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.

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

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.

[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.

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.

Lymphocyte responses to influenza and tetanus toxoid in vitro following intensive exercise and carbohydrate ingestion on consecutive days.

PubMed

Bishop, Nicolette C; Walker, Gary J; Bowley, Lee A; Evans, Kate F; Molyneux, Karen; Wallace, Fiona A; Smith, Alice C

2005-10-01

The effect of carbohydrate (CHO) ingestion on antigen- (rather than mitogen-) stimulated T-cell responses to prolonged, intensive exercise may give a more realistic insight into the effect of CHO on T-cell functional capacity and subsequent infection risk. This study investigated the effect of CHO ingestion during prolonged, intensive exercise on influenza- and tetanus toxoid-stimulated T-cell cytokine mRNA expression and proliferation. Mitogen- [phytohemagglutinin (PHA)] stimulated proliferation was assessed for comparison. Responses were assessed following exercise on consecutive mornings to determine any carryover effect. Fifteen male games players performed two exercise trials in a double-blind, randomized, crossover design. Each trial comprised 90 min of intensive, intermittent running on consecutive mornings, with either CHO (6.4% wt/vol) or placebo (PLA) beverage ingestion before, during, and after each bout of exercise. Postexercise CD3(+) cell counts were higher in PLA than CHO on both days (P < 0.05). Antigen-stimulated T-cell cytokine mRNA expression was unaffected by exercise or CHO ingestion. Before exercise on day 2, T-cell proliferative responses to PHA, influenza, and tetanus toxoid were higher in CHO than PLA by 99, 80, and 58%, respectively (P < 0.01 for PHA, P < 0.05 for influenza and tetanus toxoid). At 1 h postexercise on day 2, PHA-induced proliferation was 70% higher in CHO than PLA (P < 0.05), yet there were no differences between trials for antigen-induced proliferative responses. Therefore, mitogen-induced T-cell proliferation following strenuous exercise and CHO does not necessarily reflect responses to specific antigens and, consequently, may not provide a good model for the situation in vivo.

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.

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)

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

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.

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo

PubMed Central

1994-01-01

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta- related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation. PMID:8089189

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.

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.

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.

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.

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.

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.

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 survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line's higher tolerance of nsPEF exposure.

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 were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. © 2014 Wiley Periodicals, Inc.

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.

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.

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.

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 significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

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

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.

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis.

PubMed

Ryan, Alan J; Medh, Jheem D; McCoy, Diann M; Salome, Ronald G; Mallampalli, Rama K

2002-08-01

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.

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.

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.

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

PubMed Central

Velugula‐Yellela, Sai Rashmika; Williams, Abasha; Trunfio, Nicholas; Hsu, Chih‐Jung; Chavez, Brittany; Yoon, Seongkyu

2017-01-01

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 106 cells‐d/mL, while maintaining specific antibody production (Qp > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018 PMID:29086492

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 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.

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

PubMed

Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2016-01-28

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

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

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

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.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

PubMed

von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

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

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

Effects of human chromosome 12 on interactions between Tat and TAR of human immunodeficiency virus type 1.

PubMed Central

Alonso, A; Cujec, T P; Peterlin, B M

1994-01-01

Rates of transcriptions of the human immunodeficiency virus are greatly increased by the viral trans activator Tat. In vitro, Tat binds to the 5' bulge of the trans-activation response (TAR) RNA stem-loop, which is present in all viral transcripts. In human cells, the central loop in TAR and its cellular RNA-binding proteins are also critical for the function of Tat. Previously, we demonstrated that in rodent cells (CHO cells), but not in those which contain the human chromosome 12 (CHO12 cells), Tat-TAR interactions are compromised. In this study, we examined the roles of the bulge and loop in TAR in Tat trans activation in these cells. Whereas low levels of trans activation depended solely on interactions between Tat and the bulge in CHO cells, high levels of trans activation depended also on interactions between Tat and the loop in CHO12 cells. Since the TAR loop binding proteins in these two cell lines were identical and different from their human counterpart, the human chromosome 12 does not encode TAR loop binding proteins. In vivo binding competition studies with TAR decoys confirmed that the binding of Tat to TAR is more efficient in CHO12 cells. Thus, the protein(s) encoded on human chromosome 12 helps to tether Tat to TAR via its loop, which results in high levels of trans activation. Images PMID:8083988

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.

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.

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.

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

PubMed Central

Gaynor, P M; Greenberg, M L

1992-01-01

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed. PMID:1324908

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

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

PubMed Central

Marini, Pietro; Cascio, Maria-Grazia; King, Angela; Pertwee, Roger G; Ross, Ruth A

2013-01-01

Background and Purpose Although cannabinoid CB2 receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2-transfected CHO cells. Experimental Approach We tested the ability of well-known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2-transfected CHO cell membranes. cAMP assays were also performed in hCB2-CHO cells. Key Results The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2-CHO cells, in both [35S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB2-CHO cells when tested in the [35S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB2-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB2-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues. Conclusion and Implications Our results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution. PMID:23711022

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

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

PubMed Central

Price, B D; Morris, J D; Hall, A

1989-01-01

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829

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 with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes.

PubMed

Oron, Y; Gillo, B; Gershengorn, M C

1988-06-01

Xenopus laevis oocytes are giant cells suitable for studies of plasma membrane receptors and signal transduction pathways because of their capacity to express receptors after injection of heterologous mRNA. We studied depolarizing chloride currents evoked by acetylcholine (AcCho) in native oocytes ("intrinsic AcCho response"), by thyrotropin-releasing hormone (TRH) in oocytes injected with pituitary (GH3) cell RNA ("acquired TRH response"), and by AcCho in oocytes injected with rat brain RNA ("acquired AcCho response"). We found differences in the latencies and patterns of these responses and in the responsiveness to these agonists when applied to the animal or vegetal hemisphere, even though all of the responses are mediated by the same signal transduction pathway. The common intrinsic response to AcCho is characterized by minimal latency (0.86 +/- 0.05 sec), a rapid, transient depolarization followed by a distinct prolonged depolarization, and larger responses obtained after AcCho application at the vegetal rather than the animal hemisphere. By contrast, the acquired responses to TRH and AcCho are characterized by much longer latencies, 9.3 +/- 1.0 and 5.5 +/- 0.8 sec, respectively, and large rapid depolarizations followed by less distinct prolonged depolarizations. The responsiveness on the two hemispheres to TRH and AcCho in mRNA-injected oocytes is opposite to that for the common intrinsic AcCho response in that there is a much greater response when agonist is applied at the animal rather than the vegetal hemisphere. We suggest that the differences in these responses are caused by differences in the intrinsic properties of these receptors. Because different receptors appear to be segregated in the same oocyte in distinct localizations, Xenopus oocytes may be an important model system in which to study receptor sorting in polarized cells.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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 compared to sham treated cells. CHO cells undergoing mitosis after exposure also exhibit improper separation of chromatids which could indicate loss of function of the mitotic spindle checkpoint. Activation and loss of function of checkpoints in CHO but not Jurkat cells after nsPEF exposure suggests that activation of cell cycle checkpoints could be important in defining the character of cell line specific recovery after nsPEF exposure. Moreover, the increased sensitivity in G2/M phase exhibited by both cell lines indicates that cell cycle phase is an important consideration during nsPEF exposure, particularly when aiming to induce apoptosis.

Genistein Enhances or Reduces Glycosaminoglycan Quantity in a Cell Type-Specific Manner.

PubMed

Lan, Ying; Li, Xiulian; Liu, Xuebo; Hao, Cui; Song, Ni; Ren, Sumei; Wang, Wei; Feng, Ningchuan; Zhang, Lijuan

2018-06-27

Genistein is a natural isoflavone enriched in soybeans. It has beneficial effects for patients with mucopolysaccharidose type III through inhibiting glycosaminoglycan biosynthesis. However, other studies indicate that genistein does not always inhibit glycosaminoglycan biosynthesis. To understand the underlying molecular mechanisms, CHOK1, CHO3.1, CHO3.3, and HCT116 cells were treated with genistein and the monosaccharide compositions and quantity of all glycans from the cell lysate were measured after thorough acid hydrolysis followed by HPLC analysis. In addition, the glycosaminoglycan disaccharide compositions were obtained by stable isotope labeling coupled with LC/MS analysis. Genistein treatment reduced the amount of glycans but increased the amount of glycosaminoglycans in HCT116 cells. In contrast, genistein treatment reduced both glycan and glycosaminoglycan quantities in CHOK1, CHO3.1, and CHO3.3 cells in addition to differential changes in glycosaminoglycan disaccharide compositions. Genistein treatment reduced overall glycan quantity but glycosaminoglycan quantities were either increased or decreased in a cell type-dependent manner. © 2018 The Author(s). Published by S. Karger AG, Basel.

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

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.

[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.

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

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 isolate CHO cell variants with improved functional properties in vitro. Copyright © 2012 Wiley Periodicals, Inc.

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.

Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies[S

PubMed Central

Sengupta, Bhaswati; Narasimhulu, Chandrakala Aluganti; Parthasarathy, Sampath

2013-01-01

Generation of foam cells, an essential step for reverse cholesterol transport studies, uses the technique of receptor-dependent macrophage loading with radiolabeled acetylated LDL. In this study, we used the ability of a biologically relevant detergent molecule, lysophosphatidylcholine (lyso-PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabeled cholesterol/lyso-PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month of storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled, or radiolabeled)/lyso-PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by TLC. Such foam cells unloaded cholesterol when incubated with HDL but not with oxidized HDL. We propose that stable cholesterol or CE/lyso-PtdCho micelles would offer advantages over existing methods. PMID:24115226

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.

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

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.

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.

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.

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.

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.

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

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.

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

Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer.

PubMed

Granata, A; Nicoletti, R; Tinaglia, V; De Cecco, L; Pisanu, M E; Ricci, A; Podo, F; Canevari, S; Iorio, E; Bagnoli, M; Mezzanzanica, D

2014-01-21

Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by (1)H-magnetic spectroscopy analysis; (III) a 35-36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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

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:24632968

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

PubMed

Akhberdi, Oren; Zhang, Qian; Wang, Haichuan; Li, Yingying; Chen, Longfei; Wang, Dan; Yu, Xi; Wei, Dongsheng; Zhu, Xudong

2018-05-01

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi. Copyright © 2018 Elsevier GmbH. All rights reserved.

Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors.

PubMed

Arlauckas, Sean P; Kumar, Manoj; Popov, Anatoliy V; Poptani, Harish; Delikatny, Edward J

2017-03-07

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.

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

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.

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.

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.

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

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.

Positively Charged Residues at the Five-Fold Symmetry Axis of Cell Culture-Adapted Foot-and-Mouth Disease Virus Permit Novel Receptor Interactions

PubMed Central

Berryman, Stephen; Clark, Stuart; Kakker, Naresh K.; Silk, Rhiannon; Seago, Julian; Wadsworth, Jemma; Chamberlain, Kyle; Knowles, Nick J.

