blue-safranin to identify all the populations of mast cells. Total, active and inactive mast cells (MC) were counted in the connective tissue and the adventitious tissue underlying the pouch wall and at the base of the tumors in pouches treated with BPA-BNCT, in keeping with a previously described technique. BPA-BNCT induced a marked reduction in the total number of mast cells in the pouch (p<0.05). This reduction in the total number of mast cells was due to a reduction in mast cells at the base of the tumor (p<0.005) and it occurred at the expense of the active mast cells (p<0.05). A slight reduction that did not reach statistical significance also occurred in the amount of mast cells in the pouch wall (that corresponds to the premalignant tissue in tumor-bearing pouches), and in the adventitious tissue. In this case the

  4. The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions.

    PubMed

    Batumalaie, Kalaivani; Qvist, Rajes; Yusof, Kamaruddin Mohd; Ismail, Ikram Shah; Sekaran, Shamala Devi

    2014-05-01

    Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic β-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 % CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 μg/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 μM), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the β-cells from hamsters and its cytoprotective effect against hyperglycemia.

  5. Mapping analysis of scaffold/matrix attachment regions (s/MARs) from two different mammalian cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pilus, Nur Shazwani Mohd; Ahmad, Azrin; Yusof, Nurul Yuziana Mohd

    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 frommore » 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.« less

  6. Very High Density of Chinese Hamster Ovary Cells in Perfusion by Alternating Tangential Flow or Tangential Flow Filtration in WAVE Bioreactor™—Part II: Applications for Antibody Production and Cryopreservation

    PubMed Central

    Clincke, Marie-Françoise; Mölleryd, Carin; Samani, Puneeth K; Lindskog, Eva; Fäldt, Eric; Walsh, Kieron; Chotteau, Véronique

    2013-01-01

    A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor™ using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 × 108 cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28× more in a 1-month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013 PMID:23436783

  7. Unusual neutral oligosaccharides in mature Sindbis virus glycoproteins are synthesized from truncated precursor oligosaccharides in Chinese hamster ovary cells.

    PubMed

    Davidson, S K; Hunt, L A

    1983-03-01

    We have previously demonstrated the presence of unusual small asparaginyl-oligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-beta-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal alpha 1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.

  8. Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Utsumi, H.; Elkind, M.M.

    1983-11-01

    A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both x rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to x rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of thesemore » cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarly prevents normal V79 cells from repairing damage that is only potentially lethal.« less

  9. Effects of proliferation on the decay of thermotolerance in Chinese hamster cells.

    PubMed

    Armour, E P; Li, G C; Hahn, G M

    1985-09-01

    Development and decay of thermotolerance were observed in Chinese hamster HA-1 cells. The thermotolerance kinetics of exponentially growing and fed plateau-phase cells were compared. Following a 10-min heat exposure at 45 degrees C, cells in both growth states had similar rates of development of tolerance to a subsequent 45-min exposure at 45 degrees C. This thermotolerant state started to decay between 12 and 24 hr after the initial heat exposure. The decay appeared to initiate slightly sooner in the exponentially growing cells when compared to the fed plateau-phase cells. During the decay phase, the rate of thermotolerance decay was similar in the two growth conditions. In other experiments, cells were induced to divide at a slower rate by chronic growth (3 months) in a low concentration of fetal calf serum. Under these low serum conditions cells became more sensitive to heat and the rate of decay of thermotolerance remained the same for exponentially growing cells. Plateau-phase cells were also more sensitive, but thermotolerance decayed more rapidly in these cells. Although dramatic cell cycle perturbations were seen in the exponentially growing cells, these changes appeared not to be related to thermotolerance kinetics.

  10. Tropospheric photooxidation of CF3CH2CHO and CF3(CH2)2CHO initiated by Cl atoms and OH radicals

    NASA Astrophysics Data System (ADS)

    Antiñolo, M.; Jiménez, E.; Notario, A.; Martínez, E.; Albaladejo, J.

    2009-11-01

    The absolute rate coefficients for the tropospheric reactions of chlorine (Cl) atoms and hydroxyl (OH) radicals with CF3CH2CHO and CF3(CH2)2CHO were measured as a function of temperature (263-371 K) and pressure (50-215 Torr of He) by pulsed UV laser photolysis techniques. Vacuum UV resonance fluorescence was employed to detect and monitor the time evolution of Cl atoms. Laser induced fluorescence was used in this work as a detection of OH radicals as a function of reaction time. No pressure dependence of the bimolecular rate coefficients, kCl and kOH, was found at all temperatures. At room temperature kCl and kOH were (in 10-11 cm3 molecule-1 s-1): kCl(CF3CH2CHO) = (1.55±0.53); kCl(CF3(CH2)2CHO) = (3.39±1.38); kOH(CF3CH2CHO) = (0.259±0.050); kOH(CF3(CH2)2CHO) = (1.28±0.24). A slightly negative temperature dependence of kCl was observed for CF3CH2CHO and CF3(CH2)2CHO, and kOH(CF3CH2CHO). In contrast, kOH(CF3(CH2)2CHO) did not exhibit a temperature dependence in the studied ranged. Arrhenius expressions for these reactions were: kCl(CF3CH2CHO) =(4.4±1.0) × 10-11 exp{-(316±68)/T} cm3 molecule-1 s-1, kCl(CF3(CH2)2CHO) = (2.9±0.7) × 10-10 exp{-625±80)/T} cm3 molecule-1 s-1, kOH(CF3CH2CHO) = (7.8±2.2) × 10-12 exp{-(314±90)/T} cm3 molecule-1 s-1. The atmospheric impact of the homogeneous removal by OH radicals and Cl atoms of these fluorinated aldehydes is discussed in terms of the global atmospheric lifetimes, taking into account different degradation pathways. The calculated lifetimes show that atmospheric oxidation of CF3(CH2)xCHO are globally dominated by OH radicals, however reactions initiated by Cl atoms can act as a source of free radicals at dawn in the troposphere.

  11. Tropospheric photooxidation of CF3CH2CHO and CF3(CH2)2CHO initiated by Cl atoms and OH radicals

    NASA Astrophysics Data System (ADS)

    Antiñolo, M.; Jiménez, E.; Notario, A.; Martínez, E.; Albaladejo, J.

    2010-02-01

    The absolute rate coefficients for the tropospheric reactions of chlorine (Cl) atoms and hydroxyl (OH) radicals with CF3CH2CHO and CF3(CH2)2CHO were measured as a function of temperature (263-371 K) and pressure (50-215 Torr of He) by pulsed UV laser photolysis techniques. Vacuum UV resonance fluorescence was employed to detect and monitor the time evolution of Cl atoms. Laser induced fluorescence was used in this work for the detection of OH radicals as a function of reaction time. No pressure dependence of the bimolecular rate coefficients, kCl and kOH, was found at all temperatures. At room temperature kCl and kOH were (in 10-11 cm3 molecule-1 s-1): kCl(CF3CH2CHO) = (1.55±0.53); kCl(CF3(CH2)2CHO) = (3.39±1.38); kCl(CF3CH2CHO) = (0.259±0.050); kCl(CF3(CH2)2CHO) = (1.28±0.24). A slightly positive temperature dependence of kCl was observed for CF3CH2CHO and CF3(CH2)2CHO, and kOH(CF3CH2CHO). In contrast, kOH(CF3(CH2)2CHO) did not exhibit a temperature dependence over the range investigated. Arrhenius expressions for these reactions were: kCl(CF3CH2CHO) = (4.4±1.0)×10-11 exp{-(316±68)/T} cm3 molecule-1 s-1 kCl(CF3(CH2)2CHO) = (2.9±0.7)×10-10 exp{-(625±80)/T} cm3 molecule-1 s-1 kOH(CF3CH2CHO) = (7.8±2.2)×10-12 exp{-(314±90)/T} cm3 molecule-1 s-1 The atmospheric impact of the homogeneous removal by OH radicals and Cl atoms of these fluorinated aldehydes is discussed in terms of the global atmospheric lifetimes, taking into account different degradation pathways. The calculated lifetimes show that atmospheric oxidation of CF3(CH2)x CHO are globally dominated by OH radicals, however reactions initiated by Cl atoms can act as a source of free radicals at dawn in the troposphere.

  12. Decreased adult neurogenesis in hibernating Syrian hamster.

    PubMed

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

    2016-10-01

    Generation of new neurons from adult neural stem cells occurs in the dentate gyrus (DG) of the hippocampus and the lateral walls of the lateral ventricles. In this article, we study the neurogenesis that takes place during the hibernation of the Syrian hamster (Mesocricetus auratus). Using a variety of standard neurogenesis markers and 5-bromo-2-deoxyuridine (BrdU) incorporation, we describe a preferential decrease in the proliferation of newborn neurons in the subventricular zone (SVZ) of the hibernating hamsters (torpor) rather than in the hippocampus. Furthermore, we demonstrate that the proliferative capacity is recovered after 3-4days of torpor when arousal is triggered under natural conditions (i.e., not artificially provoked). In addition, we show that tau3R, a tau isoform with three microtubule-binding domains, is a suitable marker to study neurogenesis both in the SVZ and subgranular zone (SGZ) of the Syrian hamster brain. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

    PubMed

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J Michael; Kanerva, Anna; Hemminki, Akseli

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors.

  14. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting

    PubMed Central

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J. Michael; Kanerva, Anna; Hemminki, Akseli

    2016-01-01

    ABSTRACT Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8+ T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors. PMID:27467954

  15. Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

    PubMed

    Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

    2018-01-01

    Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

  16. IN VITRO AND IN VIVO TOXICITY: A COMPARISON OF ACRYLAMIDE, CYCLOPHOSPHAMIDE, CHLORDECONE, AND DIETHYLSTILBESTROL

    EPA Science Inventory

    Four chemicals that had been tested in an in vivo toxicological screen were tested in a Chinese hamster ovary (CHO) cytotoxicity assay. Cell density, viability, ATP concentration, rate of protein synthesis, and cellular protein concentration were decreased by exposure to acrylami...

  17. Organisation of Dietary Control for Nutrition-Training Intervention Involving Periodized Carbohydrate (CHO) Availability and Ketogenic Low CHO High Fat (LCHF) Diet.

    PubMed

    Mirtschin, Joanne G; Forbes, Sara F; Cato, Louise E; Heikura, Ida A; Strobel, Nicki; Hall, Rebecca; Burke, Louise M

    2018-02-12

    We describe the implementation of a 3-week dietary intervention in elite race walkers at the Australian Institute of Sport, with a focus on the resources and strategies needed to accomplish a complex study of this scale. Interventions involved: traditional guidelines of high carbohydrate (CHO) availability for all training sessions (HCHO); a periodized CHO diet which integrated sessions with low CHO and high CHO availability within the same total CHO intake, and a ketogenic low-CHO high-fat diet (LCHF). 7-day menus and recipes were constructed for a communal eating setting to meet nutritional goals as well as individualized food preferences and special needs. Menus also included nutrition support pre, during and post-exercise. Daily monitoring, via observation and food checklists, showed that energy and macronutrient targets were achieved: diets were matched for energy (~14.8 MJ/d) and protein (~2.1 g.kg/d), and achieved desired differences for fat and CHO: HCHO and PCHO: CHO = 8.5 g/kg/d, 60% energy; fat = 20% of energy; LCHF: 0.5 g/kg/d CHO, fat = 78% energy. There were no differences in micronutrient intakes or density between HCHO and PCHO diets; however, the micronutrient density of LCHF was significantly lower. Daily food costs per athlete were similar for each diet (~AUDS$27 ± 10). Successful implementation and monitoring of dietary interventions in sports nutrition research of the scale of the present study require meticulous planning and the expertise of chefs and sports dietitians. Different approaches to sports nutrition support raise practical challenges around cost, micronutrient density, accommodation of special needs and sustainability.

  18. Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

    PubMed Central

    Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

    2007-01-01

    We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

  19. The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

    PubMed

    Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

    1993-11-01

    The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

  20. Attenuation of cadmium-induced necrotic cell death by necrostatin-1: Potential necrostatin-1 acting sites

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hsu, T.-S.; Yang, P.-M.; Tsai, J.-S.

    2009-03-01

    Cadmium (Cd) induces necrotic death in Chinese hamster ovary (CHO) K1 cells and we have established the responsible signaling pathway. Reportedly, necrostatin-1 (Nec-1) rescues cells from necrotic death by mediating through the death domain receptor (DR) signaling pathway. We show here that Nec-1 also effectively attenuates necrotic death triggered by Cd. Two other treatments that cause necrotic cell death, one can (z-VAD-fmk/TNF-{alpha} on U937 cells) and the other cannot (etherynic acid (EA) on DLD-1 cells) be rescued by Nec-1, were also studied in parallel for comparison. Results show that Nec-1 is ineffectual in modulating intracellular calcium contents, calpain activity (amore » downstream protease), or reactive oxygen species production. It can counteract the reduction in mitochondrial membrane potential (MMP) caused by treating CHO K1 or U937 cells with necrosis-inducing agent. However, this effect was not found in EA-treated DLD-1 cells. Notably, Nec-1 elevates NF-{kappa}B activity in the presence or absence of necrosis-inducing agents. Our study shows that, in addition to DR-mediated necrosis, Nec-1 is effective in attenuating Cd-induced necrosis. It rescues cells with reduced MMP implying that mitochondrion is its major acting site.« less

  1. Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies

    PubMed Central

    Bale, Shridhar; Martiné, Alexandra; Wilson, Richard; Behrens, Anna-Janina; Le Fourn, Valérie; de Val, Natalia; Sharma, Shailendra K.; Tran, Karen; Torres, Jonathan L.; Girod, Pierre-Alain; Ward, Andrew B.; Crispin, Max; Wyatt, Richard T.

    2018-01-01

    Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan “hole” naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

  2. Comparative genotoxicity of nitrosamine drinking water disinfection byproducts in Salmonella and mammalian cells.

    PubMed

    Wagner, Elizabeth D; Hsu, Kang-Mei; Lagunas, Angelica; Mitch, William A; Plewa, Michael J

    2012-01-24

    Nitrosamine water disinfection byproducts (DBPs) are an emerging class of non-halogenated, nitrogen-containing water contaminants. Five nitrosamine DBPs were analyzed for genotoxicity (N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenylamine (NDPhA). Using Salmonella typhimurium strain YG7108 the descending rank order of mutagenicity was NDMA>NPIP>NMOR>NPYR; NDPhA was not mutagenic. We developed and calibrated an exogenous S9 mix that was highly effective in activating NDMA in Chinese hamster ovary (CHO) cells using the SCGE (Comet) assay. The descending rank order for genotoxicity was NDMA>NPIP>NMOR. NDPhA was genotoxic only at one concentration and NPYR was not genotoxic. The genotoxic potencies in S. typhimurium and CHO cells were highly correlated. Based on their comparative genotoxicity attention should be focused on the generation and occurrence of NDMA, NPIP and NMOR. Current drinking water disinfection processes may need to be modified such that the generation of nitrosamine DBPs is effectively limited in order to protect the environment and the public health. © 2011 Elsevier B.V. All rights reserved.

  3. Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

    PubMed Central

    Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

    2016-01-01

    Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

  4. Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

    PubMed

    Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

    2016-11-01

    Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

  5. Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

    PubMed

    Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

    2005-05-01

    We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

  6. Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

    PubMed

    Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

    2014-02-01

    The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

  7. Treating cell culture media with UV irradiation against adventitious agents: minimal impact on CHO performance.

    PubMed

    Yen, Sandi; Sokolenko, Stanislav; Manocha, Bhavik; Blondeel, Eric J M; Aucoin, Marc G; Patras, Ankit; Daynouri-Pancino, Farnaz; Sasges, Michael

    2014-01-01

    Sterility of cell culture media is an important concern in biotherapeutic processing. In large scale biotherapeutic production, a unit contamination of cell culture media can have costly effects. Ultraviolet (UV) irradiation is a sterilization method effective against bacteria and viruses while being non-thermal and non-adulterating in its mechanism of action. This makes UV irradiation attractive for use in sterilization of cell culture media. The objective of this study was to evaluate the effect of UV irradiation of cell culture media in terms of chemical composition and the ability to grow cell cultures in the treated media. The results showed that UV irradiation of commercial cell culture media at relevant disinfection doses impacted the chemical composition of the media with respect to several carboxylic acids, and to a minimal extent, amino acids. The cumulative effect of these changes, however, did not negatively influence the ability to culture Chinese Hamster Ovary cells, as evaluated by cell viability, growth rate, and protein titer measurements in simple batch growth compared with the same cells cultured in control media exposed to visible light. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

  8. Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9.

    PubMed

    Miao, Jinxin; Ying, Baoling; Li, Rong; Tollefson, Ann E; Spencer, Jacqueline F; Wold, William S M; Song, Seok-Hwan; Kong, Il-Keun; Toth, Karoly; Wang, Yaohe; Wang, Zhongde

    2018-05-06

    The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster ( Mesocricetus auratus ) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3⁺ and CD4⁺ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.

  9. Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.