2013-01-01

Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-Q110K). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (α5β1 and αvβ5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-Q110K substitution did not use these integrins. In contrast, the VP1-Q110K substitution appeared to result in enhanced interactions with αvβ6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use αvβ6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of αvβ6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable. PMID:23740982

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

PubMed Central

Clark, Sarah E.; Snow, Julian; Li, Jianjun; Zola, Tracey A.; Weiser, Jeffrey N.

2012-01-01

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface. PMID:22396641

Development of Efficient Charge-Selective Materials for Bulk Heterojunction Polymer Solar Cells

DTIC Science & Technology

2015-01-15

Cho, D. S. Ginger and A. K. Y. Jen, Advanced Materials, 2014, 26, 6262-6267. 17. N. Cho, C.-Z. Li, H.-L. Yip and A. K. Y. Jen, Energy...Solar Cells”, Adv. Funct. Mater. 2015, ASAP. 4. J. H. Kim, P. W. Liang, S. T. Williams, N. C. Cho, C. C. Chueh, M. S. Glaz, D. S. Ginger , A. K.-Y...Kim, P. W. Liang, S. T. Williams, N. C. Cho, C. C. Chueh, M. S. Glaz, D. S. Ginger , A. K.-Y. Jen, 2015, ASAP. 7. P. W. Liang, C. C. Chueh, X. K

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.

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.

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.

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).

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.

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.

Microbacterium rhizosphaerae sp. nov., isolated from a Ginseng field, South Korea.

PubMed

Cho, Sung-Jin; Lee, Sang-Seob

2017-01-01

A novel Gram-stain positive, aerobic, short rod-shaped, non-motile bacterium, designated strain CHO1 T , was isolated from rhizosphere soil from a ginseng agriculture field. Strain CHO1 T was observed to form yellow colonies on R2A agar medium. The cell wall peptidoglycan was found to contain alanine, glycine, glutamic acid, D-ornithine and serine. The cell wall sugars were identified as galactose, mannose, rhamnose and ribose. Strain CHO1 T was found to contain MK-11, MK-12, MK-13 as the predominant menaquinones and anteiso-C 15:0 , iso-C 16:0 , and anteiso-C 17:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, an unidentified phospholipid and three unidentified glycolipids were found to be present in strain CHO1 T . Based on 16S rRNA gene sequence analysis, strain CHO1 T was found to be closely related to Microbacterium mangrovi DSM 28240 T (97.81 % similarity), Microbacterium immunditiarum JCM 14034 T (97.45 %), Microbacterium oryzae JCM 16837 T (97.33 %) and Microbacterium ulmi KCTC 19363 T (97.10 %) and to other species of the genus Microbacterium. The DNA G+C content of CHO1 T was determined to be 70.1 mol %. The DNA-DNA hybridization values of CHO1 T with M. mangrovi DSM 28240 T , M. immunditiarum JCM 14034 T , M. oryzae JCM 16837 T and M. ulmi KCTC 19363 T were 46.7 ± 2, 32.4 ± 2, 32.0 ± 2 and 29.2 ± 2 %, respectively. On the basis of genotypic, phenotypic and phylogenetic properties, it is concluded that strain CHO1 T represents a novel species within the genus Microbacterium, for which the name Microbacterium rhizosphaerae sp. nov. is proposed. The type strain of M. rhizosphaerae is CHO1 T (= KEMB 7306-513 T  = JCM 31396 T ).

The role of lipooligosaccharide phosphorylcholine in colonization and pathogenesis of Histophilus somni in cattle.

PubMed

Elswaifi, Shaadi F; Scarratt, William K; Inzana, Thomas J

2012-06-07

Histophilus somni is a Gram-negative bacterium and member of the Pasteurellaceae that is responsible for respiratory disease and other systemic infections in cattle. One of the bacterium's virulence factors is antigenic phase variation of its lipooligosaccharide (LOS). LOS antigenic variation may occur through variation in composition or structure of glycoses or their substitutions, such as phosphorylcholine (ChoP). However, the role of ChoP in the pathogenesis of H. somni disease has not been established. In Haemophilus influenzae ChoP on the LOS binds to platelet activating factor on epithelial cells, promoting bacterial colonization of the host upper respiratory tract. However, ChoP is not expressed in the blood as it also binds C-reactive protein, resulting in complement activation and killing of the bacteria. In order to simulate the susceptibility of calves with suppressed immunity due to stress or previous infection, calves were challenged with bovine herpes virus-1 or dexamethazone 3 days prior to challenge with H. somni. Following challenge, expression of ChoP on the LOS of 2 different H. somni strains was associated with colonization of the upper respiratory tract. In contrast, lack of ChoP expression was associated with bacteria recovered from systemic sites. Histopathology of cardiac tissue from myocarditis revealed lesions containing bacterial clusters that appeared similar to a biofilm. Furthermore, some respiratory cultures contained substantial numbers of Pasteurella multocida, which were not present on preculture screens. Subsequent biofilm experiments have shown that H. somni and P. multocida grow equally well together in a biofilm, suggesting a commensal relationship may exist between the two species. Our results also showed that ChoP contributed to, but was not required for, adhesion to respiratory epithelial cells. In conclusion, expression of ChoP on H. somni LOS contributed to colonization of the bacteria to the host upper respiratory tract, but phase variable loss of ChoP expression may help the bacteria survive systemically.

The role of lipooligosaccharide phosphorylcholine in colonization and pathogenesis of Histophilus somni in cattle

PubMed Central

2012-01-01

Histophilus somni is a Gram-negative bacterium and member of the Pasteurellaceae that is responsible for respiratory disease and other systemic infections in cattle. One of the bacterium’s virulence factors is antigenic phase variation of its lipooligosaccharide (LOS). LOS antigenic variation may occur through variation in composition or structure of glycoses or their substitutions, such as phosphorylcholine (ChoP). However, the role of ChoP in the pathogenesis of H. somni disease has not been established. In Haemophilus influenzae ChoP on the LOS binds to platelet activating factor on epithelial cells, promoting bacterial colonization of the host upper respiratory tract. However, ChoP is not expressed in the blood as it also binds C-reactive protein, resulting in complement activation and killing of the bacteria. In order to simulate the susceptibility of calves with suppressed immunity due to stress or previous infection, calves were challenged with bovine herpes virus-1 or dexamethazone 3 days prior to challenge with H. somni. Following challenge, expression of ChoP on the LOS of 2 different H. somni strains was associated with colonization of the upper respiratory tract. In contrast, lack of ChoP expression was associated with bacteria recovered from systemic sites. Histopathology of cardiac tissue from myocarditis revealed lesions containing bacterial clusters that appeared similar to a biofilm. Furthermore, some respiratory cultures contained substantial numbers of Pasteurella multocida, which were not present on preculture screens. Subsequent biofilm experiments have shown that H. somni and P. multocida grow equally well together in a biofilm, suggesting a commensal relationship may exist between the two species. Our results also showed that ChoP contributed to, but was not required for, adhesion to respiratory epithelial cells. In conclusion, expression of ChoP on H. somni LOS contributed to colonization of the bacteria to the host upper respiratory tract, but phase variable loss of ChoP expression may help the bacteria survive systemically. PMID:22676226

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).

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.

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.

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

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.

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

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.

[Construction of dengue virus-specific full-length fully human antibody libraries by mammalian display technology].

PubMed

Wen, Yangming; Lan, Kaijian; Wang, Junjie; Yu, Jingyi; Qu, Yarong; Zhao, Wei; Zhang, Fuchun; Tan, Wanlong; Cao, Hong; Zhou, Chen

2013-06-01

To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)]. Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

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

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.

Pharmacological characterization of human recombinant melatonin mt1 and MT2 receptors

PubMed Central

Browning, Christopher; Beresford, Isabel; Fraser, Neil; Giles, Heather

2000-01-01

We have pharmacologically characterized recombinant human mt1 and MT2 receptors, stably expressed in Chinese hamster ovary cells (CHO-mt1 and CHO-MT2), by measurement of [3H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt1 and MT2 receptors with pKD values of 9.89 and 9.56 and Bmax values of 1.20 and 0.82 pmol mg−1 protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt1 and MT2 receptors, a number of putative antagonists had substantially higher affinities for MT2 receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt1 and CHO-MT2 cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC50 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT2 receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt1 receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt1 and CHO-MT2 cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA2 values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of Gi/Go G-proteins. This is the first report of the use of [3H]-melatonin for the characterization of recombinant mt1 and MT2 receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles. PMID:10696085

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.

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 the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products. PMID:28486471

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 pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

PubMed

Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

2011-05-01

OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

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.

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.

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.

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

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.

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.

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.

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-based production system. Copyright 2000 The International Association for Biologicals.

[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.

Molecular Effects of Doxorubicin on Choline Metabolism in Breast Cancer.

PubMed

Cheng, Menglin; Rizwan, Asif; Jiang, Lu; Bhujwalla, Zaver M; Glunde, Kristine

2017-08-01

Abnormal choline phospholipid metabolism is a hallmark of cancer. The magnetic resonance spectroscopy (MRS) detected total choline (tCho) signal can serve as an early noninvasive imaging biomarker of chemotherapy response in breast cancer. We have quantified the individual components of the tCho signal, glycerophosphocholine (GPC), phosphocholine (PC) and free choline (Cho), before and after treatment with the commonly used chemotherapeutic drug doxorubicin in weakly metastatic human MCF7 and triple-negative human MDA-MB-231 breast cancer cells. While the tCho concentration did not change following doxorubicin treatment, GPC significantly increased and PC decreased. Of the two phosphatidylcholine-specific PLD enzymes, only PLD1, but not PLD2, mRNA was down-regulated by doxorubicin treatment. For the two reported genes encoding GPC phosphodiesterase, the mRNA of GDPD6, but not GDPD5, decreased following doxorubicin treatment. mRNA levels of choline kinase α (ChKα), which converts Cho to PC, were reduced following doxorubicin treatment. PLD1 and ChKα protein levels decreased following doxorubicin treatment in a concentration dependent manner. Treatment with the PLD1 specific inhibitor VU0155069 sensitized MCF7 and MDA-MB-231 breast cancer cells to doxorubicin-induced cytotoxicity. Low concentrations of 100 nM of doxorubicin increased MDA-MB-231 cell migration. GDPD6, but not PLD1 or ChKα, silencing by siRNA abolished doxorubicin-induced breast cancer cell migration. Doxorubicin induced GPC increase and PC decrease are caused by reductions in PLD1, GDPD6, and ChKα mRNA and protein expression. We have shown that silencing or inhibiting these genes/proteins can promote drug effectiveness and reduce adverse drug effects. Our findings emphasize the importance of detecting PC and GPC individually. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells.

PubMed

Sato, T; Kiyosue, T; Arita, M

1992-01-01

We investigated the effects of ischemia-related amphipathic compounds, palmitoylcarnitine (PamCar, 0.5-50 microM) and lysophosphatidylcholine (lysoPtdCho, 5-50 microM) on sodium current (INa) of guinea-pig ventricular myocytes. The cells were perfused with low-Na+ (60 mM) Tyrode's solution, and Ca2+ and K+ currents were blocked by external Co2+ (3 mM) and internal Cs+ (140 mM), respectively. INa was elicited by depolarizing voltage steps from a holding potential of -100 mV at a temperature of 33 degrees C. PamCar (5 microM) decreased the peak INa (attained at -20 mV or -30 mV) from 6.1 +/- 2.1 nA to 3.9 +/- 1.4 nA (n = 11), or by 36.1% within 2 min, and shifted the curve of steady-state INa inactivation by 5.4 mV in the positive direction (from -76.3 +/- 4.6 mV, control to -70.9 +/- 4.0 mV, in PamCar, n = 4). Partial restoration of the amplitude and the shift of the steady-state inactivation curve of INa was attained after washout of PamCar. In contrast, lysoPtdCho at concentrations over 10 microM irreversibly depressed the INa within 0.5-3 min and the reduction of INa was followed by cell contracture or cell death (n = 9). The survival time, defined as a period from the start of lysoPtdCho application to the time of the last successful recording of the INa (before evolution of sudden changes in the holding current), depended on the concentrations of lysoPtdCho. Both PamCar and lysoPtdCho retarded the time course of activation and inactivation of INa.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

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.

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.

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

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.

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.

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.

Effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells.

PubMed

Murphy, E J; Horrocks, L A

1993-04-07

The effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells were determined. The cellular lipids were extracted on days 0, 3 and 7, following the addition of 1.2% dimethylsulfoxide to induce cellular differentiation. Proportions of ethanolamine glycerophospholipids (EtnGpl), phosphatidylinositol (PtdIns) and sphingomyelin (CerPCho) were significantly elevated following differentiation. The mole percentage of choline glycerophospholipids (ChoGpl) decreased with differentiation. The plasmalogens, both choline and ethanolamine, increased by 1.3- and 2.3-fold, respectively, during differentiation. The fatty acid composition of the phospholipid classes was also altered. PtdIns and ChoGpl had decreased proportions of polyenoic fatty acids, while these proportions were increased in EtnGpl. Both ChoGpl and EtnGpl had increased n-3/n-6 series fatty acid ratios, but this ratio was decreased in PtdIns. The mole percentage of arachidonic acid was significantly decreased in both PtdIns and ChoGpl, but elevated in EtnGpl and may be a result of the increase in ethanolamine plasmalogen. Thus, differentiation did not increase the overall mole percentage of polyenoic FA in the cells nor increase the n-6 series fatty acid proportions. We speculate plasmalogens may have a role in the differentiation process or in maintaining the cell in the differentiated state.

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

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.

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 vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion. Copyright © 2018 Bello-Morales et al.

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.

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 receptor function >DDT depletes membrane raft cholesterol content and by this way inhibits TSH receptor« less

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.

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

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

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

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

PubMed

Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

2010-11-30

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

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.

Fat adaptation in well-trained athletes: effects on cell metabolism.

PubMed

Yeo, Wee Kian; Carey, Andrew L; Burke, Louise; Spriet, Lawrence L; Hawley, John A

2011-02-01

The performance of prolonged (>90 min), continuous, endurance exercise is limited by endogenous carbohydrate (CHO) stores. Accordingly, for many decades, sports nutritionists and exercise physiologists have proposed a number of diet-training strategies that have the potential to increase fatty acid availability and rates of lipid oxidation and thereby attenuate the rate of glycogen utilization during exercise. Because the acute ingestion of exogenous substrates (primarily CHO) during exercise has little effect on the rates of muscle glycogenolysis, recent studies have focused on short-term (<1-2 weeks) diet-training interventions that increase endogenous substrate stores (i.e., muscle glycogen and lipids) and alter patterns of substrate utilization during exercise. One such strategy is "fat adaptation", an intervention in which well-trained endurance athletes consume a high-fat, low-CHO diet for up to 2 weeks while undertaking their normal training and then immediately follow this by CHO restoration (consuming a high-CHO diet and tapering for 1-3 days before a major endurance event). Compared with an isoenergetic CHO diet for the same intervention period, this "dietary periodization" protocol increases the rate of whole-body and muscle fat oxidation while attenuating the rate of muscle glycogenolysis during submaximal exercise. Of note is that these metabolic perturbations favouring the oxidation of fat persist even in the face of restored endogenous CHO stores and increased exogenous CHO availability. Here we review the current knowledge of some of the potential mechanisms by which skeletal muscle sustains high rates of fat oxidation in the face of high exogenous and endogenous CHO availability.

Micro and Nano-mediated 3D Cardiac Tissue Engineering

DTIC Science & Technology

2009-10-01

in both flow cytometry and Western Blot applications. The CD34 antigen is important in stem cell research due to its widespread use in identifying...for further characterization. We generated a pCD34 expressing CHO cell line (CHO-CD34) and analyzed pCD34 expression by flow cytometry (Figure 1A... flow cytometry using the 3G7 antibody and co-stained with an anti-CD31 antibody (AbD Serotec; FITC conjugated). CD31 (PECAM) is a pan endothelial

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 of action may be related to the stabilization of the cell cytoskeleton.

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.

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

Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

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

Hang, Howard C.; Bertozzi, Carolyn R.

2000-08-22

Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence atmore » the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.« less

Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

PubMed Central

Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

2012-01-01

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

PubMed

Seizer, Peter; Borst, Oliver; Langer, Harald F; Bültmann, Andreas; Münch, Götz; Herouy, Yared; Stellos, Konstantinos; Krämer, Björn; Bigalke, Boris; Büchele, Berthold; Bachem, Max G; Vestweber, Dietmar; Simmet, Thomas; Gawaz, Meinrad; May, Andreas E

2009-04-01

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

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

In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

PubMed

Wagner, L; Isbrucker, R; Locht, C; Arciniega, J; Costanzo, A; McFarland, R; Oh, H; Hoonakker, M; Descamps, J; Andersen, S R; Gupta, R K; Markey, K; Chapsal, J M; Lidster, K; Casey, W; Allen, D

2016-01-01

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

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 matrix. Repair experiments indicate that in CHO cells repair of DSB in loops containing multiple DSB are repaired with "slow" kinetics (t1/2 = 5 hrs) whilst DSB occurring in loops containing single DSB are repaired with "fast" kinetics (t1/2 " 10 min). Xrs- 5 cells are incapable of repairing these multiply damaged loops. This work indicates that the spatial orientation of DSB in higher order structures of chromatin are a possible factor in the repair of these lesions. By construction of a mathematical model of the process of elution from chromatin loops it was possible to postulate the size of the loops to approximate to 2.5-3 Mbp. Further evidence of a potential structural defect in the chromatin of xrs-5 cells was provided by examination of the polypeptide composition and DNA binding activity of nuclear extracts. The affinity of extracted proteins for double-stranded calf-thymus DNA was measured in nuclear extracts of xrs-5 and CHO cells. There was an alteration in the DNA binding activity of salt extractable proteins from xrs-5 as measured by a filter binding assay. By the use of SDS-PAGE and the technique of South-Western blotting, it was possible to identify the approximate molecular weights of these DNA binding proteins. Differences were found in DNA binding between proteins from CHO and xrs-5 extracts of both non-irradiated and irradiated cells. Two proteins with apparent molecular weights of 32.2 and 31.8 kDa exhibited a lower DNA binding activity in xrs-5 than proteins of similar extracts from CHO. The amount of the 32.2 kDa protein was less in the xrs-5 extracts than in CHO extracts, as measured by Coomassie blue staining. The two proteins have not yet been identified but comprise a major DNA binding activity in CHO extracts obtained by detergent-free extraction procedures. This work provides circumstantial evidence that suggests these two polypeptides may form part of the histone H1 family.

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.

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.

Effects of choline on sodium arsenite-induced neural tube defects in chick embryos.

PubMed

Song, Ge; Cui, Yi; Han, Zhong-Ji; Xia, Hong-Fei; Ma, Xu

2012-12-01

Arsenic passes through the placenta and accumulates in the neuroepithelium of embryo, whereby inducing congenital malformations such as neural tube defects (NTDs) in animals. Choline (CHO), a methyl-rich nutrient, functions as a methyl donor to participate in methyl group metabolism. Arsenic methylation has been regarded as a detoxification process and choline (CHO) is the major source of methyl-groups. However, whether CHO intake reverses the abnormal embryo development induced by sodium arsenite (SA) and the relationship between CHO intake and arsenite-induced NTDs are still unclear. In this study, we used chick embryos as animal model to investigate the effects of SA and CHO supplementation on the early development of nervous system. Our results showed that the administration of SA led to reduction in embryo viability, embryo body weight and extraembryonic vascular area, accompanied by a significantly increased incidence of the failed closure of the caudal end of the neural tube. CHO, at low dose (25 μg/μL), reversed the decrease in embryo viability and the increase in the failed closure of the caudal end of the neural tube, which were induced by SA. In addition, CHO (25 μg/μL) inhibited not only the SA-induced cell apoptosis by up-regulating Bcl-2 level, but also the global DNA methylation by increasing the expressions of DNMT1 and DNMT3a. However, less significant difference was found between the embryos co-treated with SA and CHO (50 μg/μL) and the ones treated with SA alone. Taken together, these findings suggest that low dose CHO could protect chick embryos from arsenite-induced NTDs by a possible mechanism related to the methyl metabolism. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

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

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

Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis.

PubMed

Millard, E E; Srivastava, K; Traub, L M; Schaffer, J E; Ory, D S

2000-12-08

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.

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.

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.

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.

Phosphatidylcholine and the CDP-Choline Cycle

PubMed Central

Fagone, Paolo; Jackowski, Suzanne

2012-01-01

The CDP-choline pathway of phosphatidylcholine (PtdCho) biosynthesis was first described more than 50 years ago. Investigation of the CDP-choline pathway in yeast provides a basis for understanding the CDP-choline pathway in mammals. PtdCho is considered as an intermediate in a cycle of synthesis and degradation, and the activity of a CDP-choline cycle is linked to subcellular membrane lipid movement. The components of the mammalian CDP-choline pathway include choline transport, choline kinase, phosphocholine cytidylyltransferase, and choline phosphotransferase activities. The protein isoforms and biochemical mechanisms of regulation of the pathway enzymes are related to their cell and tissue-specific functions. Regulated PtdCho turnover mediated by phospholipases or neuropathy target esterase participates in the mammalian CDP-choline cycle. Knockout mouse models define the biological functions of the CDP-choline cycle in mammalian cells and tissues. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:23010477

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

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

O'Donnell, Christopher D.; Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612; Tiwari, Vaibhav

2006-03-15

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

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 contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.

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.

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.