    PubMed Central

    Boudin, H; Grauz-Guyon, A; Faure, M P; Forgez, P; Lhiaubet, A M; Dennis, M; Beaudet, A; Rostene, W; Pelaprat, D

    1995-01-01

    In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied

  10. Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression

    PubMed Central

    Saldarriaga, Omar A.; Travi, Bruno L.; Choudhury, Goutam Ghosh; Melby, Peter C.

    2012-01-01

    IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamster's susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (−233 to −133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (−153/−142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (−153/−142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS. PMID:22517919

  11. Transient transfection of mammalian cells using a violet diode laser

    NASA Astrophysics Data System (ADS)

    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.

  12. Lymphocyte function in experimental endemic syphilis of Syrian hamsters.

    PubMed Central

    Bagasra, O; Kushner, H; Hashemi, S

    1985-01-01

    We have studied the changes in the lymph nodes, spleen and thymus that occur in inbred LSH Syrian hamsters infected with Treponema pallidum Bosnia A, the causative agent of endemic syphilis, as well as the B-cell responses of these infected animals to helper T-cell independent and dependent antigens. The lymph nodes increased significantly in weight up to 6 weeks after infection, and contained viable treponemes. No significant changes in the spleen weight were observed, and no viable treponemes could be recovered from the spleen. However, the size of the thymus decreased steadily during the course of the disease. The relative number of Ig+ cells (B cells) increased in the spleen and regional lymph nodes, whereas the relative number of T cells decreased during the course of infection. In both the spleen and lymph nodes, the relative number of macrophages increased initially and decreased thereafter in the form of a bell-shaped curve showing a peak at 4-6 weeks of infection. The ability of splenic lymphocytes from infected hamsters to mount a primary PFC response to pneumococcal polysaccharide type III (SIII), a helper T-cell independent antigen, was elevated throughout the course of infection. However, the splenic PFC response to sheep erythrocytes (SRBC), a helper T-cell dependent antigen, was increased only during the first 4 weeks of infection and progressively decreased thereafter. The PFC responses of infected lymph node lymphocytes to both SIII and SRBC were increased during the first 4 weeks and decreased thereafter. These data suggested that atrophy of the thymus seen in syphilitic infection is accompanied by the complex losses of subsets of T cells and altered B-cell functions. An early loss of suppressor T cells in both the lymph nodes and spleen occurs concomitantly with a loss of T helper cells and heterologous (treponema-unrelated) B-cell functions in the lymph nodes. Helper T cells are lost from the spleen only in the later stages of infection, whereas

  13. Radiosensitivity and thermosensitization of thermotolerant Chinese hamster cells and RIF-1 tumors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hartson-Eaton, M.; Malcolm, A.W.; Hahn, G.M.

    1984-07-01

    CHO cells subline HA-1 were made thermotolerant by a priming heat treatment(43/sup 0/C, 30 min). Later, 4, 16, or 24 hr, they were either irradiated or heated (43/sup 0/C, 30 min) and irradiated. Thermotolerance had no effect on the radiation sensitivity of the cells as measured by the D/sub 0/ value of the clonogenic survival curve. However the N value of the curve (width of shoulder) showed a significant increase at 24 hr, indicating an increased capacity to accumulate sublethal damage. The same priming treatment was given to RIF-1 tumors growing in C3H mice. Later, 24 hr, when the tumorsmore » were either irradiated or heated (43/sup 0/C, 30 min) and irradiated, it was found that thermotolerance had no effect on the radiosensitivity of the cells as measured by in vitro assay. However, thermal radiosensitization was not apparent 24 hr after the priming treatment.« less

  14. Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis.

    PubMed

    Masango, Mxolisi G; Ellis, Charlotte E; Botha, Christo J

    2015-08-01

    Diplodiosis, a neuromycotoxicosis of cattle and sheep grazing on mouldy cobs infected by Stenocarpella maydis, is considered the last major veterinary mycotoxicosis for which the causative mycotoxin is still unknown. The current study was aimed at characterizing the cell death observed in mouse neuroblastoma (Neuro-2a), Chinese hamster ovary (CHO-K1) and Madin-Darby bovine kidney (MDBK) cell lines exposed to the S. maydis metabolites (i.e. diplodiatoxin and dipmatol) by investigating the roles of necrosis and apoptosis. Necrosis was investigated using the lactate dehydrogenase (LDH) leakage and propidium iodide (PI) flow cytometry assays and apoptosis was evaluated using the caspase-3/7 and Annexin V flow cytometry assays. In addition, transmission electron microscopy (TEM) was used to correlate the cell death pathways observed in this study with their typical morphologies. Both diplodiatoxin and dipmatol (750 μM) induced necrosis and caspase-dependent apoptosis in Neuro-2a, CHO-K1 and MDBK cells. Ultrastructurally, the two mycotoxins induced mitochondrial damage, cytoplasmic vacuolation and nuclear fragmentation in the three cell lines. These findings have laid a foundation for future studies aimed at elucidating in detail the mechanism of action of the S. maydis metabolites. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.

    PubMed Central

    Ferrara, N; Winer, J; Burton, T; Rowland, A; Siegel, M; Phillips, H S; Terrell, T; Keller, G A; Levinson, A D

    1993-01-01

    Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes. Images PMID:8423215

  16. Efficiency of introns from various origins in fish cells.

    PubMed

    Bétancourt, O H; Attal, J; Théron, M C; Puissant, C; Houdebine, L M

    1993-06-01

    Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

  17. Antigenic specificity and morphologic characteristics of Chlamydia trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis.

    PubMed

    Fox, J G; Stills, H F; Paster, B J; Dewhirst, F E; Yan, L; Palley, L; Prostak, K

    1993-10-01

    Profound diarrhea associated with proliferating intestinal cells containing intraepithelial campylobacter-like organisms (ICLO) occurs in a variety of mammalian hosts, particularly swine and hamsters. Recently, intracellular bacteria were isolated from proliferative intestinal tissue of hamsters and propagated in intestine cell line 407. Oral inoculation of hamsters with cell culture lysates containing these organisms reproduced the disease in susceptible hamsters. In the present study, an intracellular bacterium from the INT 407 cell line was shown by a variety of techniques to be a member of the genus Chlamydia and has been designated Chlamydia sp. strain SFPD. McCoy cells infected with Chlamydia sp. strain SFPD demonstrated bright fluorescent-stained intracytoplasmic inclusions when examined with fluorescein-labeled species-specific C. trachomatis monoclonal antibodies. The organism also reacted to fluorescein-labeled polyclonal but not monoclonal ICLO "omega" antisera. Ultrastructural examination of the Chlamydia sp. strain SFPD from McCoy cells revealed electrondense elementary bodies and a less electron-dense reticulate-like body that was circular; both features are consistent in morphology to developmental forms of Chlamydia and do not conform to ICLO morphology. Molecular studies, 16S ribosomal sequence analysis, and sequencing of the outer membrane protein confirmed that the isolate is a C. trachomatis closely related to the mouse pneumonitis strain of C. trachomatis.

  18. Choline kinase alpha—Putting the ChoK-hold on tumor metabolism

    PubMed Central

    Arlauckas, Sean P.; Popov, Anatoliy V.; Delikatny, E. James

    2016-01-01

    It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest. PMID:27073147

  19. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth

    PubMed Central

    Opoku-Acheampong, Alexander B.; Penugonda, Kavitha; Lindshield, Brian L.

    2016-01-01

    Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth. PMID:27272436

  20. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth.

    PubMed

    Opoku-Acheampong, Alexander B; Penugonda, Kavitha; Lindshield, Brian L

    2016-01-01

    Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth.

  1. Measuring the spectrum of mutation induced by nitrogen ions and protons in the human-hamster hybrid cell line A(L)C

    NASA Technical Reports Server (NTRS)

    Kraemer, S. M.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    Astronauts can be exposed to charged particles, including protons, alpha particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human-hamster cell lines, A(L) and A(L)C. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human-hamster hybrid cell lines A(L)C and A(L). CD59(-) mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A(L) hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A(L)C, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A(L)C cells than in A(L) cells. In the case of nitrogen ions, the mutations observed in A(L)C cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59(-) mutant fraction for A(L)C cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or "skipping" mutations. We demonstrate here that A(L)C cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

  2. Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells.

    PubMed

    Pathak, R; Sarma, A; Sengupta, B; Dey, S K; Khuda-Bukhsh, A R

    2007-01-01

    To study the effects of 12C-beam of 295 keV/microm (57.24 MeV) on M5 and Chinese hamster V79 cells by using cytogenetic assays like micronuclei (MN) induction, chromosomal aberrations (CA) and apoptosis. Additionally, the relative survival of these two cell lines was tested by the colony forming ability of the cells, with a view to understanding the mechanism of cellular damages that lead to difference in cell survival. Confluent cells were irradiated with 12C-beam at various doses using 15UD Pelletron accelerator. Cell survival was studied by the colony forming ability of cells. MN assay was done by fluorescent staining. Different types of chromosomal aberrations in metaphase cells were scored at 12 h after irradiation. Apoptosis was measured at different post irradiation times as detected by nuclear fragmentation and DNA ladder was prepared after 48 h of incubation. Dose-dependent decrease in surviving fractions was found in both the cell lines. However, the surviving fractions were higher in M5 cells in comparison to V79 cells when exposed to the same radiation doses. On the other hand, induced MN frequencies, CA frequencies and apoptosis percentages were less in M5 cells than V79 cells. Very good correlations between surviving fractions and induced MN frequencies or induced total CA or induced apoptosis percentages were obtained in this study. The cell strain M5 showed relatively more radio-resistance to 12C-beam compared to Chinese hamster V79 cells in this study. As the MN formation, CA and apoptosis induction were less in M5 cells as compared to parental V79 cells, the higher cell survival in the former could possibly be attributed to their better repairing ability leading to higher cell survival.

  3. ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

    PubMed Central

    Noel, J. S.; Dewey, W. C.; Abel, J. H.; Thompson, R. P.

    1971-01-01

    Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G1. During very early G1, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G1, a granular component appeared which was intimately associated with the fibrous material. By the middle of G1, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G1 to the middle of S primarily due to the accumulation of the granular component. During the G2 period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA). PMID:4933472

  4. Evaluation of chemopreventive effects of betel leaf on the genotoxicity of pan masala.

    PubMed

    Trivedi, A H; Patel, R K; Rawal, U M; Adhvaryu, S G; Balar, D B

    1994-01-01

    The antigenotoxic effect of the aqueous extract of betel leaf (BL-ext.) against the pan masala was tested with the help of cytogenetic endpoints like chromosome aberration (CA) and sister chromatid exchange (SCE) utilizing Chinese hamster ovary (CHO) cells. Compared to the cultures treated with aqueous extract of pan masala alone, a reduction in CA and SCE frequencies in CHO cells was observed following a combined treatment with pan masala (with or without tobacco) extract and BL-ext. The protective effect of BL-ext. against the genomic damage caused by pan masala was statistically significant only after treating the cells for a longer period.

  5. Choline kinase alpha-Putting the ChoK-hold on tumor metabolism.

    PubMed

    Arlauckas, Sean P; Popov, Anatoliy V; Delikatny, E James

    2016-07-01

    It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

    PubMed Central

    Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

    2000-01-01

    The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

  7. h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

    PubMed

    Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

    2003-03-01

    1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1987-09-01

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

  9. Transcriptomics as a tool for assessing the scalability of mammalian cell perfusion systems.

    PubMed

    Jayapal, Karthik P; Goudar, Chetan T

    2014-01-01

    DNA microarray-based transcriptomics have been used to determine the time course of laboratory and manufacturing-scale perfusion bioreactors in an attempt to characterize cell physiological state at these two bioreactor scales. Given the limited availability of genomic data for baby hamster kidney (BHK) cells, a Chinese hamster ovary (CHO)-based microarray was used following a feasibility assessment of cross-species hybridization. A heat shock experiment was performed using both BHK and CHO cells and resulting DNA microarray data were analyzed using a filtering criteria of perfect match (PM)/single base mismatch (MM) > 1.5 and PM-MM > 50 to exclude probes with low specificity or sensitivity for cross-species hybridizations. For BHK cells, 8910 probe sets (39 %) passed the cutoff criteria, whereas 12,961 probe sets (56 %) passed the cutoff criteria for CHO cells. Yet, the data from BHK cells allowed distinct clustering of heat shock and control samples as well as identification of biologically relevant genes as being differentially expressed, indicating the utility of cross-species hybridization. Subsequently, DNA microarray analysis was performed on time course samples from laboratory- and manufacturing-scale perfusion bioreactors that were operated under the same conditions. A majority of the variability (37 %) was associated with the first principal component (PC-1). Although PC-1 changed monotonically with culture duration, the trends were very similar in both the laboratory and manufacturing-scale bioreactors. Therefore, despite time-related changes to the cell physiological state, transcriptomic fingerprints were similar across the two bioreactor scales at any given instance in culture. Multiple genes were identified with time-course expression profiles that were very highly correlated (> 0.9) with bioprocess variables of interest. Although the current incomplete annotation limits the biological interpretation of these observations, their full potential may be

  10. Zika virus infection of adult and fetal STAT2 knock-out hamsters.

    PubMed

    Siddharthan, Venkatraman; Van Wettere, Arnaud J; Li, Rong; Miao, Jinxin; Wang, Zhongde; Morrey, John D; Julander, Justin G

    2017-07-01

    Zika virus (ZIKV) infection was investigated in adult and fetal STAT2 knock-out (KO) hamsters. Subcutaneous injection of ZIKV of adults resulted in morbidity, mortality, and infection of the uterus, placenta, brain, spinal cord, and testicles, thus providing an opportunity to evaluate congenital ZIKV infection in a second rodent species besides mice. ZIKV-infected cells with morphologies of Sertoli cells and spermatogonia were observed in the testes, which may have implications for sexual transmission and male sterility. Neonates exposed as fetuses to ZIKV at 8 days post-coitus were not smaller than controls. Nevertheless, infectious virus and ZIKV RNA was detected in some, but not all, placentas and fetal brains of KO hamsters. STAT2 KO hamsters may be useful for addressing sexual transmission, pathogenesis, routes of fetal infection, and neurological disease outcomes, and may also be used in antiviral or vaccine studies to identify intervention strategies. Copyright © 2017. Published by Elsevier Inc.

  11. Porcine platelet lysate as a supplement for animal cell culture

    PubMed Central

    Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

    2007-01-01

    A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (<10 EU/mL). The porcine platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

  12. The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

    PubMed Central

    Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

    1990-01-01

    The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

  13. 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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    De Gregorio, Francesca; Pellegrino, Mario; Picchietti, Simona

    2011-06-01

    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 themore » 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

  14. Effects of caffeine on radiation-induced phenomena associated with cell- cycle traverse of mammalian cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Walters, R.A.; Gurley, L.R.; Tobby, R.A.

    1974-02-01

    Caffeine induced a state of G/sub 1/ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following x-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cellsmore » synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The x-rayinduced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeinetreated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed. (auth)« less

  15. Male hamster preference for odors of female hamster vaginal discharges: studies of experiential and hormonal determinants.

    PubMed

    Gregory, E; Engel, K; Pfaff, D

    1975-07-01

    Male hamsters approach sources of odors from female hamster vaginal discharges and spend significantly more time around these odor sources than around control locations in the test box. This preference for female hamster vaginal odors appears in sexually inexperienced as well as experienced males, even in individuals isolated from females since the time of weaning. Castration significantly reduces the sex odor preference, and treatment with testosterone propionate partially restores it.

  16. Pleural lesions in Syrian golden hamsters and Fischer-344 rats following intrapleural instillation of man-made ceramic or glass fibers.

    PubMed

    Everitt, J I; Bermudez, E; Mangum, J B; Wong, B; Moss, O R; Janszen, D; Rutten, A A

    1994-01-01

    The mesothelium is a target of the toxic and carcinogenic effects of certain natural mineral and man-made fibers. Long-term inhalation of a ceramic fiber (RCF-1) results in a high incidence of pleural mesotheliomas in Syrian golden hamsters but not in identically exposed Fischer-344 rats. The present study compared the histopathology of the early pleural response in rats and hamsters instilled with artificial fibers. Groups of Syrian golden hamsters and Fischer-344 rats were instilled with ceramic (RCF-1) or glass (MMVF-10) fibers directly into the pleural space. Each species received approximately equal numbers of long, thin fibers per g body weight. Fiber-induced lesions were compared 7 and 28 days postinstillation. Both hamsters and rats developed qualitatively similar dose-dependent inflammatory lesions that were not fiber-type specific. Both species developed fibrosis in conjunction with inflammation in the visceral pleura, but a striking interspecies difference was noted in the pattern of mesothelial cell response. Hamsters developed greater surface mesothelial cell proliferation and had focal aggregates of mesothelial cells embedded deep within regions of visceral pleural fibrosis. It is hypothesized from the present study that the marked fiber-induced proliferative mesothelial cell response of the hamster visceral pleura may explain the high number of pleural mesotheliomas found in long-term fiber studies in this species.

  17. Ionotropic glutamate receptor GluR2/3-immunoreactive neurons in the cat, rabbit, and hamster superficial superior colliculus.

    PubMed

    Park, Won-Mee; Kim, Min-Jeong; Jeon, Chang-Jin

    2004-06-01

    Ionotropic glutamate receptor (GluR) subtypes occur in various types of cells in the central nervous system. We studied the distribution of AMPA glutamate receptor subtype GluR2/3 in the superficial layers of cat, rabbit, and hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on this distribution. Furthermore, we compared this labeling to that of calbindin D28K and parvalbumin. Anti-GluR2/3-immunoreactive (IR) cells formed a dense band of labeled cells within the lower superficial gray layer (SGL) and upper optic layer (OL) in the cat SC. By contrast, GluR2/3-IR cells formed a dense band within the upper OL in the rabbit and within the OL in the hamster SC. Calbindin D28K-IR cells are located in three layers in the SC: one within the zonal layer (ZL) and the upper SGL in all three animals, a second within the lower OL and upper IGL in the cat, within the IGL in the rabbit and within the OL in the hamster, and a third within the deep gray layer (DGL) in all three animals. Many parvalbumin-IR neurons were found within the lower SGL and upper OL. Thus, the GluR2/3-IR band was sandwiched between the first and second layers of calbindin D28K-IR cells in the cat and rabbit SC while the distribution of GluR2/3-IR cells in the hamster matches the second layer of calbindin D28K-IR cells. The patterned distribution of GluR2/3-IR cells overlapped the tier of parvalbumin-IR neurons in cat, but only partially overlapped in hamster and rabbit. Two-color immunofluorescence revealed that more than half (55.1%) of the GluR2/3-IR cells in the hamster SC expressed calbindin D28K. By contrast, only 9.9% of GluR2/3-IR cells expressed calbindin D28K in the cat. Double-labeled cells were not found in the rabbit SC. Some (4.8%) GluR2/3-IR cells in the cat SC also expressed parvalbumin, while no GluR2/3-IR cells in rabbit and hamster SC expressed parvalbumin. In this dense band of GluR2/3, the majority of labeled cells were small to medium

  18. Immune response in the hamster: definition of a novel IgG not expressed in all hamster strains.

    PubMed Central

    Coe, J E; Schell, R F; Ross, M J

    1995-01-01

    A new IgG isotype is described in serum from Syrian hamsters. This 7S-IgG is called IgG3 and was isolated from IgG1 and IgG2 because of its great affinity for protein A. The unique antigenic determinants of IgG3 were identified with a specific rabbit antisera. IgG3 is the least expressed IgG subclass in Syrian hamsters, but serum levels increase more than 10-fold after immunization or infection. Although found in all tested outbred strains, IgG3 is expressed in only some of the commercially available inbred strains of Syrian hamsters. Five inbred hamster strains were examined, and in three strains (CB, LHC and MHA) IgG3 was not detected in normal serum or in immune serum, indicating serum levels at least 100-fold less than other normal inbred/outbred hamsters. The results of breeding experiments suggests a single gene defect is responsible for this non-expression of IgG3. Immunodeficiency was not associated with this IgG3 deficiency. Selective deficiencies of immunoglobulin classes/subclasses in experimental animals are rare. The evolution of a similar IgG3 deficiency in these three hamster strains during inbreeding suggests a novel and efficient mechanism for regulation of IgG3 synthesis in the Syrian hamster. Images Figure 2 Figure 3 Figure 5 PMID:7590875

  19. An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

    PubMed

    Fratz-Berilla, Erica J; Ketcham, Stephanie A; Parhiz, Hamideh; Ashraf, Muhammad; Madhavarao, Chikkathur N

    2017-12-01

    Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme. Published by Elsevier Inc.

  20. Intestinal transfer of choline in rat and hamster

    PubMed Central

    Sanford, P. A.; Smyth, D. H.

    1971-01-01

    1. The transfer of choline was studied with sacs of everted intestine of rat and hamster. 2. The choline transfer can be divided into two components, a diffusion process and a saturable process. The latter plays a relatively greater part at low concentrations of choline, which include the physiological concentration in the plasma. The saturable process is better seen in the hamster than in the rat. 3. Intestinal transfer of choline is influenced by substances altering the availability of energy in the cell, and by some substances chemically or pharmacologically related to choline. These findings are consistent with some kind of specific mechanism for choline transfer. 4. Part of the choline taken up by the cell appears as a metabolite not yet identified. The formation of the metabolite is a saturable process and is abolished by anaerobic conditions and by homogenization. 5. The results are also discussed in relation to parameters of transfer. PMID:5090994

  1. Molecular Prerequisites for Diminished Cold Sensitivity in Ground Squirrels and Hamsters.

    PubMed

    Matos-Cruz, Vanessa; Schneider, Eve R; Mastrotto, Marco; Merriman, Dana K; Bagriantsev, Sviatoslav N; Gracheva, Elena O

    2017-12-19

    Thirteen-lined ground squirrels and Syrian hamsters are known for their ability to withstand cold during hibernation. We found that hibernators exhibit cold tolerance even in the active state. Imaging and electrophysiology of squirrel somatosensory neurons reveal a decrease in cold sensitivity of TRPM8-expressing cells. Characterization of squirrel and hamster TRPM8 showed that the channels are chemically activated but exhibit poor activation by cold. Cold sensitivity can be re-introduced into squirrel and hamster TRPM8 by transferring the transmembrane domain from the cold sensitive rat ortholog. The same can be achieved in squirrel TRPM8 by mutating only six amino acids. Reciprocal mutations suppress cold sensitivity of the rat ortholog, supporting functional significance of these residues. Our results suggest that ground squirrels and hamsters exhibit reduced cold sensitivity, partially due to modifications in the transmembrane domain of TRPM8. Our study reveals molecular adaptations that accompany cold tolerance in two species of mammalian hibernators. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D.

    PubMed

    Bee, Jared S; Machiesky, LeeAnn M; Peng, Li; Jusino, Kristin C; Dickson, Matthew; Gill, Jeffrey; Johnson, Douglas; Lin, Hung-Yu; Miller, Kenneth; Heidbrink Thompson, Jenny; Remmele, Richard L

    2017-01-01

    Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb-1 and mAb-6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb-1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb-1 and cathepsin D was weaker than that of mAb-6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb-1 are replaced with neutral serine residues in mAb-6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140-145, 2017. © 2016 American Institute of Chemical Engineers.

  3. A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

    PubMed

    Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

    2011-08-15

    A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

    PubMed Central

    Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

    2003-01-01

    Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263

  5. Effects of Synephrine and B-Phenethylamine on Human a-Adrenoceptor Subtypes

    USDA-ARS?s Scientific Manuscript database

    Synephrine and B-phenethylamine are structurally related to ephedrine. In this study, the effects of synephrine and B-phenethylamine are investigated on a-adrenoceptor (a-AR) subtypes expressed in human embroyonic kidney (HEK293) or Chinese hamster ovary (CHO) cells, and compared to that of 1R,2S-no...

  6. Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

    PubMed Central

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

    2011-01-01

    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

  7. Comparison of cellular lethality in DNA repair-proficient or -deficient cell lines resulting from exposure to 70 MeV/n protons or 290 MeV/n carbon ions.

    PubMed

    Genet, Stefan C; Maeda, Junko; Fujisawa, Hiroshi; Yurkon, Charles R; Fujii, Yoshihiro; Romero, Ashley M; Genik, Paula C; Fujimori, Akira; Kitamura, Hisashi; Kato, Takamitsu A

    2012-11-01

    Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

  8. Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures.

    PubMed

    Nagle, W A; Soloff, B L; Moss, A J; Henle, K J

    1990-08-01

    Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.

  9. Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

    PubMed

    Mollet, Mike; Godoy-Silva, Ruben; Berdugo, Claudia; Chalmers, Jeffrey J

    2008-06-01

    Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

  10. Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

    PubMed Central

    1984-01-01

    During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole. PMID:6373793

  11. Metabolic Acidosis Increases Intracellular Calcium in Bone Cells Through Activation of the Proton Receptor OGR1

    PubMed Central

    Frick, Kevin K; Krieger, Nancy S; Nehrke, Keith; Bushinsky, David A

    2009-01-01

    Metabolic acidosis increases urine Ca without increasing intestinal absorption, leading to bone Ca loss. It is unclear how bone cells detect the increase in proton concentration. To determine which G protein-coupled proton sensing receptors are expressed in bone, PCR was performed, and products were detected for OGR1, TDAG8, G2A, and GPR4. We tested the hypothesis that the G protein-coupled proton sensor, OGR1, is an H+-sensing receptor in bone. To determine whether acid-induced bone resorption involves OGR1, we incubated mouse calvariae in neutral pH (NTL) or acidic (MET) medium ± the OGR1 inhibitor CuCl2. CuCl2 decreased MET-induced Ca efflux. We used fluorescent imaging of perfused bone cells to determine whether MET increases Cai. Perfusion with MET induced a rapid, flow-independent, increase in Cai in individual bone cells. To determine whether transfection of OGR1 into a heterologous cell type would increase Cai in response to H+, we perfused Chinese hamster ovary (CHO) cells transfected with mouse OGR1 cDNA. Perfusion with MET induced a rapid increase in Cai in OGR1-transfected CHO cells. These data indicate that OGR1 induces an increase in Cai in response to MET and is a prime candidate for an osteoblast proton sensor. PMID:18847331

  12. Metabolic acidosis increases intracellular calcium in bone cells through activation of the proton receptor OGR1.

    PubMed

    Frick, Kevin K; Krieger, Nancy S; Nehrke, Keith; Bushinsky, David A

    2009-02-01

    Metabolic acidosis increases urine Ca without increasing intestinal absorption, leading to bone Ca loss. It is unclear how bone cells detect the increase in proton concentration. To determine which G protein-coupled proton sensing receptors are expressed in bone, PCR was performed, and products were detected for OGR1, TDAG8, G2A, and GPR4. We tested the hypothesis that the G protein-coupled proton sensor, OGR1, is an H(+)-sensing receptor in bone. To determine whether acid-induced bone resorption involves OGR1, we incubated mouse calvariae in neutral pH (NTL) or acidic (MET) medium +/- the OGR1 inhibitor CuCl(2). CuCl(2) decreased MET-induced Ca efflux. We used fluorescent imaging of perfused bone cells to determine whether MET increases Ca(i). Perfusion with MET induced a rapid, flow-independent, increase in Ca(i) in individual bone cells. To determine whether transfection of OGR1 into a heterologous cell type would increase Ca(i) in response to H(+), we perfused Chinese hamster ovary (CHO) cells transfected with mouse OGR1 cDNA. Perfusion with MET induced a rapid increase in Ca(i) in OGR1-transfected CHO cells. These data indicate that OGR1 induces an increase in Ca(i) in response to MET and is a prime candidate for an osteoblast proton sensor.

  13. Role of Microvesicles in the Spread of Herpes Simplex Virus 1 in Oligodendrocytic Cells.

    PubMed

    Bello-Morales, Raquel; Praena, Beatriz; de la Nuez, Carmen; Rejas, María Teresa; Guerra, Milagros; Galán-Ganga, Marcos; Izquierdo, Manuel; Calvo, Víctor; Krummenacher, Claude; López-Guerrero, José Antonio

    2018-05-15

    Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular

  14. Hypothalamic Ventricular Ependymal Thyroid Hormone Deiodinases Are an Important Element of Circannual Timing in the Siberian Hamster (Phodopus sungorus)

    PubMed Central

    Bolborea, Matei; Wilson, Dana; Mercer, Julian G.; Ebling, Francis J. P.; Morgan, Peter J.; Barrett, Perry

    2013-01-01

    Exposure to short days (SD) induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD) phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE) cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response. PMID:23637944

  15. Dose-rate effect was observed in T98G glioma cells following BNCT.

    PubMed

    Kinashi, Yuko; Okumura, Kakuji; Kubota, Yoshihisa; Kitajima, Erika; Okayasu, Ryuichi; Ono, Koji; Takahashi, Sentaro

    2014-06-01

    It is generally said that low LET radiation produce high dose-rate effect, on the other hand, no significant dose rate effect is observed in high LET radiation. Although high LET radiations are produced in BNCT, little is known about dose-rate effect of BNCT. T98G cells, which were tumor cells, were irradiated by neutron mixed beam with BPA. As normal tissue derived cells, Chinese hamster ovary (CHO-K1) cells and DNA double strand breaks (DNA-DSBs) repair deficient cells, xrs5 cells were irradiated by the neutrons (not including BPA). To DNA-DSBs analysis, T98G cells were stained immunochemically with 53BP1 antibody. The number of DNA-DSBs was determined by counting 53BP1 foci. There was no dose-rate effect in xrs5 cells. D0 difference between 4cGy/min and 20cGy/min irradiation were 0.5 and 5.9 at the neutron and gamma-ray irradiation for CHO-K1, and 0.3 at the neutron for T98G cells. D0 difference between 20cGy/min and 80cGy/min irradiation for T98G cells were 1.2 and 0.6 at neutron irradiation plus BPA and gamma-ray. The differences between neutron irradiations at the dose rate in T98G cells were supported by not only the cell viability but also 53BP1 foci assay at 24h following irradiation to monitor DNA-DSBs. Dose-rate effect of BNCT when T98G cells include 20ppm BPA was greater than that of gamma-ray irradiation. Moreover, Dose-rate effect of the neutron beam when CHO-K1 cells did not include BPA was less than that of gamma-ray irradiation These present results may suggest the importance of dose-rate effect for more efficient BNCT and the side effect reduction. © 2013 Published by Elsevier Ltd.

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

    PubMed

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

    2006-03-05

    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.

  17. Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters.

    PubMed

    Hammerbeck, Christopher D; Brocato, Rebecca L; Bell, Todd M; Schellhase, Christopher W; Mraz, Steven R; Queen, Laurie A; Hooper, Jay W

    2016-07-15

    Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS. Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are

  18. Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters

    PubMed Central

    Hammerbeck, Christopher D.; Brocato, Rebecca L.; Bell, Todd M.; Schellhase, Christopher W.; Mraz, Steven R.; Queen, Laurie A.

    2016-01-01

    ABSTRACT Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS. IMPORTANCE Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract

  19. Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. I. Results obtained after hybridization of human cells carrying reciprocal translocations involving chromosome 1.

    PubMed

    Jongsma, A P; Burgerhout, W G

    1977-01-01

    Regional localization studies of genes coding for human PGD, PPH1, PGM1, UGPP, GuK1, Pep-C, and FH, which have been assigned to chromosome 1, were performed with man-Chinese hamster somatic cell hybrids, Informative hybrids that retained fragments of the human chromosome 1 were produced by fusion of hamster cells with human cells carrying reciprocal translocations involving chromosome 1. Analysis of the hybrids that retained one of the translocation chromosomes or de novo rearrangements involving the human 1 revealed the following gene positions: PGD and PPH1 in 1pter leads to 1p32, PGM1 in 1p32 leads to 1p22, UGPP and GuK1 in 1q21 leads to 1q42, FH in 1qter leads to 1q42, and Pep-C probably in 1q42.

  20. Effect of Lippia alba and Cymbopogon citratus essential oils on biofilms of Streptococcus mutans and cytotoxicity in CHO cells.

    PubMed

    Tofiño-Rivera, A; Ortega-Cuadros, M; Galvis-Pareja, D; Jiménez-Rios, H; Merini, L J; Martínez-Pabón, M C

    2016-12-24

    Caries is a public health problem, given that it prevails in 60 to 90% of the school-age global population. Multiple factors interact in its etiology, among them dental plaque is necessary to have lactic acid producing microorganisms like Streptococcus from he Mutans group. Existing prevention and treatment measures are not totally effective and generate adverse effects, which is why it is necessary to search for complementary strategies for their management. The study sought to evaluate the eradication capacity of Streptococcus mutans biofilms and the toxicity on eukaryotic cells of Lippia alba and Cymbopogon citratus essential oils. Essential oils were extracted from plant material through steam distillation and then its chemical composition was determined. The MBEC-high-throughput (MBEC-HTP) (Innovotech, Edmonton, Alberta, Canada) assay used to determine the eradication concentration of S. mutans ATCC 35668 strain biofilms. Cytotoxicity was evaluated on CHO cells through the MTT cell proliferation assay. The major components in both oils were Geraniol and Citral; in L. alba 18.9% and 15.9%, respectively, and in C. citratus 31.3% and 26.7%. The L. alba essential oils presented eradication activity against S. mutans biofilms of 95.8% in 0.01mg/dL concentration and C. citratus essential oils showed said eradication activity of 95.4% at 0.1, 0.01mg/dL concentrations and of 93.1% in the 0.001mg/dL concentration; none of the concentrations of both essential oils showed toxicity on CHO cells during 24h. The L. alba and C. citratus essential oils showed eradication activity against S. mutans biofilms and null cytotoxicity, evidencing the need to conduct further studies that can identify their active components and in order to guide a safe use in treating and preventing dental caries. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. Secondhand smoke induces hepatic apoptosis and fibrosis in hamster fetus.