Biphasic voltage-dependent inactivation of human NaV 1.3, 1.6 and 1.7 Na+ channels expressed in rodent insulin-secreting cells.

PubMed

Godazgar, Mahdieh; Zhang, Quan; Chibalina, Margarita V; Rorsman, Patrik

2018-05-01

Na + current inactivation is biphasic in insulin-secreting cells, proceeding with two voltage dependences that are half-maximal at ∼-100 mV and -60 mV. Inactivation of voltage-gated Na + (Na V ) channels occurs at ∼30 mV more negative voltages in insulin-secreting Ins1 and primary β-cells than in HEK, CHO or glucagon-secreting αTC1-6 cells. The difference in inactivation between Ins1 and non-β-cells persists in the inside-out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary β-cells, but not in HEK cells, inactivation of a single Na V subtype is biphasic and follows two voltage dependences separated by 30-40 mV. We propose that Na V channels adopt different inactivation behaviours depending on the local membrane environment. Pancreatic β-cells are equipped with voltage-gated Na + channels that undergo biphasic voltage-dependent steady-state inactivation. A small Na + current component (10-15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na + channel component is completely inactivated at -90 to -80 mV and is therefore inactive in the β-cell. It has been proposed that the biphasic inactivation reflects the contribution of different Na V α-subunits. We tested this possibility by expression of TTX-resistant variants of the Na V subunits found in β-cells (Na V 1.3, Na V 1.6 and Na V 1.7) in insulin-secreting Ins1 cells and in non-β-cells (including HEK and CHO cells). We found that all Na V subunits inactivated at 20-30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. Na V 1.7 inactivated at 15--20 mV more negative membrane potentials than Na V 1.3 and Na V 1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of Na V inactivation (separated by 30 mV) were also observed following expression of a single type of Na V α-subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic Na V inactivation in insulin-secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition. © 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

PubMed

Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-04-01

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

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 in the visualization of the receptor. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:7826341

Regulation of Muscle Glycogen Metabolism during Exercise: Implications for Endurance Performance and Training Adaptations

PubMed Central

Hearris, Mark A.; Hammond, Kelly M.; Fell, J. Marc; Morton, James P.

2018-01-01

Since the introduction of the muscle biopsy technique in the late 1960s, our understanding of the regulation of muscle glycogen storage and metabolism has advanced considerably. Muscle glycogenolysis and rates of carbohydrate (CHO) oxidation are affected by factors such as exercise intensity, duration, training status and substrate availability. Such changes to the global exercise stimulus exert regulatory effects on key enzymes and transport proteins via both hormonal control and local allosteric regulation. Given the well-documented effects of high CHO availability on promoting exercise performance, elite endurance athletes are typically advised to ensure high CHO availability before, during and after high-intensity training sessions or competition. Nonetheless, in recognition that the glycogen granule is more than a simple fuel store, it is now also accepted that glycogen is a potent regulator of the molecular cell signaling pathways that regulate the oxidative phenotype. Accordingly, the concept of deliberately training with low CHO availability has now gained increased popularity amongst athletic circles. In this review, we present an overview of the regulatory control of CHO metabolism during exercise (with a specific emphasis on muscle glycogen utilization) in order to discuss the effects of both high and low CHO availability on modulating exercise performance and training adaptations, respectively. PMID:29498691

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

PubMed

Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

1997-10-15

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

Untargeted LC-MS/MS Profiling of Cell Culture Media Formulations for Evaluation of High Temperature Short Time Treatment Effects.

PubMed

Floris, Patrick; McGillicuddy, Nicola; Albrecht, Simone; Morrissey, Brian; Kaisermayer, Christian; Lindeberg, Anna; Bones, Jonathan

2017-09-19

An untargeted LC-MS/MS platform was implemented for monitoring variations in CHO cell culture media upon exposure to high temperature short time (HTST) treatment, a commonly used viral clearance upstream strategy. Chemically defined (CD) and hydrolysate-supplemented media formulations were not visibly altered by the treatment. The absence of solute precipitation effects during media treatment and very modest shifts in pH values observed indicated sufficient compatibility of the formulations evaluated with the HTST-processing conditions. Unsupervised chemometric analysis of LC-MS/MS data, however, revealed clear separation of HTST-treated samples from untreated counterparts as observed from analysis of principal components and hierarchical clustering sample grouping. An increased presence of Maillard products in HTST-treated formulations contributed to the observed differences which included organic acids, observed particularly in chemically defined formulations, and furans, pyridines, pyrazines, and pyrrolidines which were determined in hydrolysate-supplemented formulations. The presence of Maillard products in media did not affect cell culture performance with similar growth and viability profiles observed for CHO-K1 and CHO-DP12 cells when cultured using both HTST-treated and untreated media formulations.

Mutagens in cooked foods - metabolism and genetic toxicity

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

Felton, J.S.; Bjeldanes, L.F.; Hatch, F.T.

1984-02-17

Recently developed in our laboratories is an efficient extraction procedure incorporating XAD resin adsorption which yields from 200/sup 0/C grilled ground beef an extract containing 230 Salmonella TA1538 revertants per g fresh weight of original ground beef. These mutagenic components are specific for frameshift-sensitive Salmonella strains and have an absolute requirement for metabolic activation. Normal-phase HPLC separation of methanol-extractable metabolites generated from reaction of 2-amino-3-methylimidazo (4,5-f)quinoline (IQ), a mutagenic component of broiled food with rat liver microsomes resulted in one direct-acting mutagenic peak and a second more polar peak still requiring metabolic activation. Two potent thermally-produced bacterial mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)more » indole (Trp-P-2) and IQ, were examined in mammalian cells. In excision repair-deficient CHO cells, Trp-P-2 exposure caused cytotoxicity, mutagenicity, sister chromatid exchange, and chromosomal aberrations at concentrations more than 30-fold lower than those for IQ. In normal repair-proficient CHO cells Trp-P-2 was one-half as active and IQ was inactive. Relative to Trp-P-2, IQ is much more potent in the Salmonella bacterial system than in mammalian CHO cells.« less

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.

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.

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.

Monitoring leachables from single-use bioreactor bags for mammalian cell culture by dispersive liquid-liquid microextraction followed by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry.

PubMed

Dorival-García, N; Bones, J

2017-08-25

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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

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

Correlation analysis of expressions of PTEN and p53 with the value obtained by magnetic resonance spectroscopy and apparent diffusion coefficient in the tumor and the tumor-adjacent area in magnetic resonance imaging for glioblastoma.

PubMed

Li, Yunyun; Ji, Feng; Jiang, Yuzhi; Zhao, Ting; Xu, Chongfu

2018-01-01

To explore the correlation of the expression levels of phosphate and tension homology deleted on chromosome ten (PTEN) and p53 of glioblastoma multiforme (GBM) with the value obtained by magnetic resonance spectroscopy (MRS) and apparent diffusion coefficient (ADC) in the tumor and the tumor-adjacent area in magnetic resonance imaging (MRI). A total of 38 patients were operated for GBM. All the patients had received diffusion-weighted imaging (DWI) and MRS prior to surgery. ADC of water molecules and values of metabolite indexes of MRS, including n-acetyl aspartate (NAA), choline (Cho) and creatine (Cr), were recorded, and the ratios of Cho/NAA, Cho/Cr and NAA/Cr were calculated. Hematoxylin-eosin (H&E) staining was done to examine the morphology of tumor and of tumor-adjacent tissues; immunohistochemistry (IHC) was performed to examine the expressions of PTEN and p53 in the tumor and the tumor-adjacent area. Finally, the correlations of the expressions of PTEN and p53 with ADC, Cho/NAA, Cho/Cr and NAA/Cr of the tumor and the tumor-adjacent area were analyzed. H&E staining showed that GBM tissues had disordered morphology, different sizes of cells, large cell nuclei and significant cell heterogeneity. IHC indicated that the expression level of p53 protein in the tumor was significantly higher than in the tumor-adjacent tissues (p<0.05). The expression level of PTEN protein was high in the tumor-adjacent tissues, but significantly deficient in the tumor. DWI showed that the signal of DWI in the tumor was significantly increased, but ADC was decreased compared with the tumor-adjacent area. MRS indicated that the wave band of Cho in the tumor was significantly increased, NAA was significantly lowered, and Cr section was decreased compared with the tumor-adjacent area, while NAA/Cr in the tumor was significantly decreased compared with the tumoradjacent area (p<0.05). Correlation analysis indicated that PTEN levels in the tumor and the tumor-adjacent area were positively correlated with ADC in the corresponding area, while p53 in the tumor and the tumor-adjacent area was negatively correlated with ADC in the corresponding area. Cho/NAA and Cho/Cr in the tumor were positively correlated with p53 in the tumor, but negatively correlated with PTEN in the tumor. However, NAA/Cr of the tumor was irrelevant to the levels of PTEN and p53. The test results of DWI and MRS of patients with GBM can accurately reflect the inactivation or mutation of PTEN and p53.

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

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 elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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.

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.

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.

The permeability of EUDRAGIT RL and HEMA-MMA microcapsules to glucose and inulin.

PubMed

Douglas, J A; Sefton, M V

1990-10-05

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA-MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 degrees C were of the same order of magnitude for both capsules, based on release measurements: approximately 5 x 10(-6) cm/s for EUDRAGIT RL and approximately 2 x 10(-6) for HEMA-MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 +/- 4 x 10(-8) cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10(-6) cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 microm HEMA-MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.

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.

Preemptive Approach to Improving Survival in Epithelial Ovarian Cancer

DTIC Science & Technology

2012-06-01

Metab 2008;93:3471–7. 692[87] Cho JH, Lee JG, Yang YI, Kim JH, Ahn JH, Baek NI, Lee KT, Choi JH. Eupatilin, a die- 693tary flavonoid , induces G2/M cell...Metab 2008;93:3471–7. 692[87] Cho JH, Lee JG, Yang YI, Kim JH, Ahn JH, Baek NI, Lee KT, Choi JH. Eupatilin, a die- 693tary flavonoid , induces G2/M

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

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

PubMed Central

Fan, Shaoan; Wu, Feng; Martiniuk, Frank; Hale, Martha L; Ellington, Andrew D; Tchou-Wong, Kam-Meng

2008-01-01

AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION: We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication. PMID:19009652

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 investigations into both epigenetic modifications and transcription factor interactions will help gain a better understanding of what characteristics make a higher producing CHO cell line. Discovery of these characteristics will aid in better genetic engineering strategies for future recombinant cell lines, improving productivity and shortening the clonal selection process.

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.

Regional gene mapping using mixed radiation hybrids and reverse chromosome painting.