    PubMed

    Huang, Chien-Wei; Horng, Chi-Ting; Huang, Chih-Yang; Cho, Ta-Hsiung; Tsai, Yi-Chang; Chen, Li-Jeng; Hsu, Tsai-Ching; Tzang, Bor-Show

    2016-09-01

    Secondhand smoke (SHS) is an important health issue worldwide. Inhaling SHS during pregnancy could cause abnormalities in the internal tissues of newborns, which may then impair fetal development and even cause severe intrauterine damage and perinatal death. However, the understanding of cytopathic mechanisms of SHS by maternal passive smoking on fetus liver during pregnancy is still limited. This study analyzed the effects of high-dose SHS (SHSH) on fetus liver using a maternal passive smoking animal model. Experiments showed that hepatic matrix metalloproteinase-9 activity and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells were significantly increased in livers from fetuses of hamsters treated with SHSH. Similarly, expressions of both extrinsic and intrinsic apoptotic molecules were significantly higher in livers from fetuses of hamsters exposed to SHSH. Additionally, significantly increased inflammatory proteins, including transforming growth factor β, inducible nitric oxide synthase, and interleukin 1β, and fibrotic signaling molecules, including phosphorylated Smad2/3, SP1, and α-smooth muscle actin, were observed in the fetus livers from hamsters treated with SHSH. This study revealed that SHSH not only increased apoptosis through intrinsic and extrinsic pathways in the livers of fetuses from hamsters exposed to SHSH but also augmented hepatic fibrosis via Smad2/3 signaling. © The Author(s) 2015.

  2. Evaluation of eight cephalosporins in hamster colitis model.

    PubMed Central

    Ebright, J R; Fekety, R; Silva, J; Wilson, K H

    1981-01-01

    Eight commonly used cephalosporins were evaluated in the hamster colitis mode. They were all found to cause hemorrhagic cecitis and death within 10 days of being given as subcutaneous or oral challenges. Necropsy findings were indistinguishable from clindamycin-induced cecitis. Bacteria-free cecal filtrate obtained from hamsters dying of cephalosporin-induced cecitis contained toxin similar or identical to hat produced by Clostridium difficile isolated from the cecum of a hamster. Daily oral administration of poorly absorbed cephalosporins protected hamsters from clindamycin-induced cecitis and death as long as the cephalosporins were continued. The absorbable cephalosporins were ineffective in protecting hamsters from clindamycin-induced cecitis. This difference probably relates to the lower concentrations of absorbable cephalosporins maintained in the ceca of the hamsters. The possible correlation of these findings to human cases of cephalosporin-induced pseudomembranous colitis is discussed. PMID:6973951

  3. [Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

    PubMed

    Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

    2011-05-01

    To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

  4. Measuring and Modeling Sonoporation Dynamics in Mammalian Cells via Calcium Imaging

    NASA Astrophysics Data System (ADS)

    Kumon, R. E.; Parikh, P.; Sabens, D.; Aehle, M.; Kourennyi, D.; Deng, C. X.

    2007-05-01

    In this study, calcium imaging via the fluorescent indicator Fura-2 is used to characterize the sonoporation of Chinese Hamster Ovarian (CHO) cells in the presence of Optison™ microbubbles. Evolution of the calcium concentration within cells is determined from real-time fluorescence intensity measurements before, during, and after exposure to a 1 MHz ultrasound tone burst (0.2 s, 0.45 MPa). To relate microscopic sonoporation parameters to the measurements, an analytical model that includes sonoporation and plasma membrane transport is developed, assuming rapid mixing (uniform spatial distribution) in the cell. Fitting the measured data to the model provides estimated values for the poration area as a function of poration relaxation rate as well as plasma membrane pump and leakage rates. A modified compartment model that includes the effects of sonoporation, buffering proteins, and transport across the plasma membrane, endoplasmic reticulum, and mitochondria is also investigated. Numerical 3solutions of this model show a variety of behaviors for the calcium dynamics of the cell.

  5. Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, Part 1: early and late spectral changes are indicative of the presence of apoptosis in cell cultures

    NASA Astrophysics Data System (ADS)

    Mulvey, Christine S.; Zhang, Kexiong; Liu, Wei-Han Bobby; Waxman, David J.; Bigio, Irving J.

    2011-11-01

    Apoptosis, a form of programmed cell death with unique morphological and biochemical features, is dysregulated in cancer and is activated by many cancer chemotherapeutic drugs. Noninvasive assays for apoptosis in cell cultures can aid in screening of new anticancer agents. We have previously demonstrated that elastic scattering spectroscopy can monitor apoptosis in cell cultures. In this report we present data on monitoring the detailed time-course of scattering changes in a Chinese hamster ovary (CHO) and PC-3 prostate cancer cells treated with staurosporine to induce apoptosis. Changes in the backscattering spectrum are detectable within 10 min, and continue to progress up to 48 h after staurosporine treatment, with the magnitude and kinetics of scattering changes dependent on inducer concentration. Similar responses were observed in CHO cells treated with several other apoptosis-inducing protocols. Early and late scattering changes were observed under conditions shown to induce apoptosis via caspase activity assay and were absent under conditions where apoptosis was not induced. Finally, blocking caspase activity and downstream apoptotic morphology changes prevented late scattering changes. These observations demonstrate that early and late changes in wavelength-dependent backscattering correlate with the presence of apoptosis in cell cultures and that the late changes are specific to apoptosis.

  6. Pharmacokinetics and pharmacodynamics of tetra(m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: comparison with clinical measurements.

    PubMed

    Glanzmann, T; Forrer, M; Blant, S A; Woodtli, A; Grosjean, P; Braichotte, D; van den Bergh, H; Monnier, P; Wagnières, G

    2000-08-01

    The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of

  7. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

    PubMed

    Bai, Xingwen; Bao, Huifang; Li, Pinghua; Wei, Wei; Zhang, Meng; Sun, Pu; Cao, Yimei; Lu, Zengjun; Fu, Yuanfang; Xie, Baoxia; Chen, Yingli; Li, Dong; Luo, Jianxun; Liu, Zaixin

    2014-07-24

    Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site-directed mutant

  8. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor

    PubMed Central

    2014-01-01

    Background Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Results Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site

  9. In vivo but not in vitro leptin enhances lymphocyte proliferation in Siberian hamsters (Phodopus sungorus).

    PubMed

    Demas, Gregory E

    2010-04-01

    Mounting an immune response requires a relatively substantial investment of energy and marked reductions in energy availability can suppress immune function and presumably increase disease susceptibility. We have previously demonstrated that a moderate reduction in energy stores by partial surgical lipectomy impairs humoral immunity of Siberian hamsters (Phodopus sungorus) and is mediated, in part, by changes in the adipose tissue hormone leptin. The goals of the present study were to assess the role of leptin in cell-mediated immunity and to determine if the potential effects of leptin on immunity are via the direct actions of this hormone on lymphocytes, or indirect, via the sympathetic nervous system (SNS). In Experiment 1, hamsters received osmotic minipumps containing either murine leptin (0.5 microl/h) or vehicle alone for 10 days and splenocyte proliferation in response to the T-cell mitogen Concanavalin A (Con A) was determined. In Experiment 2, Con A-induced splenocyte proliferation was tested in the presence or absence of leptin in vitro. In Experiment 3, exogenous leptin was administered to intact or sympathetically denervated hamsters. Hamsters treated with in vivo leptin displayed increased splenocyte proliferation compared with control hamsters receiving vehicle. In contrast, in vitro leptin had no effect on splenocyte proliferation. Sympathetic denervation attenuated, but did not block, leptin-induced increases in immunity. Taken together, these results are consistent with the idea that leptin can enhance cell-mediated immunity; the SNS appears to contribute, least in part, to leptin-induced increases in immunity. Importantly, these findings confirm previous studies that leptin serves as an important endocrine link between energy balance and immunity. (c) 2009 Elsevier Inc. All rights reserved.

  10. Effects of red mold dioscorea on oral carcinogenesis in DMBA-induced hamster animal model.

    PubMed

    Hsu, Wei-Hsuan; Lee, Bao-Hong; Pan, Tzu-Ming

    2011-06-01

    Monascus-fermented products offer valuable therapeutic benefits and have been extensively used for centuries in East Asia. Dioscorea has been proved to have anti-cancer effect. The aim of this study is to investigate the anti-tumor ability of the ethanol extract of red mold dioscorea (RMDE) on 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. We induced oral squamous cell carcinoma (OSCC) in the buccal pouch of male Syrian golden hamsters by painting with 0.5% DMBA three times a week for 14 weeks. From 9 to 14 weeks, a dose of 50, 100, and 200 mg RMDE per kg body weight were painting with the hamsters for 6 weeks on days alternate to the DMBA application. The results demonstrated that RMDE decreased nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E(2) (PGE(2)) overexpression in hamster buccal pouches in the DMBA treatment group and increased p53, serum tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) to significantly stimulate caspase-8 and -3 activities, indicating that RMDE reduced oxidative damage causing by DMBA and induced apoptosis in oral cancer cells. Therefore, RMDE may have therapeutic potentials against OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Flow-through electroporation based on constant voltage for large-volume transfection of cells.

    PubMed

    Geng, Tao; Zhan, Yihong; Wang, Hsiang-Yu; Witting, Scott R; Cornetta, Kenneth G; Lu, Chang

    2010-05-21

    Genetic modification of cells is a critical step involved in many cell therapy and gene therapy protocols. In these applications, cell samples of large volume (10(8)-10(9)cells) are often processed for transfection. This poses new challenges for current transfection methods and practices. Here we present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to approximately 20 mL/min) based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, we dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. We tested the delivery of pEFGP-C1 plasmids encoding enhanced green fluorescent protein into Chinese hamster ovary (CHO-K1) cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, approximately 75% of the viable CHO cells were transfected after the procedure. We also generalize the guidelines for scaling up these flow-through electroporation devices. We envision that this technique will serve as a generic and low-cost tool for a variety of clinical applications requiring large volume of transfected cells. Copyright 2010 Elsevier B.V. All rights reserved.

  12. [Cardiac neuronal depopulation in hamsters (Mesocricetus auratus) chronically infected with Trypanosoma cruzi].

    PubMed

    Chapadeiro, E; Silva, E L; Silva, A C; Fernandes, P; Ramirez, L E

    1999-01-01

    The aim of this study was to obtain an experimental animal model of destruction of cardiac neurons in order to investigate the behavior of the cardiac nervous system of hamsters chronically infected with Trypanosoma cruzi. We counted the neuronal cells of the cardiac autonomic nervous plexus in hamsters inoculated with 35,000 blood forms of three different T. cruzi strains and killed 5, 8 and 10 months after infection. We showed for the first time severe neuronal destruction in an experimental animal model with characteristics similar to those observed in human Chagas'disease.

  13. Application of magnetic carriers to two examples of quantitative cell analysis

    NASA Astrophysics Data System (ADS)

    Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

    2017-04-01

    The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

  14. Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells.

    PubMed

    Lefler, David; Mukhin, Yurii V; Pettus, Tobiah; Leeb-Lundberg, L M Fredrik; Garnovskaya, Maria N; Raymond, John R

    2003-04-01

    Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.

  15. Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model.

    PubMed

    Dong, Xianglin; Xu, Tao; Ma, Shaolin; Wen, Hao

    2015-06-01

    The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues.

  16. Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model

    PubMed Central

    DONG, XIANGLIN; XU, TAO; MA, SHAOLIN; WEN, HAO

    2015-01-01

    The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues. PMID:26136958

  17. Damage and Repair of DNA in 5-Bromodeoxyuridine-Labeled Chinese Hamster Cells Exposed to Fluorescent Light

    PubMed Central

    Ben-Hur, E.; Elkind, M. M.

    1972-01-01

    Illumination of Chinese hamster cells with fluorescent light after 5-bromodeoxyuridine incorporation leads to extensive single-strand breakage in the DNA of the exposed cells. The rate of production of single-strand breaks is dependent on the extent to which thymine is replaced by 5-bromouracil. At least some of the breaks observed with alkaline gradients are probably produced in vivo and are probably not contingent upon alkaline hydrolysis since breakage can be demonstrated with neutral gradients also. Cells are able to rejoin most of the single-strand breaks within 60 min; however, damage to the DNA-containing material (the “complex”) initially released from cells is repaired more slowly. Cysteamine protects against single-strand breakage with a dose-modifying factor of 2.8. A comparison is made between the production of single-strand breaks by fluorescent light and X-rays, and the significance of such breaks relative to cell survival is discussed. PMID:5063839

  18. Effect of caffeine on the ultraviolet light induction of SV40 virus from transformed hamster cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zamansky, G.B.; Kleinman, L.F.; Little, J.B.

    1976-01-01

    The effect of caffeine on the uv light induction of SV40 virus from two transformed hamster cell lines heterogeneous for the induction of infectious virus was studied. The amount of virus induced was significantly increased in both cell lines when exposure to uv light was followed by treatment with caffeine. Caffeine in the absence of uv irradiation did not stimulate virus induction, nor did it stimulate SV40 replication in a lytic infection. There was an apparent difference in the concentrations of caffeine which maximally stimulated SV40 virus induction in the two cell lines. This effect could not be explained bymore » differences in cell survival after exposure to uv light and caffeine. Since caffeine is known to cause the accumulation of gaps formed in DNA during postreplication repair of uv-irradiated rodent cells, our results support the hypothesis that the formation of gaps or breaks in DNA is an important early step in virus induction.« less

  19. Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

    PubMed

    Gros, Laurent; Renodon-Cornière, Axelle; de Saint Vincent, Bruno Robert; Feder, Marcin; Bujnicki, Janusz M; Jacquemin-Sablon, Alain

    2006-11-01

    By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

  20. Induction of tissue transglutaminase by dexamethasone: its correlation to receptor number and transglutaminase-mediated cell death in a series of malignant hamster fibrosarcomas.

    PubMed Central

    Johnson, T S; Scholfield, C I; Parry, J; Griffin, M

    1998-01-01

    Treatment of the hamster fibrosarcoma cell lines (Met B, D and E) and BHK-21 hamster fibroblast cells with the glucocorticoid dexamethasone led to a powerful dose-dependent mRNA-synthesis-dependent increase in transglutaminase activity, which can be correlated with dexamethasone-responsive receptor numbers in each cell line. Increasing the number of dexamethasone-responsive receptors by transfection of cells with the HG1 glucocorticoid receptor protein caused an increase in transglutaminase activity that was proportional to the level of transfected receptor. In all experiments the levels of the tissue transglutaminase-mediated detergent-insoluble bodies was found to be comparable with increases in transglutaminase activity. Despite an increase in detergent-insoluble body formation, an increase in apoptosis as measured by DNA fragmentation was not found. Incubation of cells with the non-toxic competitive transglutaminase substrate fluorescein cadaverine led to the incorporation of this fluorescent amine into cellular proteins when cells were damaged after exposure to trypsin during cell passage. These cross-linked proteins containing fluorescein cadaverine were shown to be present in the detergent-insoluble bodies, indicating that the origin of these bodies is via activation of tissue transglutaminase after cell damage by trypsinization rather than apoptosis per se, since Met B cells expressing the bcl-2 cDNA were not protected from detergent-insoluble body formation. We describe a novel mechanism of cell death related to tissue transglutaminase expression and cell damage. PMID:9512467

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Radiobiological response of U251MG, CHO-K1 and V79 cell lines to accelerator-based boron neutron capture therapy

    PubMed Central

    Sato, Eisuke; Zaboronok, Alexander; Yamamoto, Tetsuya; Nakai, Kei; Taskaev, Sergey; Volkova, Olga; Mechetina, Ludmila; Taranin, Alexander; Kanygin, Vladimir; Isobe, Tomonori; Mathis, Bryan J; Matsumura, Akira

    2018-01-01

    Abstract In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2–3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5–3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm−2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear–quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P < 0.01) and confirmed that the neutron source and irradiation regimen were sufficient for control over cell colony formation. We believe our study will serve as a model for ongoing in vitro experiments on neutron capture therapy to advance in this area for further development of accelerator-based BNCT into the clinical phase. PMID:29281044

  3. Histiocytic Sarcoma and Bilateral Facial Vein Thrombosis in a Siberian Hamster (Phodopus sungorus).

    PubMed

    Coble, Dondrae J; Shoemaker, Margaret; Harrington, Bonnie; Dardenne, Adrienne D; Bolon, Brad

    2015-04-01

    A 21-mo-old, male Siberian hamster (Phodopus sungorus) presented with left-sided facial swelling, proptosis of the left eye, and blepharospasm of the right eye. The hamster had been used only for breeding. Because of the poor prognosis, the hamster was euthanized without additional diagnostic assays or treatments. Routine gross pathologic evaluation demonstrated exophthalmos and presumptive hyphema of the left eye, bilateral facial edema, freely movable nodules within the mesentery, white foci within the liver, and a large mass effacing the cranial pole of the right kidney. On histologic evaluation, the mesenteric nodules and liver foci expressed histiocytic marker CD163 and thus were diagnosed as sites of histiocytic sarcoma, whereas the kidney mass was a well-differentiated renal cell carcinoma. The facial swelling resulted from bilateral, chronic, severe, branching thrombi in many facial veins. Additional age-related histopathologic findings were observed in other organs, including diffuse glomerulopathy, nesidioblastosis (pancreatic islet neoformation), and multiple foci of severe cartilage degeneration in the axial skeleton. To our knowledge, this report provides the first description of histiocytic sarcoma in a Siberian hamster.