PubMed

Lin, J Y; Bedford, J S

1997-11-01

We describe a new approach for low-resolution physical mapping using pooled DNA probe from mixed (non-clonal) populations of human-CHO cell hybrids and reverse chromosome painting. This mapping method is based on a process in which the human chromosome fragments bearing a complementing gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irradiations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on normal human metaphase spreads using biotinylated DNA from this population of "enriched" hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose location is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented the defect responsible for the hypersensitivity to ionizing radiation in CHO irs-20 cells. This method represents an efficient alternative to conventional concordance analysis in somatic cell hybrids where detailed chromosome analysis of numerous hybrid clones is necessary. Using this approach, it is possible to localize a gene for which there is no prior sequence or linkage information to a subchromosomal region, thus facilitating association with known mapping landmarks (e.g. RFLP, YAC or STS contigs) for higher-resolution mapping.

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.

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.

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.

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts

PubMed Central

Bushinsky, David A.

2010-01-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO3−])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco2)] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Cai). To determine whether Resp increases Cai, as does Met, we imaged Cai in primary cultures of bone cells. pH for Met = 7.07 ([HCO3−] = 11.8 mM) and for Resp = 7.13 (Pco2 = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Cai in individual bone cells; however, Met stimulated Cai to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Cai in Met than Resp. Both Met and Resp induced a marked, transient increase in Cai in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Cai by Met in primary bone cells is not a function of OGR1 alone, but must involve H+ receptors other than OGR1, or pathways sensitive to Pco2, HCO3−, or total CO2 that modify the effect of H+ in primary bone cells. PMID:20504884

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts.

PubMed

Frick, Kevin K; Bushinsky, David A

2010-08-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO(3)(-)])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco(2))] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Ca(i)). To determine whether Resp increases Ca(i), as does Met, we imaged Ca(i) in primary cultures of bone cells. pH for Met = 7.07 ([HCO(3)(-)] = 11.8 mM) and for Resp = 7.13 (Pco(2) = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Ca(i) in individual bone cells; however, Met stimulated Ca(i) to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Ca(i) in Met than Resp. Both Met and Resp induced a marked, transient increase in Ca(i) in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Ca(i) by Met in primary bone cells is not a function of OGR1 alone, but must involve H(+) receptors other than OGR1, or pathways sensitive to Pco(2), HCO(3)(-), or total CO(2) that modify the effect of H(+) in primary bone cells.

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

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.

Interaction between EphrinB1 and CNK1 Found to Play Role in Tumor Progression | Poster

Cancer.gov

By Nancy Parrish, Staff Writer The family of proteins known as ephrins plays a critical role in a variety of biological processes. In a recent article in the Journal of Biological Chemistry, Hee Jun Cho, Ph.D., and colleagues report on the interaction between proteins CNK1 and ephrinB1 that promotes cell movement. Their findings may have an important implication in developing new therapeutics for reducing metastases in certain cancers. “Eph and ephrin signaling has become an area of intense interest due to the influence these molecules exert on the control of cell adhesion and cell movement,” Cho said. “This signaling affects the formation of tissues during development and has been shown to play an instructive role in angiogenesis, as well as tumor cell invasion.”

Folic acid conjugated mPEG-PEI600 as an efficient non-viral vector for targeted nucleic acid delivery.

PubMed

Xu, Zhenhua; Jin, Jiefu; Siu, Leo K S; Yao, Hong; Sze, Johnny; Sun, Hongzhe; Kung, Hsiang-Fu; Poon, Wai Sang; Ng, Samuel S M; Lin, Marie C

2012-04-15

In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. (1)H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine2000 and Linear PEI 22 kDa (L-PEI 22 kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. Copyright © 2012 Elsevier B.V. All rights reserved.

Scavenger receptor B1 (SR-B1) profoundly excludes high density lipoprotein (HDL) apolipoprotein AII as it nibbles HDL-cholesteryl ester.

PubMed

Gillard, Baiba K; Bassett, G Randall; Gotto, Antonio M; Rosales, Corina; Pownall, Henry J

2017-05-26

Reverse cholesterol transport (transfer of macrophage-cholesterol in the subendothelial space of the arterial wall to the liver) is terminated by selective high density lipoprotein (HDL)-cholesteryl ester (CE) uptake, mediated by scavenger receptor class B, type 1 (SR-B1). We tested the validity of two models for this process: "gobbling," i.e. one-step transfer of all HDL-CE to the cell and "nibbling," multiple successive cycles of SR-B1-HDL association during which a few CEs transfer to the cell. Concurrently, we compared cellular uptake of apoAI with that of apoAII, which is more lipophilic than apoAI, using HDL-[ 3 H]CE labeled with [ 125 I]apoAI or [ 125 I]apoAII. The studies were conducted in CHO-K1 and CHO-ldlA7 cells (LDLR -/- ) with (CHO-SR-B1) and without SR-B1 overexpression and in human Huh7 hepatocytes. Relative to CE, both apoAI and apoAII were excluded from uptake by all cells. However, apoAII was more highly excluded from uptake (2-4×) than apoAI. To distinguish gobbling versus nibbling mechanisms, media from incubations of HDL with CHO-SR-B1 cells were analyzed by non-denaturing PAGE, size-exclusion chromatography, and the distribution of apoAI, apoAII, cholesterol, and phospholipid among HDL species as a function of incubation time. HDL size gradually decreased, i.e. nibbling, with the concurrent release of lipid-free apoAI; apoAII was retained in an HDL remnant. Our data support an SR-B1 nibbling mechanism that is similar to that of streptococcal serum opacity factor, which also selectively removes CE and releases apoAI, leaving an apoAII-rich remnant. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses

PubMed Central

Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D

1999-01-01

The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents. PMID:10204994

Elevated Myo-Inositol, Choline, and Glutamate Levels in the Associative Striatum of Antipsychotic-Naive Patients With First-Episode Psychosis: A Proton Magnetic Resonance Spectroscopy Study With Implications for Glial Dysfunction

PubMed Central

Plitman, Eric; de la Fuente-Sandoval, Camilo; Reyes-Madrigal, Francisco; Chavez, Sofia; Gómez-Cruz, Gladys; León-Ortiz, Pablo; Graff-Guerrero, Ariel

2016-01-01

Glial disturbances are highly implicated in the pathophysiology of schizophrenia and may be linked with glutamatergic dysregulation. Myo-inositol (mI), a putative marker of glial cells, and choline (Cho), representative of membrane turnover, are both present in larger concentrations within glial cells than in neurons, and their elevation is often interpreted to reflect glial activation. Proton magnetic resonance spectroscopy (1H-MRS) allows for the evaluation of mI, Cho, glutamate, glutamate + glutamine (Glx), and N-acetylaspartate (NAA). A collective investigation of these measures in antipsychotic-naive patients experiencing their first nonaffective episode of psychosis (FEP) can improve the understanding of glial dysfunction and its implications in the early stages of schizophrenia. 3-Tesla 1H-MRS (echo time = 35ms) was performed in 60 antipsychotic-naive patients with FEP and 60 age- and sex-matched healthy controls. mI, Cho, glutamate, Glx, and NAA were estimated using LCModel and corrected for cerebrospinal fluid composition within the voxel. mI, Cho, and glutamate were elevated in the FEP group. After correction for multiple comparisons, mI positively correlated with grandiosity. The relationships between mI and glutamate, and Cho and glutamate, were more positive in the FEP group. These findings are suggestive of glial activation in the absence of neuronal loss and may thereby provide support for the presence of a neuroinflammatory process within the early stages of schizophrenia. Dysregulation of glial function might result in the disruption of glutamatergic neurotransmission, which may influence positive symptomatology in patients with FEP. PMID:26320195

Elevated Myo-Inositol, Choline, and Glutamate Levels in the Associative Striatum of Antipsychotic-Naive Patients With First-Episode Psychosis: A Proton Magnetic Resonance Spectroscopy Study With Implications for Glial Dysfunction.

PubMed

Plitman, Eric; de la Fuente-Sandoval, Camilo; Reyes-Madrigal, Francisco; Chavez, Sofia; Gómez-Cruz, Gladys; León-Ortiz, Pablo; Graff-Guerrero, Ariel

2016-03-01

Glial disturbances are highly implicated in the pathophysiology of schizophrenia and may be linked with glutamatergic dysregulation. Myo-inositol (mI), a putative marker of glial cells, and choline (Cho), representative of membrane turnover, are both present in larger concentrations within glial cells than in neurons, and their elevation is often interpreted to reflect glial activation. Proton magnetic resonance spectroscopy ((1)H-MRS) allows for the evaluation of mI, Cho, glutamate, glutamate + glutamine (Glx), and N-acetylaspartate (NAA). A collective investigation of these measures in antipsychotic-naive patients experiencing their first nonaffective episode of psychosis (FEP) can improve the understanding of glial dysfunction and its implications in the early stages of schizophrenia. 3-Tesla (1)H-MRS (echo time = 35 ms) was performed in 60 antipsychotic-naive patients with FEP and 60 age- and sex-matched healthy controls. mI, Cho, glutamate, Glx, and NAA were estimated using LCModel and corrected for cerebrospinal fluid composition within the voxel. mI, Cho, and glutamate were elevated in the FEP group. After correction for multiple comparisons, mI positively correlated with grandiosity. The relationships between mI and glutamate, and Cho and glutamate, were more positive in the FEP group. These findings are suggestive of glial activation in the absence of neuronal loss and may thereby provide support for the presence of a neuroinflammatory process within the early stages of schizophrenia. Dysregulation of glial function might result in the disruption of glutamatergic neurotransmission, which may influence positive symptomatology in patients with FEP. © The Author 2015. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transformation with TT8 and HB12 RNAi Constructs in Model Forage (Medicago sativa, Alfalfa) Affects Carbohydrate Structure and Metabolic Characteristics in Ruminant Livestock Systems.

PubMed

Li, Xinxin; Zhang, Yonggen; Hannoufa, Abdelali; Yu, Peiqiang

2015-11-04

Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative impact on feed digestibility. TT8 and HB12 genes were shown to have low expression levels in low-lignin tissues of alfalfa, but to date, there has been no study on the effect of down-regulation of these two genes in alfalfa on nutrient chemical profiles and availability in ruminant livestock systems. The objectives of this study were to investigate the effect of transformation of alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO nutritive value in ruminant livestock systems. The results showed that transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded CHO fractions (RDCA4, P = 0.06; RDCB1, P < 0.01) and totally degraded CHO fraction (TRDCHO, P = 0.08). Both HB12 and TT8 populations had significantly higher in vitro digestibility of neutral detergent fiber (NDF) at 30 h of incubation (ivNDF30) compared to the control (P < 0.01). The TT8 populations had highest ivDM30 and ivNDF240. Transformation of alfalfa with TT8 and HB12 RNAi constructs induced molecular structure changes. Different CHO functional groups had different sensitivities and different responses to the transformation. The CHO molecular structure changes induced by the transformation were associated with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8 RNAi could improve forage quality by increasing the availability of both NDF and DM. Further study is needed on the relationship between the transformation-induced structure changes at a molecular level and nutrient utilization in ruminant livestock systems when lignification is much higher.

Carbohydrate availability and exercise training adaptation: too much of a good thing?

PubMed

Bartlett, Jonathan D; Hawley, John A; Morton, James P

2015-01-01

Traditional nutritional approaches to endurance training have typically promoted high carbohydrate (CHO) availability before, during and after training sessions to ensure adequate muscle substrate to meet the demands of high daily training intensities and volumes. However, during the past decade, data from our laboratories and others have demonstrated that deliberately training in conditions of reduced CHO availability can promote training-induced adaptations of human skeletal muscle (i.e. increased maximal mitochondrial enzyme activities and/or mitochondrial content, increased rates of lipid oxidation and, in some instances, improved exercise capacity). Such data have led to the concept of 'training low, but competing high' whereby selected training sessions are completed in conditions of reduced CHO availability (so as to promote training adaptation), but CHO reserves are restored immediately prior to an important competition. The augmented training response observed with training-low strategies is likely regulated by enhanced activation of key cell signalling kinases (e.g. AMPK, p38MAPK), transcription factors (e.g. p53, PPARδ) and transcriptional co-activators (e.g. PGC-1α), such that a co-ordinated up-regulation of both the nuclear and mitochondrial genomes occurs. Although the optimal practical strategies to train low are not currently known, consuming additional caffeine, protein, and practising CHO mouth-rinsing before and/or during training may help to rescue the reduced training intensities that typically occur when 'training low', in addition to preventing protein breakdown and maintaining optimal immune function. Finally, athletes should practise 'train-low' workouts in conjunction with sessions undertaken with normal or high CHO availability so that their capacity to oxidise CHO is not blunted on race day.

Asymmetrical flow field-flow fractionation coupled with multi-angle laser light scattering for stability comparison of virus-like particles in different solution environments.

PubMed

Chen, Yi; Zhang, Yan; Zhou, Yuefang; Luo, Jian; Su, Zhiguo

2016-06-08

The stabilities of two commercially available virus like particles, CHO-HBsAg expressed by Chinese hamster ovary (CHO) cells and Hans-HBsAg expressed by Hansenula polymorpha (Hans), were compared using AF4-MALLS under different treatment processes. The initial molecular weight and hydrodynamic diameter of CHO-HBsAg measured with AF4-MALLS were 4727kDa and 29.4nm, while those of Hans-HBsAg were 3039kDa and 22.8nm respectively. In salt solution of 2M ammonium sulfate, the molecular weight and size of CHO-HBsAg had little change, and its antigenicity remained 95%, while those of Hans-HBsAg changed greatly, resulting in aggregation and 75% antigenicity loss. In freeze-thaw operations, Hans-HBsAg aggregated heavily. Most of the aggregates precipitated and the rest soluble aggregates reached 10(5)-10(6)kDa in molecular weight. The antigenicity of Hans-HBsAg decreased to 26.9% after five freeze-thaw cycles. For CHO-HBsAg, there was no obvious aggregation in freeze-thaw, and the antigenicity retained above 98%. In heating process, Hans-HBsAg gradually aggregated to large particles with temperature and the antigenicity decreased to 10% when the temperature reached 80°C. In contrast, CHO-HBsAg would not aggregate with temperature, remained 92% antigenicity at 80°C. The study demonstrated that CHO-HBsAg appeared to be a superior vaccine antigen in term of particle stability and constant antigenicity, which are important in production, transportation and storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser1270

PubMed Central

Barauna, Valerio Garrone; Campos, Luciene Cristina Gastalho; Miyakawa, Ayumi Aurea; Krieger, Jose Eduardo

2011-01-01

Objectives We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser1270 are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results Western blotting analysis showed that SS (18 h, 15 dyn/cm2) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser1270 compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser1270, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser1270. PMID:21901117

Comparison among conventional and advanced MRI, 18F-FDG PET/CT, phenotype and genotype in glioblastoma.

PubMed

Valentini, Maria Consuelo; Mellai, Marta; Annovazzi, Laura; Melcarne, Antonio; Denysenko, Tetyana; Cassoni, Paola; Casalone, Cristina; Maurella, Cristiana; Grifoni, Silvia; Fania, Piercarlo; Cistaro, Angelina; Schiffer, Davide

2017-10-31

Glioblastoma (GB) is a highly heterogeneous tumor. In order to identify in vivo the most malignant tumor areas, the extent of tumor infiltration and the sites giving origin to GB stem cells (GSCs), we combined positron emission tomography/computed tomography (PET/CT) and conventional and advanced magnetic resonance imaging (MRI) with histology, immunohistochemistry and molecular genetics. Prior to dura opening and tumor resection, forty-eight biopsy specimens [23 of contrast-enhancing (CE) and 25 of non-contrast enhancing (NE) regions] from 12 GB patients were obtained by a frameless image-guided stereotactic biopsy technique. The highest values of 2-[18F]-fluoro-2-deoxy-D-glucose maximum standardized uptake value ( 18 F-FDG SUV max ), relative cerebral blood volume (rCBV), Choline/Creatine (Cho/Cr), Choline/N-acetylaspartate (Cho/NAA) and Lipids/Lactate (LL) ratio have been observed in the CE region. They corresponded to the most malignant tumor phenotype, to the greatest molecular spectrum and stem cell potential. On the contrary, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the CE region were very variable. 18 F-FDG SUV max , Cho/Cr and Cho/NAA ratio resulted the most suitable parameters to detect tumor infiltration. In edematous areas, reactive astrocytes and microglia/macrophages were influencing variables. Combined MRI and 18 F-FDG PET/CT allowed to recognize the specific biological significance of the different identified areas of GB.

Comparison among conventional and advanced MRI, 18F-FDG PET/CT, phenotype and genotype in glioblastoma

PubMed Central

Valentini, Maria Consuelo; Mellai, Marta; Annovazzi, Laura; Melcarne, Antonio; Denysenko, Tetyana; Cassoni, Paola; Casalone, Cristina; Maurella, Cristiana; Grifoni, Silvia; Fania, Piercarlo; Cistaro, Angelina; Schiffer, Davide

2017-01-01

Glioblastoma (GB) is a highly heterogeneous tumor. In order to identify in vivo the most malignant tumor areas, the extent of tumor infiltration and the sites giving origin to GB stem cells (GSCs), we combined positron emission tomography/computed tomography (PET/CT) and conventional and advanced magnetic resonance imaging (MRI) with histology, immunohistochemistry and molecular genetics. Prior to dura opening and tumor resection, forty-eight biopsy specimens [23 of contrast-enhancing (CE) and 25 of non-contrast enhancing (NE) regions] from 12 GB patients were obtained by a frameless image-guided stereotactic biopsy technique. The highest values of 2-[18F]-fluoro-2-deoxy-D-glucose maximum standardized uptake value (18F-FDG SUVmax), relative cerebral blood volume (rCBV), Choline/Creatine (Cho/Cr), Choline/N-acetylaspartate (Cho/NAA) and Lipids/Lactate (LL) ratio have been observed in the CE region. They corresponded to the most malignant tumor phenotype, to the greatest molecular spectrum and stem cell potential. On the contrary, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the CE region were very variable. 18F-FDG SUVmax, Cho/Cr and Cho/NAA ratio resulted the most suitable parameters to detect tumor infiltration. In edematous areas, reactive astrocytes and microglia/macrophages were influencing variables. Combined MRI and 18F-FDG PET/CT allowed to recognize the specific biological significance of the different identified areas of GB. PMID:29207673

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.

Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

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

Miles, K.; Anthony, D.T.; Rubin, L.L.

1987-09-01

The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximalmore » response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.« less

[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.

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.

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

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.

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.

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

PubMed

Saldova, Radka; Kilcoyne, Michelle; Stöckmann, Henning; Millán Martín, Silvia; Lewis, Amanda M; Tuite, Catherine M E; Gerlach, Jared Q; Le Berre, Marie; Borys, Michael C; Li, Zheng Jian; Abu-Absi, Nicholas R; Leister, Kirk; Joshi, Lokesh; Rudd, Pauline M

2017-03-01

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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

A comparison of normal versus low dietary carbohydrate intake on substrate oxidation during and after moderate intensity exercise in women.

PubMed

Patterson, Rachel; Potteiger, Jeffrey A

2011-12-01

We compared the effects of consuming a 2-day low-carbohydrate (CHO) diet (low-CHO; 20% CHO, 40% protein, 40% fat) versus an isocaloric 2-day moderate-CHO diet (mod-CHO; 55% CHO, 15% protein, 30% fat) on substrate oxidation during and after exercise in ten active, young women. Subjects were 24.9 ± 6.2% body fat with a VO(2max) of 68.8 ± 13.8 ml/kg FFM/min. For 2 days prior to exercise, subjects consumed either the mod-CHO or the low-CHO diet and then completed treadmill exercise at 55% of VO(2max) until 350 kcal of energy was expended. During exercise and for 2 h post-exercise, expired gases were analyzed to determine oxidation rates for CHO (CHO-OX) and fat (FAT-OX). Significant differences (p < 0.05) were found between diets for CHO-OX and FAT-OX (mg/kg FFM/min) during exercise, 1 h post-ex, and 2 h post-ex. During exercise, FAT-OX was higher (low-CHO 8.7 ± 2.2 vs. mod-CHO 6.2 ± 2.2) and CHO-OX was lower (low-CHO 25.1 ± 5.6 vs. mod-CHO 31.1 ± 6.2) following the low-CHO diet. A similar trend was observed during 1 h post-ex for FAT-OX (low-CHO 2.2 ± 0.5 vs. mod-CHO 1.6 ± 0.5) and CHO-OX (low-CHO 2.5 ± 1.2 vs. mod-CHO 4.1 ± 1.9), as well as 2 h post-ex for FAT-OX (low-CHO vs. 1.9 ± 0.5 mod-CHO 1.7 ± 0.4) and CHO-OX (low-CHO 2.5 ± 0.9 vs. mod-CHO 3.1 ± 1.1). Significant positive correlations were observed between VO(2max) and CHO-OX during exercise and post-exercise, as well as significant negative correlations between VO(2max) and FAT-OX post-exercise in the low-CHO condition. Waist circumference and FAT-OX exhibited a significant negative correlation during exercise in the low-CHO condition. Dietary macronutrient intake influenced substrate oxidation in active young women during and after moderate intensity exercise.

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.

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.

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

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

[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.

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.

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.

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.

Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

PubMed

Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

2013-12-01

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Olive oil and walnut breakfasts reduce the postprandial inflammatory response in mononuclear cells compared with a butter breakfast in healthy men.