  4. Laguna Negra Virus Infection Causes Hantavirus Pulmonary Syndrome in Turkish Hamsters (Mesocricetus brandti).

    PubMed

    Hardcastle, K; Scott, D; Safronetz, D; Brining, D L; Ebihara, H; Feldmann, H; LaCasse, R A

    2016-01-01

    Laguna Negra virus (LNV) is a New World hantavirus associated with severe and often fatal cardiopulmonary disease in humans, known as hantavirus pulmonary syndrome (HPS). Five hamster species were evaluated for clinical and serologic responses following inoculation with 4 hantaviruses. Of the 5 hamster species, only Turkish hamsters infected with LNV demonstrated signs consistent with HPS and a fatality rate of 43%. Clinical manifestations in infected animals that succumbed to disease included severe and rapid onset of dyspnea, weight loss, leukopenia, and reduced thrombocyte numbers as compared to uninfected controls. Histopathologic examination revealed lung lesions that resemble the hallmarks of HPS in humans, including interstitial pneumonia and pulmonary edema, as well as generalized infection of endothelial cells and macrophages in major organ tissues. Histologic lesions corresponded to the presence of viral antigen in affected tissues. To date, there have been no small animal models available to study LNV infection and pathogenesis. The Turkish hamster model of LNV infection may be important in the study of LNV-induced HPS pathogenesis and development of disease treatment and prevention strategies. © The Author(s) 2015.

  5. Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

    PubMed Central

    Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

    2014-01-01

    NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

  6. Mutagenic activities of heterocyclic amines in Chinese hamster lung cells in culture

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Terada, M.; Nagao, M.; Nakayasu, M.

    1986-01-01

    A mutation assay system with Chinese hamster lung cells (CHL) using diphtheria toxin resistance as a selective marker has been established. The mutagenic activities of heterocyclic amines, originally isolated from pyrolyzates of amino acids and proteins, broiled fish and fried beef were assayed in cultured CHL cells in the absence and presence of a metabolic activation system, with diphtheria toxin resistance as a marker. All the heterocyclic amines tested except 3-amino-1,4-dimethyl-5H-pyrido (4,3-b)indole (Trp-P-1) required the presence of a metabolic activation system for mutagenicity on CHL cells. 3-Amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2) was the most mutagenic among the heterocyclic amines tested. Other compounds weremore » also mutagenic in the following order of decreasing potency: Trp-P-1, 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 2-amino-9H-pyrido(2,3-b)indole (A..cap alpha..C), 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) and 2-aminodipyrido(1,2--a:3',2'-d)imidazole (Glu-P-2).« less

  7. Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

    PubMed

    Lee, Y J; Hou, Z Z; Curetty, L; Armour, E P; al-Saadi, A; Bernstein, J; Corry, P M

    1992-04-01

    Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.

  8. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

    Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

    1988-01-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  9. Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

    PubMed Central

    Kadan, M J; Sturm, S; Anderson, W F; Eglitis, M A

    1992-01-01

    Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. PMID:1312632

  10. Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene

    NASA Technical Reports Server (NTRS)

    Kraemer, S. M.; Vannais, D. B.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

  11. Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Verma, S.P.; Sonwalkar, N.

    1991-04-01

    The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between {minus}10 and 5{degree}C (low-temperature transition), 10 and 22{degree}C (middle-temperature transition), and 32 and 40{degree}C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14{degree}C). Second, the middle-temperature transition shifts up to the range of about 20-32{degree}C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of aboutmore » 15-40{degree}C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.« less

  12. Sewage sludge hazardous assessment: chemical evaluation and cytological effects in CHO-k1 cells.

    PubMed

    Bonomo, M M; Morozesk, M; Duarte, I D; Rocha, L D; Fernandes, M N; Matsumoto, S T

    2016-06-01

    Application of sewage sludge in agricultural lands is a growing practice in several countries due to its numerous benefits to soil and crops, where chemical and pathogen levels are determined by corresponding legislation. However, the presence of contaminants in residues must always be controlled before application due to their dangerous effects over the ecosystem and potential risks to human health. The main objective of this study was to integrate biological and chemical analysis in order to help elucidating the residue potential toxic, cytotoxic, and mutagenic effects. We evaluate samples of sewage sludge before and after the sanitizing treatment with lime in cytokinesis-block assay using CHO-k1 culture cells. The sanitizing treatment promoted a decrease in pathogen levels, which is the main purpose of this process. Even with chemical levels below the established by environmental agencies, results showed sewage sludge ability to enhance genotoxic and mutagenic effects, proving that residue should be handled with caution in order to minimize its environmental and human risk.

  13. Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation

    PubMed Central

    Wang, Wenchao; Li, Zhiyuan; Liu, Juanjuan; Yang, Xingxing; Ji, Xinmiao; Luo, Yan; Hu, Chen; Hou, Yubin; He, Qianqian; Fang, Jun; Wang, Junfeng; Liu, Qingsong; Li, Guohui; Lu, Qingyou; Zhang, Xin

    2016-01-01

    Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs. PMID:27223425

  14. Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus.

    PubMed

    Antle, Michael C; LeSauter, Joseph; Silver, Rae

    2005-06-09

    The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.

  15. Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

    PubMed

    Aguirre-Vázquez, Alain; Sampayo-Reyes, Adriana; González-Escalante, Laura; Hernández, Alba; Marcos, Ricard; Castorena-Torres, Fabiola; Lozano-Garza, Gerardo; Taméz-Guerra, Reyes; de León, Mario Bermúdez

    2017-01-01

    Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

  16. Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

    PubMed

    Albrecht, Simone; Kaisermayer, Christian; Gallagher, Clair; Farrell, Amy; Lindeberg, Anna; Bones, Jonathan

    2018-06-01

    Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MS E discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control. © 2018 Wiley Periodicals, Inc.

  17. Circadian rhythms accelerate wound healing in female Siberian hamsters

    PubMed Central

    Cable, Erin J.; Onishi, Kenneth G.; Prendergast, Brian J.

    2017-01-01

    Circadian rhythms (CRs) provide temporal regulation and coordination of numerous physiological traits, including immune function. CRs in multiple aspects of immune function are absent in rodents that have been rendered circadian-arrhythmic through various methods. In Siberian hamsters, circadian arrhythmia can be induced by disruptive light treatments (DPS). Here we examined CRs in wound healing, and the effects of circadian disruption on wound healing in DPS-arrhythmic hamsters. Circadian entrained/rhythmic (RHYTH) and behaviorally-arrhythmic (ARR) female hamsters were administered a cutaneous wound either 3 h after light onset (ZT03) or 2 h after dark onset (ZT18); wound size was quantified daily using image analyses. Among RHYTH hamsters, ZT03 wounds healed faster than ZT18 wounds, whereas in ARR hamsters, circadian phase did not affect wound healing. In addition, wounds healed slower in ARR hamsters. The results document a clear CR in wound healing, and indicate that the mere presence of organismal circadian organization enhances this aspect of immune function. Faster wound healing in CR-competent hamsters may be mediated by CR-driven coordination of the temporal order of mechanisms (inflammation, leukocyte trafficking, tissue remodeling) underlying cutaneous wound healing. PMID:27998755

  18. V79 Chinese-hamster cells rendered resistant to high cadmium concentration also become resistant to oxidative stress.

    PubMed Central

    Mello-Filho, A C; Chubatsu, L S; Meneghini, R

    1988-01-01

    Chinese hamster cells (V79) resistant to high concentrations of Cd2+ in the medium were obtained by using the procedure of Beach & Palmiter [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2110-2114], which in mouse led to amplification of metallothionein (MT) genes and to an enrichment in cellular MT. The Cd-resistant V79 clones isolated were significantly more resistant than parental cells to oxidative stress by extracellular H2O2 or a mixture of H2O2 and superoxide anion (O2-) generated by xanthine oxidase plus acetaldehyde. On a per-cell basis, there was no difference between the two cells in their total H2O2-decomposing or O2-(-)dismutating activity. The most likely explanation is that an enrichment in MT content in the Cd-resistant cells was responsible for this effect, because of the antioxidant properties already described for this protein. Images Fig. 2. PMID:2851992

  19. EMODIN EFFICACY ON THE AKT, MAPK, ERK AND DNMT EXPRESSION PATTERN DURING DMBA-INDUCED ORAL CARCINOMA IN GOLDEN SYRIAN HAMSTERS.

    PubMed

    Manimaran, Asokan; Manoharan, Shanmugam; Neelakandan, Mani

    2016-01-01

    The present study has evaluated the Emodin efficacy on the Akt, MAPK, ERK and DNMT expression pattern during 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinoma in golden Syrian hamsters, in order to explore its antitumor potential. Oral tumors were developed in the buccal pouches of golden Syrian hamsters using the carcinogen, DMBA. While the incidence of tumor formation was 100% in hamsters treated with DMBA alone, the tumor formation was not noticed in DMBA+ Emodin treated hamsters. Also, Emodin reduced the severity of precancerous pathological lesions such as dysplasia, in the hamsters treated with DMBA. Emodin administration corrected the abnormalities in the expression pattern of Akt, MAPK, ERK and DNMT in the buccal mucosa of hamsters treated with DMBA. The present study thus suggests that the tumor preventive potential of Emodin is partly related to its modulating effect on the Akt, MAPK, ERK and DNMT expression pattern, as these molecular markers have a pivotal role in the process of cell proliferation, inflammation, invasion, and apoptosis.

  20. Judgement on "hit or non-hit" of CHO cells exposed to accelerated heavy-ions (Fe- or Ar-ions) using division delay and CR-39 plastics as an indicator.

    PubMed

    Mehnati, P; Yatagai, F; Tsuzuki, T; Hanaoka, F; Sasaki, H

    2001-03-01

    The cell killing effect of ionizing radiation depends on the degree of linear energy transfer (LET). The relative biological effectiveness (RBE) reaches a maximum at LET of around 100-200 keV/micron and decreases at higher levels. The ion clusters produced by high-LET radiation are not uniformly distributed. The incidence of non-hit cell events is higher in high LET irradiation than in the cases of low-LET irradiation. This fact could explain the decrease in the cell killing effect at higher levels of LET irradiation. Since the cell killing effect may be related to the nuclear traversal of heavy-ions, it is necessary to establish methods to distinguish the hit cells from the non-hit cells, especially in case with high LET irradiation. Using time-lapse photography, we first examined the hit events by observing the division delay in the cells caused by high-LET irradiation. In addition, we explored the use of CR-39 plastics to detect the exact position of heavy-ion traversal on the surface of a flask where cells were growing. When Chinese hamster ovary (CHO-K1) cells were exposed to 4 Gy of accelerated Fe-ions (2000 keV/micron) or Ar (1640 keV/micron)-ions, the surviving fraction decreased to about 30% in both cases of irradiation. Eighty percent of the irradiated cells, suffered a division delay in contrast to the remaining 20% of the cells which showed a normal division time (12-13 hrs). The later 20% of the cells is considered to be a population of cells which were not actually traversed by heavy-ions. The difference between the higher values of the surviving fraction (approximately 30%) and the non-hit cell population (20%) indicates that some hit cells can grow even after being hit by heavy-ions. The fraction of recovered cells determined by the time-lapse photography method was 10%, and this value closely correlated with the difference between the surviving fraction and the non-hit cells. We used the Poisson distribution of the hit-events by heavy-ions among

  1. Phosphatidylcholine catabolism in the MCF-7 cell cycle.

    PubMed

    Lin, Weiyang; Arthur, Gilbert

    2006-10-01

    The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

  2. Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

    PubMed

    Pacis, Efren; Yu, Marcella; Autsen, Jennifer; Bayer, Robert; Li, Feng

    2011-10-01

    The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process. Copyright © 2011 Wiley Periodicals, Inc.

  3. Topical Olive Leaf Extract Improves Healing of Oral Mucositis in Golden Hamsters.

    PubMed

    Showraki, Najmeh; Mardani, Maryam; Emamghoreishi, Masoumeh; Andishe-Tadbir, Azadeh; Aram, Alireza; Mehriar, Peiman; Omidi, Mahmoud; Sepehrimanesh, Masood; Koohi-Hosseinabadi, Omid; Tanideh, Nader

    2016-12-01

    Oral mucositis (OM) is a common side effect of anti-cancer drugs and needs significant attention for its prevention. This study aimed to evaluate the healing effects of olive leaf extract on 5-fluorouracil-induced OM in golden hamster. OM was induced in 63 male golden hamsters by the combination of 5-fluorouracil injections (days 0, 5 and 10) and the abrasion of the cheek pouch (days 3 and 4). On day 12, hamsters were received topical olive leaf extract ointment, base of ointment, or no treatment (control) for 5 days. Histopathology evaluations, blood examinations, and tissue malondialdehyde level measurement were performed 1, 3 and 5 days after treatments. Histopathology score and tissue malondialdehyde level were significantly lower in olive leaf extract treated group in comparison with control and base groups ( p = 0.000). Significant decreases in white blood cell, hemoglobin, hematocrit , and mean corpuscular volume and an increase in mean corpuscular hemoglobin concentration were observed in olive leaf extract treated group in comparison with control and base groups ( p < 0.05). Our findings demonstrated that daily application of olive leaf extract ointment had healing effect on 5-fluorouracil induced OM in hamsters. Moreover, the beneficial effect of olive leaf extract on OM might be due to its antioxidant and anti-inflammatory properties.

  4. neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

    PubMed

    Buzard, G S; Enomoto, T; Hongyo, T; Perantoni, A O; Diwan, B A; Devor, D E; Reed, C D; Dove, L F; Rice, J M

    1999-10-01

    Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Voelker, D.R.

    1989-12-01

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)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 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({supmore » 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.« less

  6. Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system.

    PubMed

    Lee, Joon Chul; Chang, Ho Nam; Oh, Duk Jae

    2005-01-01

    Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.

  7. Homologous Recombination Repair Protects Against Particulate Chromate-induced Chromosome Instability in Chinese Hamster Cells

    PubMed Central

    Stackpole, Megan M.; Wise, Sandra S.; Duzevik, Eliza Grlickova; Munroe, Ray C.; Thompson, W. Douglas; Thacker, John; Thompson, Larry H.; Hinz, John M.; Wise, John Pierce

    2008-01-01

    Particulate hexavalent chromium [Cr(VI)] compounds are well-established human carcinogens. Cr(VI)-induced tumors are characterized by chromosomal instability (CIN); however, the mechanisms of this effect are unknown. We investigated the hypothesis that homologous recombination (HR) repair of DNA double strand breaks protect cells from Cr(VI)-induced CIN by focusing on the XRCC3 and RAD51C genes, which play an important role in cellular resistance to DNA double strand breaks. We used Chinese hamster cells defective in each HR gene (irs3 for RAD51C and irs1SF for XRCC3) and compared with their wildtype parental and cDNA-complemented controls. We found that the intracellular Cr ion levels varied among the cell lines after particulate chromate treatment. Importantly, accounting for differences in Cr ion levels, we discovered that XRCC3 and RAD51C cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, relative to wild-type and cDNA-complimented cells. We also observed the emergence of high levels of chromatid exchanges in the two mutant cell lines. For example, 1 ug/cm2 lead chromate induced 20 and 32 exchanges in XRCC3- and RAD51C-deficient cells, respectively, whereas no exchanges were detected in the wildtype and cDNA-complemented cells. These observations suggest that HR protects cells from Cr(VI)-induced CIN, consistent with the ability of particulate Cr(VI) to induce double strand breaks. PMID:17662313

  8. Induction of carcinomas and sarcomas by 9,10-dimethyl-1,2-benzanthracene administration into the hamster maxillary sinus.

    PubMed

    Yura, Y; Tsujimoto, H; Kusaka, J; Harada, K; Yoshida, H; Sato, M

    1995-03-01

    To determine whether the local administration of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the hamster maxillary sinus induced carcinoma at the injected site, hamsters were injected with 30 microliters of 0.5% solution of DMBA in dimethyl sulfoxide (DMSO) through the infraorbital foramen into the maxillary sinus once weekly for 10 weeks (Group 2). Another group of hamsters (Group 1) received similar injections of 30 microliters of DMSO only. In a third group of animals (Group 3), a roll of oxycellulose was inserted into the maxillary sinus and 40 microliters of a 2% solution of DMBA in DMSO was injected once. Sinonasal carcinomas were demonstrated in 73% (8/11) of the hamsters in Group 2 and sarcomas were shown in 73% (8/11) of the hamsters in Group 3, as well as some carcinomas. No tumors were seen in the Group 1 hamsters. Histologic examination revealed squamous cell carcinomas arising from the surface epithelium and submucous glands of the nasal cavity and maxillary sinus. These findings indicate that the intrasinal administration of a 0.5% solution of DMBA in DMSO is a reliable method for inducing maxillary sinus cancer.

  9. Chemoprevention by Quercetin of Oral Squamous Cell Carcinoma by Suppression of the NF-κB Signaling Pathway in DMBA-treated Hamsters.