PubMed

Jiménez-Gómez, Yolanda; López-Miranda, José; Blanco-Colio, Luis M; Marín, Carmen; Pérez-Martínez, Pablo; Ruano, Juan; Paniagua, Juan A; Rodríguez, Fernando; Egido, Jesús; Pérez-Jiménez, Francisco

2009-06-01

Inflammation is crucial in all stages of atherosclerosis, and few studies have investigated the effect of dietary fat on markers of inflammation related to this disease during the postprandial period. To evaluate the chronic effects of dietary fat on the postprandial expression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) in healthy subjects. 20 healthy men followed three different diets for 4 weeks each, according to a randomized crossover design: Western diet: 15% protein, 47% carbohydrates (CHO), 38% fat (22% saturated fatty acid (SFA)); Mediterranean diet: 15% protein, 47% CHO, 38% fat (24% monounsaturated fatty acid (MUFA)); CHO-rich and n-3 diet: 15% protein, 55% CHO, <30% fat (8% polyunsaturated fatty acid (PUFA)). After 12-h fast, volunteers were given a breakfast with a fat composition similar to that consumed in each of the diets-butter breakfast: 35% SFA; olive oil breakfast: 36% MUFA; walnut breakfast: 16% PUFA, 4% alpha-linolenic acid (LNA). The butter breakfast induced a higher increase in tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) expression than the olive oil or walnut breakfasts (P=0.014) in PBMCs. Moreover, we found a higher postprandial response in the mRNA of interleukin (IL)-6 with the intake of butter and olive oil breakfasts than with the walnut breakfast (P=0.025) in these cells. However, the effects of the three fatty breakfasts on the plasma concentrations of these proinflammatory parameters showed no significant differences (P=N.S.). Consumption of a butter-enriched meal elicits greater postprandial expression of proinflammatory cytokine mRNA in PBMCs, compared to the olive oil and walnut breakfasts.

PD-L1 Detection in Tumors Using [(64)Cu]Atezolizumab with PET.

PubMed

Lesniak, Wojciech G; Chatterjee, Samit; Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Pomper, Martin G; Nimmagadda, Sridhar

2016-09-21

The programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pair is a major immune checkpoint pathway exploited by cancer cells to develop and maintain immune tolerance. With recent approvals of anti-PD-1 and anti-PD-L1 therapeutic antibodies, there is an urgent need for noninvasive detection methods to quantify dynamic PD-L1 expression in tumors and to evaluate the tumor response to immune modulation therapies. To address this need, we assessed [(64)Cu]atezolizumab for the detection of PD-L1 expression in tumors. Atezolizumab (MPDL3208A) is a humanized, human and mouse cross-reactive, therapeutic PD-L1 antibody that is being investigated in several cancers. Atezolizumab was conjugated with DOTAGA and radiolabeled with copper-64. The resulting [(64)Cu]atezolizumab was assessed for in vitro and in vivo specificity in multiple cell lines and tumors of variable PD-L1 expression. We performed PET-CT imaging, biodistribution, and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-hPD-L1) and in controls (CHO). Specificity of [(64)Cu]atezolizumab was further confirmed in orthotopic tumor models of human breast cancer (MDAMB231 and SUM149) and in a syngeneic mouse mammary carcinoma model (4T1). We observed specific binding of [(64)Cu]atezolizumab to tumor cells in vitro, correlating with PD-L1 expression levels. Specific accumulation of [(64)Cu]atezolizumab was also observed in tumors with high PD-L1 expression (CHO-hPD-L1 and MDAMB231) compared to tumors with low PD-L1 expression (CHO, SUM149). Collectively, these studies demonstrate the feasibility of using [(64)Cu]atezolizumab for the detection of PD-L1 expression in different tumor types.

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.

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.

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.

Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.

PubMed

Bressel, Tatiana A B; de Queiroz, Jana Dara Freires; Gomes Moreira, Susana Margarida; da Fonseca, Jéssyca T; Filho, Edson A; Guastaldi, Antônio Carlos; Batistuzzo de Medeiros, Silvia Regina

2017-11-28

Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials. Laser beam irradiation at various fluences (132, 210, or 235 J/cm 2 ) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm 2 was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay. The best titanium surface was that produced by laser beam irradiation at 235 J/cm 2 fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity. The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae

PubMed Central

2004-01-01

The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Prüger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121–28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5–3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells. PMID:15588231

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine.

PubMed

Cong, Wei; Jin, Hong; Jiang, Chengda; Yan, Weiyao; Liu, Mingqiu; Chen, Jiulian; Zuo, Xiaoping; Zheng, Zhaoxin

2010-01-01

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10(9) CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID(50) of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 x 10(9) CFU of p3D-NT56/S. cho were protected in 9 days. INRA, EDP Sciences, 2010.

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

PubMed Central

Cong, Wei; Jin, Hong; Jiang, Chengda; Yan, Weiyao; Liu, Mingqiu; Chen, Jiulian; Zuo, Xiaoping; Zheng, Zhaoxin

2010-01-01

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 109 CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID50 of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 109 CFU of p3D-NT56/S. cho were protected in 9 days. PMID:20167192

The importance of dietary carbohydrates.

PubMed

Sánchez-Castillo, Claudia P; Hudson, Geoffrey J; Englyst, Hans N; Dewey, Peter; James, W Philip T

2002-12-01

Forty years ago carbohydrates (CHO) were regarded as a simple energy source whereas they are now recognized as important food components. The human diet contains a wide range of CHO, the vast majority of which are of plant origin. Modern techniques based on chemical classification of dietary CHO replaced the traditional "by difference" measurement. They provide a logical basis for grouping into categories of specific nutritional importance. The physiological effects of dietary CHO are highly dependent on the rate and extent of digestion and absorption in the small intestine and fermentation in the large intestine, interactions which promote human health. Current knowledge of the fate of dietary CHO means that the potentially undesirable properties of many modern foods could be altered by using processing techniques that yield foods with more intact plant cell wall structures. Such products would more closely resemble the foods in the pre-agriculture diet with respect to the rate of digestion and absorption of CHO in the small intestine. The potentially detrimental physiological consequences of eating sugars and starch that are rapidly digested and absorbed in the small intestine suggest that, as fibre, the form, as well as the amount of starch should be considered. Increasing consumer awareness of the relationship between diet and health has led to demands for more widespread nutrition labelling. The entry "carbohydrate" is required in most countries, and the value is usually obtained "by difference" and used in the calculation of energy content. However, the value provides no nutritional information per se. Food labels should provide values that aid consumers in selecting a healthy diet.

Comparison of [(11)C]Choline ([(11)C]CHO) and [(18)F]Bombesin (BAY 86-4367) as Imaging Probes for Prostate Cancer in a PC-3 Prostate Cancer Xenograft Model.

PubMed

Schwarzenböck, Sarah Marie; Schmeja, Philipp; Kurth, Jens; Souvatzoglou, Michael; Nawroth, Roman; Treiber, Uwe; Kundt, Guenther; Berndt, Sandra; Graham, Keith; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Ziegler, Sibylle I; Dinkelborg, Ludger; Wester, Hans-Jürgen; Krause, Bernd Joachim

2016-06-01

Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [(18)F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [(11)C]Choline ([(11)C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT). We carried out a dual-tracer small animal PET/CT study comparing [(18)F]Bombesin and [(11)C]CHO. The androgen-independent human prostate tumor cell line PC-3 was implanted subcutaneously in the flanks of nu/nu NMRI mice (n = 10) (PET/CT measurements of two [(11)C]Choline mice could not be analyzed due to technical reasons). [(18)F]Bombesin and [(11)C]CHO PET/CT imaging was performed about 3-4 weeks after the implantation of PC-3 cells on two separate days. After the intravenous tail vein injection of 14 MBq [(18)F]Bombesin and 37 MBq [(11)C]CHO, respectively, a dynamic study over 60 min was acquired in list mode using an Inveon animal PET/CT scanner (Siemens Medical Solutions). The sequence of [(18)F]Bombesin and [(11)C]CHO was randomized. Image analysis was performed using summed images as well as dynamic data. To calculate static and dynamic tumor-to-muscle (T/M), tumor-to-blood (T/B), liver-to-blood (L/B), and kidney-to-blood (K/B) ratios, 4 × 4 × 4 mm(3) volumes of interest (VOIs) of tumor, muscle (thigh), liver, kidney, and blood derived from transversal slices were used. The mean T/M ratio of [(18)F]Bombesin and [(11)C]CHO was 6.54 ± 2.49 and 1.35 ± 0.30, respectively. The mean T/B ratio was 1.83 ± 0.79 for [(18)F]Bombesin and 0.55 ± 0.10 for [(11)C]CHO. The T/M ratio as well as the T/B ratio for [(18)F]Bombesin were significantly higher compared to those for [(11)C]CHO (p < 0.001, respectively). Kidney and liver uptake was statistically significantly lower for [(18)F]Bombesin (K/B 3.41 ± 0.81, L/B 1.99 ± 0.38) compared to [(11)C]CHO [K/B 7.91 ± 1.85 (p < 0.001), L/B 6.27 ± 1.99 (p < 0.001)]. The magnitudes of the time course of T/M and T/B ratios (T/M and T/Bdyn ratios) were statistically significantly different (showing a higher uptake of [(18)F]Bombesin compared to [(11)C]CHO); additionally, also the change of the T/M and T/B ratios over time was significantly different between both tracers in the dynamic analysis (p < 0.001, respectively). Furthermore, there was a statistically significantly different change of the K/B and L/B ratios over time between the two tracers in the dynamic analysis (p = 0.026 and p < 0.001, respectively). [(18)F]Bombesin (BAY 86-4367) visually and semi-quantitatively outperforms [(11)C]CHO in the PC-3 prostate cancer xenograft model. [(18)F]Bombesin tumor uptake was significantly higher compared to [(11)C]CHO. [(18)F]Bombesin showed better imaging properties compared to the clinically utilized [(11)C]CHO due to a higher tumor uptake as well as a lower liver and kidney uptake.

Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.

PubMed Central

Whitbeck, J C; Peng, C; Lou, H; Xu, R; Willis, S H; Ponce de Leon, M; Peng, T; Nicola, A V; Montgomery, R I; Warner, M S; Soulika, A M; Spruce, L A; Moore, W T; Lambris, J D; Spear, P G; Cohen, G H; Eisenberg, R J

1997-01-01

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells. PMID:9223502

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.

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.

Proton MRS detects Metabolic Changes in Hormone Sensitive and Resistant Human Prostate Cancer Model CWR22 and CWR22r

PubMed Central

Le, H. Carl; Lupu, Mihaela; Kotedia, Khushali; Rosen, Neal; Solit, David; Koutcher, Jason A.