    PubMed

    Zhang, Wen; Yin, Gang; Dai, Jianguo; Sun, Y U; Hoffman, Robert M; Yang, Zhijian; Fan, Yuan

    2017-08-01

    The aim of this study was to investigate the effects of the flavonoid quercetin on chemoprevention of oral squamous cell carcinoma (OSCC). The study involved molecular signaling pathways in 7,12-dimethylbenz(a) anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. DMBA (0.5%) was painted at the right buccal pouches of hamsters for 14 weeks to induce carcinoma. DMBA-treated hamsters received simultaneous doses of quercetin. Animals without DMBA induction were used as normal controls. The incidence of OSCC and the severity of pre-malignant lesions were determined histologically. Apoptosis in the pouch tissue was determined by TUNEL staining. The mRNA and protein expression of NF-κB p50 and p65, as well as Bcl-2 and Bax genes were analyzed using RT-PCR and Western blotting, respectively. Quercetin, at various doses, significantly reduced OSCC incidence and severity of hyperplasia and dysplasia compared to the DMBA-induction-only group (p<0.01). Apoptosis was induced by quercetin treatment compared to the DMBA-induction-only group (p<0.01). mRNA and protein expression of NF-κB p50, p65 as well as Bcl-2 genes were significantly suppressed by quercetin at high doses compared to DMBA induction only (p<0.05). However, mRNA and protein expression of the Bax gene was increased by quercetin treatment at medium and high doses, compared to the DMBA-induction-only group (p<0.05). Quercetin significantly reduced body-weight loss compared to the DMBA-induction-only group (p<0.05). Quercetin reduced tumor incidence and induced apoptosis through modulation of NF-κB signaling and its target genes Bcl-2 and Bax in the DMBA-induced carcigenesis hamster model, suggesting the potential of quercetin as a candidate for OSCC chemoprevention. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  10. Reciprocal Translocation Observed in End-of-Production Cells of a Commercial CHO-Based Process.

    PubMed

    Rouiller, Yolande; Kleuser, Beate; Toso, Emiliano; Palinksy, Wolf; Rossi, Mara; Rossatto, Paola; Barberio, Davide; Broly, Hervé

    2015-01-01

    During the validation of an additional working cell bank derived from a validated master cell bank to support the commercial production continuum of a recombinant protein, we observed an unexpected chromosomal location of the gene of interest in some end-of-production cells. This event-identified by fluorescence in situ hybridization and multicolour chromosome painting as a reciprocal translocation involving a chromosome region containing the gene of interest with its integral coding and flanking sequences-was unique, occurred probably during or prior to multicolour chromosome painting establishment, and was transmitted to the descending generations. Cells bearing the translocation had a transient and process-independent selective advantage, which did not affect process performance and product quality. However, this first report of a translocation affecting the gene of interest location in Chinese Hamster Ovary cells used for producing a biotherapeutic indicates the importance of the demonstration of the integrity of the gene of interest in end-of-production cells. The expression of recombinant therapeutic proteins in mammalian cells depends on the establishment of a cell line with the gene of interest integrated in the host genome and stably expressed over time. Before being used for commercial production, cell lines are submitted to a qualification program in order to ensure their phenotypic and genotypic characteristics and the efficacy and safety of the product. During the production life cycle of a therapeutic protein, additional cells banks have to be validated after exhaustion of the current qualified cell bank in order to support the commercial production continuum of the recombinant protein. It is during the validation of an additional working cell bank derived from a validated master cell bank that we detected a different chromosome bearing the gene of interest in a portion of cells at the end of the upstream production phase. In our case, this event did

  11. Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone.

    PubMed

    Wijnhoven, Hanneke J H; Ennen, Leo; Rodenburg, Richard J T; Dekhuijzen, P N Richard

    2006-01-01

    Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model.

  12. Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone

    PubMed Central

    Wijnhoven, Hanneke JH; Ennen, Leo; Rodenburg, Richard JT; Dekhuijzen, PN Richard

    2006-01-01

    Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model. PMID:18046906

  13. Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization--a case study.

    PubMed

    Chen, Dayue; Nims, Raymond; Dusing, Sandra; Miller, Pamela; Luo, Wen; Quertinmont, Michelle; Parekh, Bhavin; Poorbaugh, Josh; Boose, Jeri Ann; Atkinson, E Morrey

    2008-11-01

    An adventitious agent contamination occurred during a routine 9 CFR bovine viral screening test at BioReliance for an Eli Lilly Chinese Hamster Ovary (CHO) cell-derived Master Cell Bank (MCB) intended for biological production. Scientists from the sponsor (Eli Lilly and Company) and the testing service company (BioReliance) jointly conducted a systematic investigation in an attempt to determine the root cause of the contamination. Our investigation resulted in the identification of the viral nature of the contaminant. Subsequent experiments indicated that the viral contaminant was a non-enveloped and non-hemadsorbing virus. Transmission electron microscopy (TEM) revealed that the viral contaminant was 25-30 nm in size and morphologically resembled viruses of the family Picornaviridae. The contaminant virus was readily inactivated when exposed to acidic pH, suggesting that the viral contaminant was a member of rhinoviruses. Although incapable of infecting CHO cells, the viral contaminant replicated efficiently in Vero cell with a life cycle of approximately 16 h. Our investigation provided compelling data demonstrating that the viral contaminant did not originate from the MCB. Instead, it was introduced into the process during cell passaging and a possible entry point was proposed. We identified the viral contaminant as an equine rhinitis A virus using molecular cloning and DNA sequencing. Finally, our investigation led us to conclude that the source of the viral contaminant was the equine serum added to the cell growth medium in the 9 CFR bovine virus test.

  14. Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains.

    PubMed

    Raymond, Gregory J; Raymond, Lynne D; Meade-White, Kimberly D; Hughson, Andrew G; Favara, Cynthia; Gardner, Donald; Williams, Elizabeth S; Miller, Michael W; Race, Richard E; Caughey, Byron

    2007-04-01

    In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

  15. Caffeine induces metformin anticancer effect on fibrosarcoma in hamsters.

    PubMed

    Popović, D J; Lalošević, D; Miljković, D; Popović, K J; Čapo, I; Popović, J K

    2018-04-01

    We investigated the effect of metformin and caffeine on fibrosarcoma in hamsters. 32 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups, with a minimum of 6 animals per group. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group started peroral treatment with metformin 500 mg/kg daily, the second with caffeine 100 mg/kg daily and the third with a combination of metformin 500 mg/kg and caffeine 100 mg/kg daily, via a gastric probe 3 days before tumor inoculation. After 2 weeks, when the tumors were approximately 2 cm in the control group, all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were pathohistologically (HE) and immunohistochemically (Ki-67, CD 31, COX IV, GLUT-1, iNOS) assessed and the main organs toxicologically analyzed, including the control animals that had received metformin and caffeine. Tumor volume was determined using the formula LxS2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified. Images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances were determined by the Student's t-test. The combination of metformin and caffeine inhibited fibrosarcoma growth in hamsters without toxicity. Administration of metformin with caffeine might be an effective and safe approach in novel nontoxic adjuvant anticancer treatment.

  16. Long-term carcinogenicity study in Syrian golden hamster of particulate emissions from coal- and oil-fired power plants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Persson, S.A.; Ahlberg, M.; Berghem, L.

    1988-04-01

    Male Syrian golden hamsters were given 15 weekly intratracheal instillations with suspensions of coal fly ash or oil fly ash. Controls were instilled with saline containing gelatine (0.5 g/100 mL) or to check particle effects with suspensions of hematite (Fe/sub 2/O/sub 3/). The common weekly dose was 4.5 mg/hamster. In addition, one subgroup of hamsters was treated with oil fly ash at a weekly dose of 3.0 mg/hamster and another with coal fly ash at a weekly dose of 6.0 mg/hamster. Other groups of hamsters were treated with suspensions of benzo(a)pyrene (BaP) or with suspensions on coal fly ash, oilmore » fly ash, or Fe/sub 2/O/sub 3/ coated with BaP. The mass median aerodynamic diameters of the coal and oil fly ashes were 4.4 microns and 28 microns, respectively. Hamsters treated with oil fly ash showed a higher frequency of bronchiolar-alveolar hyperplasia than hamsters in the other treatment groups. Squamous dysplasia and squamous metaplasia were most frequent in animals treated with suspensions of BaP or BaP-coated particles. The earliest appearance of a tumor, the highest incidence of tumors, and the highest incidence of malignant tumors were observed in hamsters treated with oil fly ash coated with BaP. Squamous cell carcinoma and adenosquamous carcinoma were the most frequent malignant tumors. No malignant tumors and only few benign tumors were observed in hamsters instilled with suspensions of fly ash not coated with BaP. The present study gives no indication that coal fly ash could create more serious health problems than oil fly ash.« less

  17. DNA homology and immunological cross-reactivity between Aeromonas hydrophila cytotonic toxin and cholera toxin.

    PubMed Central

    Schultz, A J; McCardell, B A

    1988-01-01

    DNA colony hybridization with three 18- to 20-base-long synthetic oligonucleotide probes for cholera toxin (CT) was used to screen 12 clinical isolates of Aeromonas hydrophila. Under stringent hybridizing (overnight at 40 degrees C) and washing (1 h at 50 degrees C) conditions, nine strains reacted with the 32P-labeled CT probes. Concentrated (10x) cell-free supernatants or lysates from eight cultures, heated at 56 degrees C for 20 min, produced cytotonic effects in Y-1 mouse adrenal cells and Chinese hamster ovary (CHO) cells and caused a 1.5- to 22-fold increase in production of cyclic AMP in CHO cells. Preincubation with anti-CT reduced the CHO cell titer of cell lysates by 10-fold. In the GM1 ganglioside enzyme-linked immunosorbent assay, heated supernatants and lysates gave readings equivalent to 3.5 to 100 ng of CT. Three proteins with molecular weights of 89,900, 37,000, and 11,000 reacted with anti-CT on immunoblots of cell lysates from sodium dodecyl sulfate-polyacrylamide gels. These results suggest that there is DNA homology and immunological cross-reactivity between CT and the A. hydrophila cytotonic toxin. Images PMID:2830300

  18. The products of the thermal decomposition of CH{sub 3}CHO

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vasiliou, AnGayle; National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, Colorado 80401; Piech, Krzysztof M.

    2011-07-07

    We have used a heated 2 cm x 1 mm SiC microtubular ({mu}tubular) reactor to decompose acetaldehyde: CH{sub 3}CHO +{Delta}{yields} products. Thermal decomposition is followed at pressures of 75-150 Torr and at temperatures up to 1675 K, conditions that correspond to residence times of roughly 50-100 {mu}s in the {mu}tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: vacuum ultraviolet photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH{sub 3}CHO, we have studied three isotopologues, CH{sub 3}CDO, CD{sub 3}CHO, and CD{sub 3}CDO. We have identified the thermal decomposition productsmore » CH{sub 3} (PIMS), CO (IR, PIMS), H (PIMS), H{sub 2} (PIMS), CH{sub 2}CO (IR, PIMS), CH{sub 2}=CHOH (IR, PIMS), H{sub 2}O (IR, PIMS), and HC{identical_to}CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH{sub 3}CHO; namely, radical decomposition: CH{sub 3}CHO +{Delta}{yields} CH{sub 3}+[HCO]{yields} CH{sub 3}+ H + CO; elimination: CH{sub 3}CHO +{Delta}{yields} H{sub 2}+ CH{sub 2}=C=O; isomerization/elimination: CH{sub 3}CHO +{Delta}{yields}[CH{sub 2}=CH-OH]{yields} HC{identical_to}CH + H{sub 2}O. An interesting result is that both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH{sub 2}=C:, as an intermediate in the decomposition of vinyl alcohol: CH{sub 2}=CH-OH +{Delta}{yields}[CH{sub 2}=C:]+ H{sub 2}O {yields} HC{identical_to}CH + H{sub 2}O.« less

  19. Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

    PubMed

    Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

    2010-07-01

    Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

  20. Heat and cold acclimation in helium-cold hypothermia in the hamster.

    NASA Technical Reports Server (NTRS)

    Musacchia, X. J.

    1972-01-01

    A study was made of the effects of acclimation of hamsters to high (34-35 C) and low (4-5 C) temperatures for periods up to 6 weeks on the induction of hypothermia in hamsters. Hypothermia was achieved by exposing hamsters to a helox mixture of 80% helium and 20% oxygen at 0 C. Hypothermic induction was most rapid (2-3 hr) in heat-acclimated hamsters and slowest (6-12 hr) in cold-acclimated hamsters. The induction period was intermediate (5-8 hr) in room temperature nonacclimated animals (controls). Survival time in hypothermia was relatable to previous temperature acclimations. The hypothesis that thermogenesis in cold-acclimated hamsters would accentuate resistance to induction of hypothermia was substantiated.

  1. Induction of lyme arthritis in LSH hamsters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schmitz, J.L.; Schell, R.F.; Hejka, A.

    1988-09-01

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

  2. Relative biological effectiveness of accelerated heavy ions for induction of morphological transformation in Syrian hamster embryo cells.

    PubMed

    Han, Z B; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

    1998-09-01

    Syrian hamster embryo cells were used to study the morphological transformation induced by accelerated heavy ions with different linear energy transfer (LET) ranging from 13 to 400 keV/micron. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), then inoculated to culture dishes. Morphologically altered colonies were scored as transformants. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to X-rays first increased with LET, reached a maximum value of about 7 at 100 keV/micron, then decreased with the further increase of LET. Our findings confirmed that high LET heavy ions are much more effective than X-rays for the induction of in vitro cell transformation.

  3. Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

    PubMed

    Bol, S A; van Steeg, H; van Oostrom, C T; Tates, A D; Vrieling, H; de Groot, A J; Mullenders, L H; van Zeeland, A A; Jansen, J G

    1999-05-01

    The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

  4. Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

    PubMed Central

    Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Münz, Markus

    2015-01-01

    Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct. PMID:26510188

  5. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    PubMed

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media.

    PubMed

    Meunier, Sarah M; Todorovic, Biljana; Dare, Emma V; Begum, Afroza; Guillemette, Simon; Wenger, Andrew; Saxena, Priyanka; Campbell, J Larry; Sasges, Michael; Aucoin, Marc G

    2016-01-01

    Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.

  7. Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media

    PubMed Central

    Meunier, Sarah M.; Todorovic, Biljana; Dare, Emma V.; Begum, Afroza; Guillemette, Simon; Wenger, Andrew; Saxena, Priyanka; Campbell, J. Larry; Sasges, Michael; Aucoin, Marc G.

    2016-01-01

    Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm2. Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm2, there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media. PMID:26975046

  8. Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs.

    PubMed

    Zucchelli, Silvia; Patrucco, Laura; Persichetti, Francesca; Gustincich, Stefano; Cotella, Diego

    2016-01-01

    Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation. Among well-established techniques (codon optimization and improvement of mRNA secondary structure), we describe SINEUPs, a family of antisense long non-coding RNAs that are able to increase translation of partially overlapping protein-coding mRNAs. By exploiting their modular structure, SINEUP molecules can be designed to target virtually any mRNA of interest, and thus to increase the production of secreted proteins. Thus, synthetic SINEUPs represent a new versatile tool to improve the production of secreted proteins in biomanufacturing processes.

  9. Effects of porcine pancreatic enzymes on the pancreas of hamsters. Part 2: carcinogenesis studies.

    PubMed

    Nozawa, Fumiaki; Yalniz, Mehmet; Saruc, Murat; Standop, Jens; Egami, Hiroshi; Pour, Parviz M

    2012-09-10

    Our previous study suggested that porcine pancreatic extract in hamsters with peripheral insulin resistance, normalizes insulin output, islet size and pancreatic DNA synthetic rate. It also inhibited the growth of human pancreatic cancer cells in nude mice. To examine the potential value of the porcine pancreatic extract in controlling pancreatic carcinogenesis in this model, the present experiment was performed. Hamsters were fed a high fat diet and four weeks later when insulin resistance emerges, they were divided into two groups. One group received 1 g/kg BW of porcine pancreatic extract in drinking water and the other group received tap water. One week later, when insulin output normalizes in porcine pancreatic extract-treated hamsters, a single subcutaneous injection of N-nitrosobis-(2-oxopropyl) amine (BOP) at a dose of 40 mg/kg BW was given to all hamsters. The experiment was terminated at 43 weeks after the porcine pancreatic extract treatment. The number and size of pancreatic tumors, blood glucose, insulin, amylase and lipase levels, the average size of islets and the number of insulin cells/islets were determined. The incidence of pancreatic cancer was significantly lower in the porcine pancreatic extract group (P=0.043), as well as the plasma insulin level and the size of the islets in the porcine pancreatic extract group were significantly lower (P<0.001) than in the control group. No significantly differences were found in the glucose level between the groups. These results show that porcine pancreatic extract has a potential to inhibit pancreatic cancer growth.

  10. Directed Student Inquiry: Modeling in Roborovsky Hamsters

    ERIC Educational Resources Information Center

    Elwess, Nancy L.; Bouchard, Adam

    2007-01-01

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

  11. Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

    PubMed

    Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

    1997-07-18

    The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

  12. Pineal melatonin synthesis in Syrian hamsters: A summary

    NASA Astrophysics Data System (ADS)

    Rollag, M. D.

    1982-12-01

    During the past decade there has been ample documentation of the proposition that the pineal gland mediates photoperiodic influences upon reproductive behavior of hamsters. It is commonly hypothesized that the pineal gland expresses its activity by transformation of photoperiodic information into an hormonal output, that hormone being melatonin. If this hypothesis is correct, there must be some essential diffrence in melatonin's output when hamsters are exposed to different photoperiodic environments. The experiments summarized in this communication analyze pineal melatonin contents in Syrian hamsters maintained in a variety of photoperiodic conditions during different physiological states. The results demonstrate that adult hamsters have a daily surge in pineal melatonin content throughout their lifetime when exposed to simulated annual photoperiodic cycles. There is some fluctuation in the amount of pineal melatonin produced during different physiological states and photoperiodic environments, but these fluctuations seem small when compared to those normally found for other regulatory hormones. When hamsters are exposed to different photoperiodic regimens, the daily melatonin surge maintains a relatively constant phase relationship with respect to the onset of daily activity. There is a concomitant change in its phase relationship with respect to light-dark transitions.

  13. The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

    PubMed

    Sánchez, O; Montesino, R; Toledo, J R; Rodríguez, E; Díaz, D; Royle, L; Rudd, P M; Dwek, R A; Gerwig, G J; Kamerling, J P; Harvey, D J; Cremata, J A

    2007-08-15

    We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

  14. Fasting-induced daily torpor in desert hamsters (Phodopus roborovskii).

    PubMed

    Chi, Qing-Sheng; Wan, Xin-Rong; Geiser, Fritz; Wang, De-Hua

    2016-09-01

    Daily torpor is frequently expressed in small rodents when facing energetically unfavorable ambient conditions. Desert hamsters (Phodopus roborovskii, ~20g) appear to be an exception as they have been described as homeothermic. However, we hypothesized that they can use torpor because we observed reversible decreases of body temperature (Tb) in fasted hamsters. To test this hypothesis we (i) randomly exposed fasted summer-acclimated hamsters to ambient temperatures (Tas) ranging from 5 to 30°C or (ii) supplied them with different rations of food at Ta 23°C. All desert hamsters showed heterothermy with the lowest mean Tb of 31.4±1.9°C (minimum, 29.0°C) and 31.8±2.0°C (minimum, 29.0°C) when fasted at Ta of 23°C and 19°C, respectively. Below Ta 19°C, the lowest Tb and metabolic rate increased and the proportion of hamsters using heterothermy declined. At Ta 5°C, nearly all hamsters remained normothermic by increasing heat production, suggesting that the heterothermy only occurs in moderately cold conditions, perhaps to avoid freezing at extremely low Tas. During heterothermy, Tbs below 31°C with metabolic rates below 25% of those during normothermia were detected in four individuals at Ta of 19°C and 23°C. Consequently, by definition, our observations confirm that fasted desert hamsters are capable of shallow daily torpor. The negative correlation between the lowest Tbs and amount of food supply shows that heterothermy was mainly triggered by food shortage. Our data indicate that summer-acclimated desert hamsters can express fasting-induced shallow daily torpor, which may be of significance for energy conservation and survival in the wild. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Inhaled ozone as a mutagen. II - Effect on the frequency of chromosome aberrations observed in irradiated Chinese hamsters.

    NASA Technical Reports Server (NTRS)

    Zelac, R. E.; Cromroy, H. L.; Bolch, W. E., Jr.; Dunavant, B. G.; Bevis, H. A.

    1971-01-01

    Exposure-adjusted break frequencies for chromosome aberrations produced in Chinese hamster circulating blood lymphocytes were the quantitative indicator of damage from 5 hrs of exposure to X-radiation and/or to ozone. Radiation produced 5.51 x 0.0001 breaks/cell rad for cells withdrawn 2 weeks after exposure, a reasonable value when compared with data from in vivo exposure of human lymphocytes and Chinese hamster bone marrow cells. Animals exposed to the two agents simultaneously exhibited more than 70% of the total breaks anticipated assuming the expected equal contributions to be additive. Extending to humans, at presently permitted levels, exposure to ozone would be much more detrimental than exposure to radiati*n.

  16. Pleural dosimetry and pathobiological responses in rats and hamsters exposed subchronically to MMVF 10a fiberglass.

    PubMed

    Bermudez, Edilberto; Mangum, James B; Moss, Owen R; Wong, Brian A; Everitt, Jeffrey I

    2003-07-01

    Interspecies differences in pulmonary and pleural responses to the inhalation of natural mineral and synthetic vitreous fibers have been observed in chronic and subchronic studies. However, the reasons for these differences are not clearly understood. There are also fiber-specific differences in the outcome of chronic inhalation exposure to natural mineral and synthetic vitreous fibers. Whether these differences are dependent upon the ability of these fibers to translocate to the pleural space is unknown. The present study was conducted to compare retained fiber burdens and selected pathological responses in the pleural compartments of rats and hamsters following subchronic inhalation of MMVF 10a fiberglass, a fiber negative for tumorigenesis or fibrosis in chronic studies. Fischer 344 rats and Syrian golden hamsters were exposed for 4 or 12 weeks by nose-only inhalation at nominal aerosol mass concentrations of 45 mg/m3 (610 WHO fibers/cc). Pulmonary fiber burdens and pulmonary inflammatory responses were greater in rats than in hamsters. The total number of fibers in the lung was approximately three orders of magnitude greater than in the pleural compartment. Pleural burdens in the hamster (160 fibers/cm2 surface area) were significantly greater than burdens in similarly exposed rats (60 fibers/cm2 surface area) following 12 weeks of exposure. With time postexposure, pleural burdens decreased in hamsters but were essentially unchanged in rats. Pleural inflammatory responses in both species were minimal. In rats, pleural inflammation was characterized by increased numbers of macrophages and increases in mesothelial cell replication during the period of fiber exposure. In contrast, hamsters had increased numbers of macrophages and lymphocytes, and mesothelial-cell replication indices were elevated on the parietal pleura of the costal wall and diaphragm, with some of these responses persisting through 12 weeks of postexposure recovery. Taken together, the results

  17. Widespread extrahippocampal NAA/(Cr+Cho) abnormalities in TLE with and without mesial temporal sclerosis.

    PubMed

    Mueller, Susanne G; Ebel, Andreas; Barakos, Jerome; Scanlon, Cathy; Cheong, Ian; Finlay, Daniel; Garcia, Paul; Weiner, Michael W; Laxer, Kenneth D

    2011-04-01

    MR spectroscopy has demonstrated extrahippocampal NAA/(Cr+Cho) reductions in medial temporal lobe epilepsy with (TLE-MTS) and without (TLE-no) mesial temporal sclerosis. Because of the limited brain coverage of those previous studies, it was, however, not possible to assess differences in the distribution and extent of these abnormalities between TLE-MTS and TLE-no. This study used a 3D whole brain echoplanar spectroscopic imaging (EPSI) sequence to address the following questions: (1) Do TLE-MTS and TLE-no differ regarding severity and distribution of extrahippocampal NAA/(Cr+Cho) reductions? (2) Do extrahippocampal NAA/(Cr+Cho) reductions provide additional information for focus lateralization? Forty-three subjects (12 TLE-MTS, 13 TLE-no, 18 controls) were studied with 3D EPSI. Statistical parametric mapping (SPM2) was used to identify regions of significantly decreased NAA/(Cr+Cho) in TLE groups and in individual patients. TLE-MTS and TLE-no had widespread extrahippocampal NAA/(Cr+Cho) reductions. NAA/(Cr+Cho) reductions had a bilateral fronto-temporal distribution in TLE-MTS and a more diffuse, less well defined distribution in TLE-no. Extrahippocampal NAA/(Cr+Cho) decreases in the single subject analysis showed a large inter-individual variability and did not provide additional focus lateralizing information. Extrahippocampal NAA/(Cr+Cho) reductions in TLE-MTS and TLE-no are neither focal nor homogeneous. This reduces their value for focus lateralization and suggests a heterogeneous etiology of extrahippocampal spectroscopic metabolic abnormalities in TLE.

  18. Biological effects of mixed-ion beams. Part 1: Effect of irradiation of the CHO-K1 cells with a mixed-ion beam containing the carbon and oxygen ions.

    PubMed

    Czub, Joanna; Banaś, Dariusz; Braziewicz, Janusz; Buraczewska, Iwona; Jaskóła, Marian; Kaźmierczak, Urszula; Korman, Andrzej; Lankoff, Anna; Lisowska, Halina; Szefliński, Zygmunt; Wojewódzka, Maria; Wójcik, Andrzej

    2018-05-30

    Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Characteristics of 263K Scrapie Agent in Multiple Hamster Species

    PubMed Central

    Barbian, Kent D.; Race, Brent; Favara, Cynthia; Gardner, Don; Taubner, Lara; Porcella, Stephen; Race, Richard

    2009-01-01

    Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)–resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species. PMID:19193264

  20. Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization

    PubMed Central

    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 (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). 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. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation

  1. Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells.

    PubMed

    Zwanenburg, T S; van Zeeland, A A; Natarajan, A T

    1985-01-01

    Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations. In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor. The significance of these results is discussed.

  2. TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

    PubMed

    Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

    2004-07-16

    Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

  3. Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI*

    PubMed Central

    Wood, Peta; Mulay, Vishwaroop; Darabi, Masoud; Chan, Karen Cecilia; Heeren, Joerg; Pol, Albert; Lambert, Gilles; Rye, Kerry-Anne; Enrich, Carlos; Grewal, Thomas

    2011-01-01

    The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes. PMID:21525007

  4. Effects of porcine pancreatic enzymes on the pancreas of hamsters. Part 1: basic studies.

    PubMed

    Saruc, Murat; Nozawa, Fumiaki; Yalniz, Mehmet; Itami, Atsushi; Pour, Parviz M

    2012-09-10

    Porcine pancreatic enzymes (PPE) extracted from glandular stomach has been used for the treatment of pancreatic cancer patients. Unfortunately, no information is available on the in vitro and in vivo effect on the pancreas and other tissues. We used Syrian Golden hamsters, a unique pancreatic cancer model, to obtain basic information on PPE for its eventual use for the treatment of pancreatic cancer. PPE was used in different concentrations in vitro and in vivo. The stability of the enzyme in the water solution was investigated. It was given to the hamsters by gavage in concentrations of 1g/kg and 400 mg/kg for short periods and in aqueous solution for 65 days. Plasma enzyme and insulin, the size of islets and the number of the insulin cells per islet were examined. The enzyme activity of PPE was maintained in water solution for at least 24 hours. Due to its content of calcium chloride it showed a high toxicity to normal and malignant hamster pancreatic cancer cells and human pancreatic cancer cell lines in vitro. PPE did not alter the plasma pancreatic enzyme levels regardless of the dose, duration and application route. On the contrary, PPE reduced their levels significantly. Remarkably, it also reduced the level of insulin, the size of the islets and the number of insulin cells in the islets significantly. The results imply that PPE does not enter the blood circulation but it appears to slow down the function of both the exocrine and endocrine pancreas.

  5. NADE (p75NTR-associated cell death executor) suppresses cellular growth in vivo.

    PubMed

    Tong, Xiangjun; Xie, Dong; Roth, Wilfried; Reed, John; Koeffler, H Phillip

    2003-06-01

    NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74. It was reported to mediate nerve growth factor-induced apoptosis. We independently isolated human NADE (pHGR74) from breast cancer cell lines. Expression of NADE in various human cancer cell lines, and human and murine tissues was examined. NADE was highly expressed in human endocrine-related organs and embryotic murine tissues. Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo. We used the yeast two-hybrid system to search for NADE binding protein. Dynactin was identified as a candidate. The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE. Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF. Thus, human and murine NADE appear to have different functions.

  6. Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

    PubMed

    Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

    1994-01-01

    Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural

  7. Short photoperiod-induced ovarian regression is mediated by apoptosis in Siberian hamsters (Phodopus sungorus)

    PubMed Central

    Moffatt-Blue, C S; Sury, J J; Young, Kelly A

    2009-01-01

    Siberian hamster reproduction is mediated by photoperiod-induced changes in gonadal activity. However, little is known about how photoperiod induces cellular changes in ovarian function. We hypothesized that exposing female hamsters to short (inhibitory) as opposed to long (control) photoperiods would induce an apoptosis-mediated disruption of ovarian function. Ovaries and plasma from hamsters exposed to either long (LD, 16 h light:8 h darkness) or short (SD, 8 h light:16 h darkness) days were collected during diestrus II after 3, 6, 9 and 12 weeks and processed for histology or RIA respectively. Apoptosis was assessed by in situ TUNEL and active caspase-3 protein immunolabeling. No significant differences were observed among LD hamsters for any parameter; therefore, these control data were pooled. SD exposure induced a decline in preantral follicles (P < 0.05), early antral/antral follicles (P < 0.01) and corpora lutea (P < 0.01) by week 12 as compared with LD. Terminal atretic follicles appeared by SD week 9; by week 12, these had become the predominant ovarian structures. Estradiol concentrations decreased by weeks 9 and 12 SD when compared with both LD and week-3 SD hamsters (P < 0.05); however, no changes were observed for progesterone. TUNEL-positive follicles in SD ovaries increased at week 3 and subsequently declined by week 12 as compared with LD ovaries (P < 0.01). Active capsase-3 protein immunostaining peaked at SD week 3 as compared with all other groups (P < 0.01). TUNEL and capsase-3 immunolabeling were localized to granulosa cells of late-preantral and early-antral/antral follicles. These data indicate that SD exposure rapidly induces follicular apoptosis in Siberian hamsters, which ultimately disrupts both estradiol secretion and folliculogenesis, resulting in the seasonal loss of ovarian function. PMID:16595728

  8. Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

    PubMed

    Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

    2015-02-15

    We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Differential homologous desensitization of the human histamine H3 receptors of 445 and 365 amino acids expressed in CHO-K1 cells.

    PubMed

    García-Gálvez, Ana-Maricela; Escamilla-Sánchez, Juan; Flores-Maldonado, Catalina; Contreras, Rubén-Gerardo; Arias, Juan-Manuel; Arias-Montaño, José-Antonio

    2018-01-01

    Histamine H 3 receptors (H 3 Rs) signal through Gα i/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H 3 R (hH 3 R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH 3 R 445 and hH 3 R 365 ) are widely expressed in the human brain. We previously showed that the hH 3 R 445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH 3 R 365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH 3 R 445 . In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH 3 R 445 and hH 3 R 365 , respectively), there were no differences in receptor affinity for selective H 3 R ligands or for agonist-induced [ 35 S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H 3 R agonist RAMH (1 μM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH 3 R 445 and hH 3 R 365 , respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH 3 R 365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Blood Vessel Normalization in the Hamster Oral Cancer Model for Experimental Cancer Therapy Studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ana J. Molinari; Romina F. Aromando; Maria E. Itoiz

    Normalization of tumor blood vessels improves drug and oxygen delivery to cancer cells. The aim of this study was to develop a technique to normalize blood vessels in the hamster cheek pouch model of oral cancer. Materials and Methods: Tumor-bearing hamsters were treated with thalidomide and were compared with controls. Results: Twenty eight hours after treatment with thalidomide, the blood vessels of premalignant tissue observable in vivo became narrower and less tortuous than those of controls; Evans Blue Dye extravasation in tumor was significantly reduced (indicating a reduction in aberrant tumor vascular hyperpermeability that compromises blood flow), and tumor bloodmore » vessel morphology in histological sections, labeled for Factor VIII, revealed a significant reduction in compressive forces. These findings indicated blood vessel normalization with a window of 48 h. Conclusion: The technique developed herein has rendered the hamster oral cancer model amenable to research, with the potential benefit of vascular normalization in head and neck cancer therapy.« less

  11. Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

    PubMed

    Kerst, G; Beschorner, U; Unsöld, B; von Hahn, T; Schreiber, R; Greger, R; Gerlach, U; Lang, H J; Kunzelmann, K; Bleich, M

    2001-10-01

    KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

  12. SNARE-mediated trafficking of {alpha}{sub 5}{beta}{sub 1} integrin is required for spreading in CHO cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Skalski, Michael; Coppolino, Marc G.

    2005-10-07

    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 cellmore » spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of {alpha}{sub 5}{beta}{sub 1} 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.« less

  13. Molecular analysis of peroxisome proliferation in the hamster.

    PubMed

    Choudhury, Agharul I; Sims, Helen M; Horley, Neill J; Roberts, Ruth A; Tomlinson, Simon R; Salter, Andrew M; Bruce, Mary; Shaw, P Nicholas; Kendall, David; Barrett, David A; Bell, David R

    2004-05-15

    Three novel P450 members of the cytochrome P450 4A family were cloned as partial cDNAs from hamster liver, characterised as novel members of the CYP4A subfamily, and designated CYP4A17, 18, and 19. Hamsters were treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, methylclofenapate (MCP) or Wy-14,643, and shown to develop hepatomegaly and induction of CYP4A17 RNA, and concomitant induction of lauric acid 12- hydroxylase. This treatment also resulted in hypolipidaemia, which was most pronounced in the VLDL fraction, with up to 50% reduction in VLDL-triglycerides; by contrast, blood cholesterol concentration was unaffected by this treatment. These data show that hamster is highly responsive to induction of CYP4A by peroxisome proliferators. To characterise the molecular basis of peroxisome proliferation, the hamster PPARalpha was cloned and shown to encode a 468-amino-acid protein, which is highly similar to rat and mouse PPARalpha proteins. The level of expression of hamster PPARalpha in liver is intermediate between mouse and guinea pig. These results fail to support the hypothesis that the level of PPARalpha in liver is directly responsible for species differences in peroxisome proliferation.

  14. Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

    PubMed

    Hunt, L A

    1980-08-01

    To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

  15. Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

    PubMed Central

    Hunt, L A

    1980-01-01

    To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein. Images PMID:6255177

  16. Hamster and Murine Models of Severe Destructive Lyme Arthritis

    PubMed Central

    Munson, Erik; Nardelli, Dean T.; Du Chateau, Brian K.; Callister, Steven M.; Schell, Ronald F.

    2012-01-01

    Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology. PMID:22461836

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

    PubMed Central

    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

  18. The adaptive immune response does not influence hantavirus disease or persistence in the Syrian hamster

    PubMed Central

    Prescott, Joseph; Safronetz, David; Haddock, Elaine; Robertson, Shelly; Scott, Dana; Feldmann, Heinz

    2013-01-01

    Pathogenic New World hantaviruses cause severe disease in humans characterized by a vascular leak syndrome, leading to pulmonary oedema and respiratory distress with case fatality rates approaching 40%. Hantaviruses infect microvascular endothelial cells without conspicuous cytopathic effects, indicating that destruction of the endothelium is not a mechanism of disease. In humans, high levels of inflammatory cytokines are present in the lungs of patients that succumb to infection. This, along with other observations, suggests that disease has an immunopathogenic component. Currently the only animal model available to study hantavirus disease is the Syrian hamster, where infection with Andes virus (ANDV), the primary agent of disease in South America, results in disease that closely mimics that seen in humans. Conversely, inoculation of hamsters with a passaged Sin Nombre virus (SNV), the virus responsible for most cases of disease in North America, results in persistent infection with high levels of viral replication. We found that ANDV elicited a stronger innate immune response, whereas SNV elicited a more robust adaptive response in the lung. Additionally, ANDV infection resulted in significant changes in the blood lymphocyte populations. To determine whether the adaptive immune response influences infection outcome, we depleted hamsters of CD4+ and CD8+ T cells before infection with hantaviruses. Depletion resulted in inhibition of virus-specific antibody responses, although the pathogenesis and replication of these viruses were unaltered. These data show that neither hantavirus replication, nor pathogenesis caused by these viruses, is influenced by the adaptive immune response in the Syrian hamster. PMID:23600567

  19. Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

    PubMed

    Schrader, Thorsten; Münter, Klaus; Kleine-Ostmann, Thomas; Schmid, Ernst

    2008-12-01

    The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

  20. The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

    PubMed

    Usaj, Marko; Kanduser, Masa

    2012-09-01

    The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

  1. Antipodocalyxin Antibody chPcMab-47 Exerts Antitumor Activity in Mouse Xenograft Models of Colorectal Adenocarcinomas.

    PubMed

    Kaneko, Mika K; Kunita, Akiko; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Ogasawara, Satoshi; Ohishi, Tomokazu; Abe, Shinji; Itai, Shunsuke; Harada, Hiroyuki; Kawada, Manabu; Nishioka, Yasuhiko; Fukayama, Masashi; Kato, Yukinari

    2017-08-01

    Podocalyxin (PODXL) is expressed in several cancers, including brain tumors and colorectal cancers. PODXL overexpression is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells, and produced a clone PcMab-47 that could be used for investigating PODXL expression by flow cytometry and immunohistochemical analysis. Herein, we produced a human-mouse chimeric PcMab-47 (chPcMab-47) and investigated its antitumor activity against PODXL-expressing tumors. chPcMab-47 reacted with LN229, LN229/PODXL, and Chinese hamster ovary (CHO)/PODXL cells, but it did not react with CHO-K1 or PODXL-knockout LN229 cell line (PDIS-13). chPcMab-47 exerted antitumor activity against a mouse xenograft model using CHO/PODXL. Furthermore, chPcMab-47 was reactive with colorectal cancer cell lines such as HCT-15, Caco-2, HCT-8, and DLD-1. chPcMab-47 also exhibited antitumor activity against a mouse xenograft model using HCT-15. These results suggest that chPcMab-47 could be useful for antibody therapy against PODXL-expressing cancers.

  2. Characterization of the Host Response to Pichinde Virus Infection in the Syrian Golden Hamster by Species-Specific Kinome Analysis*

    PubMed Central

    Falcinelli, Shane; Gowen, Brian B.; Trost, Brett; Napper, Scott; Kusalik, Anthony; Johnson, Reed F.; Safronetz, David; Prescott, Joseph; Wahl-Jensen, Victoria; Jahrling, Peter B.; Kindrachuk, Jason

    2015-01-01

    The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV1) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens. PMID:25573744

  3. A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

    PubMed

    Courtès, Franck; Ebel, Bruno; Guédon, Emmanuel; Marc, Annie

    2016-05-01

    to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors. Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10(6) cells ml(-1), while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10(7) cells ml(-1). Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l(-1). Considering the LDH content of viable cells measured to be 110 U per 10(9) cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R(2) ≥ 0.95) for the three types of cell populations. The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

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

    PubMed

    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. (c) 2010 American Institute of Chemical Engineers

  5. [The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

    PubMed

    Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

    1995-01-01

    A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

  6. Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization and defense against bacterial endotoxin in hamsters

    PubMed Central

    Lei, Wei; Nguyen, Heidi; Brown, Naoko; Ni, Hua; Kiffer-Moreira, Tina; Reese, Jeff; Millán, José Luis; Paria, Bibhash C.

    2013-01-01

    Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types, and their hormonal regulation, cell-type and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5 and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; and both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone is the major uterine Akp2 inducer, both progesterone and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. In contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection. PMID:23929901

  7. Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation.

    PubMed

    Periyannan Rajeswari, Prem Kumar; Joensson, Haakan N; Andersson-Svahn, Helene

    2017-01-01

    The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

    PubMed Central

    Mak, P; Wójcik, K; Thogersen, I B; Dubin, A

    1996-01-01

    Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3. PMID:8890190

  9. [Rabies in the common hamster (Cricetus cricetus) in Slovakia].

    PubMed

    Svrcek, S; Ondrejka, R; Mlynarcíková, K; Svec, J

    1984-11-01

    The trials were conducted within the full-scale research on the ecology of lyssa virus. In a period of the mass outbreak of common hamster population in the East Slovakian region, 283 hamsters were examined for rabies. Using the direct immunofluorescence method (DIFM), the rabies antigen was detected in the brain of five hamsters. Three virus strains (denoted as 3 O, 7 E, 9 E) were isolated by means of the inoculation test on sucking mice. On the basis of the detection of the nucleo-protein antigen by DIFM, or its inhibition, detection of the Babes-Negri bodies, determination of the neutralization index, titration on mice and determination of incubation time, the isolated strains were identified as the street strains of rabies virus. As determined by further detailed studies on biological characteristics (determination of the invasiveness index on animals with different susceptibility to rabies virus, determination of pathogenicity for different species of laboratory animals, different weight categories, with different methods of administration, invasiveness index), the "hamster" strains are included among those of intermediate virulence or reduced virulence. At intramuscular administration, the most virulent of the three "hamster" strains studied (3 O) induces a fatal course of rabies in common fox and cat; for wolves, dogs and rabbits it is apathogenic. This strain is also contained in the salivary glands of foxes and cats. In immunofluorescent detection of the rabies nucleoprotein antigen, the "hamster" strains formed a mixed picture of fluorescing particles, characteristic of street strains.

  10. Investigation of triamterene as an inhibitor of the TGR5 receptor: identification in cells and animals.

    PubMed

    Li, Yingxiao; Cheng, Kai Chun; Niu, Chiang-Shan; Lo, Shih-Hsiang; Cheng, Juei-Tang; Niu, Ho-Shan

    2017-01-01

    G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5. We transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo. Triamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na + /Ca 2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats

  11. Anti-GD2-ch14.18/CHO coated nanoparticles mediate glioblastoma (GBM)-specific delivery of the aromatase inhibitor, Letrozole, reducing proliferation, migration and chemoresistance in patient-derived GBM tumor cells.

    PubMed

    Tivnan, Amanda; Heilinger, Tatjana; Ramsey, Joanne M; O'Connor, Gemma; Pokorny, Jenny L; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Boyd, Andrew W; Kim, Ella L; Lode, Holger N; Cryan, Sally-Ann; Prehn, Jochen H M

    2017-03-07

    Aromatase is a critical enzyme in the irreversible conversion of androgens to oestrogens, with inhibition used clinically in hormone-dependent malignancies. We tested the hypothesis that targeted aromatase inhibition in an aggressive brain cancer called glioblastoma (GBM) may represent a new treatment strategy. In this study, aromatase inhibition was achieved using third generation inhibitor, Letrozole, encapsulated within the core of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). PLGA-NPs were conjugated to human/mouse chimeric anti-GD2 antibody ch14.18/CHO, enabling specific targeting of GD2-positive GBM cells. Treatment of primary and recurrent patient-derived GBM cells with free-Letrozole (0.1 μM) led to significant decrease in cell proliferation and migration; in addition to reduced spheroid formation. Anti-GD2-ch14.18/CHO-NPs displayed specific targeting of GBM cells in colorectal-glioblastoma co-culture, with subsequent reduction in GBM cell numbers when treated with anti-GD2-ch14.18-PLGA-Let-NPs in combination with temozolomide. As miR-191 is an estrogen responsive microRNA, its expression, fluctuation and role in Letrozole treated GBM cells was evaluated, where treatment with premiR-191 was capable of rescuing the reduced proliferative phenotype induced by aromatase inhibitor. The repurposing and targeted delivery of Letrozole for the treatment of GBM, with the potential role of miR-191 identified, provides novel avenues for target assessment in this aggressive brain cancer.

  12. Anti-GD2-ch14.18/CHO coated nanoparticles mediate glioblastoma (GBM)-specific delivery of the aromatase inhibitor, Letrozole, reducing proliferation, migration and chemoresistance in patient-derived GBM tumor cells

    PubMed Central

    Tivnan, Amanda; Heilinger, Tatjana; Ramsey, Joanne M; O’Connor, Gemma; Pokorny, Jenny L; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Boyd, Andrew W; Kim, Ella L; Lode, Holger N; Cryan, Sally-Ann; Prehn, Jochen H.M

    2017-01-01

    Aromatase is a critical enzyme in the irreversible conversion of androgens to oestrogens, with inhibition used clinically in hormone-dependent malignancies. We tested the hypothesis that targeted aromatase inhibition in an aggressive brain cancer called glioblastoma (GBM) may represent a new treatment strategy. In this study, aromatase inhibition was achieved using third generation inhibitor, Letrozole, encapsulated within the core of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). PLGA-NPs were conjugated to human/mouse chimeric anti-GD2 antibody ch14.18/CHO, enabling specific targeting of GD2-positive GBM cells. Treatment of primary and recurrent patient-derived GBM cells with free-Letrozole (0.1 μM) led to significant decrease in cell proliferation and migration; in addition to reduced spheroid formation. Anti-GD2-ch14.18/CHO-NPs displayed specific targeting of GBM cells in colorectal-glioblastoma co-culture, with subsequent reduction in GBM cell numbers when treated with anti-GD2-ch14.18-PLGA-Let-NPs in combination with temozolomide. As miR-191 is an estrogen responsive microRNA, its expression, fluctuation and role in Letrozole treated GBM cells was evaluated, where treatment with premiR-191 was capable of rescuing the reduced proliferative phenotype induced by aromatase inhibitor. The repurposing and targeted delivery of Letrozole for the treatment of GBM, with the potential role of miR-191 identified, provides novel avenues for target assessment in this aggressive brain cancer. PMID:28178667

  13. Absence of C-type natriuretic peptide receptors in hamster glomeruli.

    PubMed

    Luk, J K; Wong, E F; Wong, N L

    1994-01-01

    The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

  14. Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

    PubMed

    Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

    2015-01-01

    Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth

  15. Removal of Transmissible Spongiform Encephalopathy Prion from Large Volumes of Cell Culture Media Supplemented with Fetal Bovine Serum by Using Hollow Fiber Anion-Exchange Membrane Chromatography

    PubMed Central

    Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

    2015-01-01

    Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco’s modified Eagle’s medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using

  16. [The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action].

    PubMed

    Kirillova, T V; Rozanov, Iu M; Spivak, I M

    1992-01-01

    A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 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.

  17. Melatonin alleviates hyperthyroidism induced oxidative stress and neuronal cell death in hippocampus of aged female golden hamster, Mesocricetus auratus.

    PubMed

    Rao, Geeta; Verma, Rakesh; Mukherjee, Arun; Haldar, Chandana; Agrawal, Neeraj Kumar

    2016-09-01

    Oxidative stress is a well known phenomenon under hyperthyroid condition that induces various physiological and neural problems with a higher prevalence in females. We, therefore investigated the antioxidant potential of melatonin (Mel) on hyperthyroidism-induced oxidative stress and neuronal cell death in the hippocampus region of brain (cognition and memory centre) of aged female golden hamster, Mesocricetus auratus. Aged female hamsters were randomly divided into four experimental groups (n=7); group-I: control, group-II: Melatonin (5mgkg(-1)day(-1), i.p., for one week), group-III: Hyperthyroid (100μg kg(-1)day(-1), i.p., for two weeks) and group-IV- Hyper+Mel. Hormonal profiles (thyroid and melatonin), activity of antioxidant enzymes (SOD, CAT and GPX), lipid peroxidation level (TBARS) and the specific apoptotic markers (Bax/Bcl-2 ratio and Caspase-3) expression were evaluated. A significant increase in the profile of total thyroid hormone (tT3 and tT4) in hyperthyroidic group as compared to control while tT3 significantly decreased in melatonin treated hyperthyroidic group. However, Mel level significantly decreased in hyperthyroidic group but increased in melatonin treated hyperthyroidic group. Further, the number of immune-positive cells for thyroid hormone receptor-alpha (TR-α) decreased in the hippocampus of hyperthyroidic group and increased in melatonin treated hyperthyroidic group. Profiles of antioxidant enzymes showed a significant decrease in hyperthyroidic group with a simultaneous increase in lipid peroxidation (TBARS). Melatonin treatment to hyperthyroidic group lead to decreased TBARS level with a concomitant increase in antioxidant enzyme activity. Moreover, increased expression of Bax/Bcl-2 ratio and Caspase-3, in hyperthyroidic group had elevated neuronal cell death in hippocampal area and melatonin treatment reduced its expression in hyperthyroidic group. Our findings thus indicate that melatonin reduced the hyperthyroidism

  18. Histopathology of Lyme arthritis in LSH hamsters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hejka, A.; Schmitz, J.L.; England, D.M.

    1989-05-01

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

  19. The adaptive immune response does not influence hantavirus disease or persistence in the Syrian hamster.

    PubMed

    Prescott, Joseph; Safronetz, David; Haddock, Elaine; Robertson, Shelly; Scott, Dana; Feldmann, Heinz

    2013-10-01

    Pathogenic New World hantaviruses cause severe disease in humans characterized by a vascular leak syndrome, leading to pulmonary oedema and respiratory distress with case fatality rates approaching 40%. Hantaviruses infect microvascular endothelial cells without conspicuous cytopathic effects, indicating that destruction of the endothelium is not a mechanism of disease. In humans, high levels of inflammatory cytokines are present in the lungs of patients that succumb to infection. This, along with other observations, suggests that disease has an immunopathogenic component. Currently the only animal model available to study hantavirus disease is the Syrian hamster, where infection with Andes virus (ANDV), the primary agent of disease in South America, results in disease that closely mimics that seen in humans. Conversely, inoculation of hamsters with a passaged Sin Nombre virus (SNV), the virus responsible for most cases of disease in North America, results in persistent infection with high levels of viral replication. We found that ANDV elicited a stronger innate immune response, whereas SNV elicited a more robust adaptive response in the lung. Additionally, ANDV infection resulted in significant changes in the blood lymphocyte populations. To determine whether the adaptive immune response influences infection outcome, we depleted hamsters of CD4(+) and CD8(+) T cells before infection with hantaviruses. Depletion resulted in inhibition of virus-specific antibody responses, although the pathogenesis and replication of these viruses were unaltered. These data show that neither hantavirus replication, nor pathogenesis caused by these viruses, is influenced by the adaptive immune response in the Syrian hamster. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  20. Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA, V and X Secretory Phospholipases A2 (sPLA2).

    PubMed

    Kuksis, Arnis; Pruzanski, Waldemar

    2017-06-01

    Biologically active F- and E/D-type-prostane ring isomers (F 2 -IP and E 2 /D 2 -IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA 2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA 2 , which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1-4 h (37 °C) in the absence or presence of recombinant human sPLA 2 (1-2.5 µg/ml). In the absence of exogenously added sPLA 2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL 3 , respectively. In the presence of group V or group X sPLA 2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA 2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8-24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA 2 . A co-location of sPLA 2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.