2010-01-01

17-Allylamino, 17-Demethoxygeldanamycin (17-AAG), an effective inhibitor of the heat shock protein hsp90, preferentially inhibiting tumor hsp90 compared to hsp90 from normal cells (1), has shown promising results against several cancers, including hormone resistant prostate cancer. Levels of several oncogenic proteins critical to tumor growth and progression, such as AR (androgen receptor) and HER2/neu, were reduced 4 hours post 17-AAG treatment. Post treatment metabolic changes have also been observed in several tumor cell lines. In this study total choline (t-cho) distributions in hormone sensitive CWR22 and hormone resistant CWR22r prostate cancer xenograft tumors in mice were measured before, 4 hours and 48 hours after a single bolus 17-AAG treatment at 100 mg/kg using proton MRS. Our results show that tumor t-cho levels declined 4 hours after the treatment for CWR22 (P = 0.001) and 48 hours post treatment for CWR22r (P=0.003). Metabolic changes, in particular of t-cho intensity detected by 1H MRSI, are consistent with the observed immunohistochemistry changes, tumor growth inhibition for CWR22r (P=0.01 at 14 days post treatment) and a constant PSA level versus increasing PSA for control CWR22 (P=0.01). Metabolic changes in t-cho by proton MRSI can be used as an early biomarker of response for advanced stage prostate cancer in targeted therapy such as 17-AAG. PMID:19780165

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+/-11 and 75+/-33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. Bmax values around 3-4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, Kd values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [3H]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells.

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.

Effects of indirubin and isatin on cell viability, mutagenicity, genotoxicity and BAX/ERCC1 gene expression.

PubMed

Fogaça, Manoela Viar; Cândido-Bacani, Priscila de Matos; Benicio, Lucas Milanez; Zapata, Lara Martinelli; Cardoso, Priscilla de Freitas; de Oliveira, Marcelo Tempesta; Calvo, Tamara Regina; Varanda, Eliana Aparecida; Vilegas, Wagner; de Syllos Cólus, Ilce Mara

2017-12-01

Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 μM) or ISA (0.5 to 50 μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD 50 - 1 g/kg b.w.) and submitted to comet assay in vivo. IRN reduced the viability of CHO-K1 (24 h; 5 to 200 μM) and HeLa cells (10 to 200 μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 μM; HeLa: 5 and 10 μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine: An anticancer agent targeting hypoxic cells

PubMed Central

Seow, Helen A.; Penketh, Philip G.; Shyam, Krishnamurthy; Rockwell, Sara; Sartorelli, Alan C.

2005-01-01

To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 μM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NAD(P)H: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues. PMID:15964988

β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

PubMed

Raschi, Elena; Chighizola, Cecilia B; Grossi, Claudia; Ronda, Nicoletta; Gatti, Rita; Meroni, Pier Luigi; Borghi, M Orietta

2014-12-01

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

PubMed

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

PubMed Central

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

2016-01-01

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible. PMID:26942051

Fluorescent carbohydrate probes for cell lectins

NASA Astrophysics Data System (ADS)

Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

2001-09-01

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

Role of Copper and Cholesterol Association in the Neurodegenerative Process

PubMed Central

Morel, Gustavo R.; de Alaniz, María J. T.; Castillo, Omar; Marra, Carlos A.

2013-01-01

Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho) as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified) contents in plasma and brain zones (cortex and hippocampus), markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms). The ratio beta-amyloid (1-42)/(1-40) was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test) was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus) with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma Aβ1-42/Aβ1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage. PMID:24288650

Pharmacological characterization of the bifunctional opioid ligand H-Dmt-Tic-Gly-NH-Bzl (UFP-505).

PubMed

Dietis, N; McDonald, J; Molinari, S; Calo, G; Guerrini, R; Rowbotham, D J; Lambert, D G

2012-02-01

While producing good-quality analgesia, µ-opioid (MOP) receptor activation produces a number of side-effects including tolerance. Simultaneous blockade of δ-opioid (DOP) receptors has been shown to reduce tolerance to morphine. Here, we characterize a prototype bifunctional opioid H-Dmt-Tic-Gly-NH-Bzl (UFP-505). We measured receptor binding affinity in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, k-opioid (KOP), nociceptin/orphanin (NOP) receptors. For activation, we measured the binding of GTPγ(35)S to membranes from CHO(hMOP), CHO(hDOP), rat cerebrocortex, and rat spinal cord. In addition, we assessed 'end organ' responses in the guinea pig ileum and mouse vas deferens. UFP-505 bound to CHO(hMOP) and CHO(hDOP) with (binding affinity) pK(i) values of 7.79 and 9.82, respectively. There was a weak interaction at KOP and NOP (pK(i) 6.29 and 5.86). At CHO(hMOP), UFP-505 stimulated GTPγ(35)S binding with potency (pEC(50)) of 6.37 and in CHO(hDOP) reversed the effects of a DOP agonist with affinity (pK(b)) of 9.81 (in agreement with pK(i) at DOP). UFP-505 also stimulated GTPγ(35)S binding in rat cerebrocortex and spinal cord with pEC(50) values of 6.11-6.53. In the guinea pig ileum (MOP-rich preparation), UFP-505 inhibited contractility with pEC(50) of 7.50 and in the vas deferens (DOP-rich preparation) reversed the effects of a DOP agonist with an affinity (pA(2)) of 9.15. We have shown in a range of preparations and assays that UFP-505 behaves as a potent MOP agonist and DOP antagonist; a MOP/DOP bifunctional opioid. Further studies in dual expression systems and whole animals with this prototype are warranted.

An alternative mechanism for radioprotection by dimethyl sulfoxide; possible facilitation of DNA double-strand break repair.

PubMed

Kashino, Genro; Liu, Yong; Suzuki, Minoru; Masunaga, Shin-ichiro; Kinashi, Yuko; Ono, Koji; Tano, Keizo; Watanabe, Masami

2010-01-01

The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, i.e., 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in CHO, but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

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.

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

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.

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 rights reserved.

Induction of steatohepatitis (NASH) with insulin resistance in wildtype B6 mice by a western-type diet containing soybean oil and cholesterol.

PubMed

Henkel, Janin; Coleman, Charles Dominic; Schraplau, Anne; Jӧhrens, Korinna; Weber, Daniela; Castro, José Pedro; Hugo, Martin; Schulz, Tim Julius; Krämer, Stephanie; Schürmann, Annette; Püschel, Gerhard Paul

2017-03-21

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g. high-fat diets) or overweight and insulin resistance (e.g. methionine-choline-deficient diets) or they are based on monogenetic defects (e.g. ob/ob mice). In the current study, a western-type diet containing soybean oil with high n 6-PUFA and 0.75% cholesterol (SOD+Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast a soybean oil-containing western-type diet without cholesterol (SOD) induced only mild steatosis but neither hepatic inflammation nor fibrosis, weight gain or insulin resistance. Another high-fat diet mainly consisting of lard and supplemented with fructose in drinking water (LAD+Fru) resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD+Cho but livers were devoid of inflammation and fibrosis. Although both LAD+Fru- and SOD+Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD+Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. Summarizing, dietary cholesterol in SOD+Cho diet may trigger hepatic inflammation and fibrosis. SOD+Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

DOE PAGES

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; ...

2016-11-14

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

PubMed Central

Hasim, Sahar; Allison, David P.; Retterer, Scott T.; Hopke, Alex; Wheeler, Robert T.; Doktycz, Mitchel J.

2016-01-01

ABSTRACT Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system. PMID:27849179

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

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

Hasim, Sahar; Allison, David P.; Retterer, Scott T.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is alteredmore » or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in this paper in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. Finally, AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.« less

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

PubMed

Yang, William C; Lu, Jiuyi; Kwiatkowski, Chris; Yuan, Hang; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

2014-01-01

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers.

Effects of arsenic on modification of promyelocytic leukemia (PML): PML responds to low levels of arsenite

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

Hirano, Seishiro, E-mail: seishiro@nies.go.jp; Graduate School of Pharmaceutical Sciences, Chiba University; Watanabe, Takayuki

2013-12-15

Inorganic arsenite (iAs{sup 3+}) is a two-edged sword. iAs{sup 3+} is a well-known human carcinogen; nevertheless, it has been used as a therapeutic drug for acute promyelocytic leukemia (APL), which is caused by a fusion protein comprising retinoic acid receptor-α and promyelocytic leukemia (PML). PML, a nuclear transcription factor, has a RING finger domain with densely positioned cysteine residues. To examine PML-modulated cellular responses to iAs{sup 3+}, CHO-K1 and HEK293 cells were each used to establish cell lines that expressed ectopic human PML. Overexpression of PML increased susceptibility to iAs{sup 3+} in CHO-K1 cells, but not in HEK293 cells. Exposuremore » of PML-transfected cells to iAs{sup 3+} caused PML to change from a soluble form to less soluble forms, and this modification of PML was observable even with just 0.1 μM iAs{sup 3+} (7.5 ppb). Western blot and immunofluorescent microscopic analyses revealed that the biochemical changes of PML were caused at least in part by conjugation with small ubiquitin-like modifier proteins (SUMOylation). A luciferase reporter gene was used to investigate whether modification of PML was caused by oxidative stress or activation of antioxidant response element (ARE) in CHO-K1 cells. Modification of PML protein occurred faster than activation of the ARE in response to iAs{sup 3+}, suggesting that PML was not modified as a consequence of oxidative stress-induced ARE activation. - Highlights: • PML was found in nuclear microspecles in response to arsenite. • Arsenite triggers SUMOylation of PML. • Arsenite modifies PML at as low as 0.1 μM. • Modification of PML is not caused by ARE activation.« less

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.

Inhibition of homophilic dimerization and disruption of cell adhesion by P-cadherin-specific small molecules from SPR-based assays.

PubMed

Senoo, Akinobu; Nagatoishi, Satoru; Moberg, Anna; Babol, Linnea Nygren; Mitani, Tomoya; Tashima, Takumi; Kudo, Shota; Tsumoto, Kouhei

2018-05-09

The inhibitor for the homophilic dimerization of P-cadherin was discovered by SPR-based screening using fragment compounds. Our SPR assays identified a specific P-cadherin binder, which was able to inhibit the cell adhesion of living CHO cells that expressed P-cadherin.

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

PubMed

Liu, J; Wang, Y; Szalay, A A; Escher, A

2000-01-01

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright 2000 John Wiley & Sons, Ltd.

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: potential role of the NFkappa B pathway.

PubMed

Ismail, Wan Iryani W; King, Judy A; Anwar, Khawar; Pillay, Tahir S

2013-08-01

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO-IR) and 3T3-L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β-subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate-1 (IRS-1) on serine 307 (S307) in both CHO-IR cells and 3T3-L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin-stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling-1 and -3 (SOCS-1 and -3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO-IR cells were pre-treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β-subunit and phosphorylation of IRS-1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. Copyright © 2013 Wiley Periodicals, Inc.

Induction of immunoglobulin G1, interleukin-6 and interleukin-10 by Taenia crassiceps metacestode carbohydrates

PubMed Central

Dissanayake, Senarath; Khan, Nasir; Shahin, Allen; Wijesinghe, Shanaka; Lukic, Miodrag

2002-01-01

T helper type 2 (Th2) -polarized immune responses are characteristically dominant in helminth infections. Two murine models that show a Th1 to Th2 polarization with infection progression are those of Schistosoma mansoni and Taenia crassiceps. In both, an early Th1 response is replaced by a late Th2 response. We report that the nucleic acid-, protein- and lipid-free carbohydrate fraction of T. crassiceps metacestodes (denoted T-CHO) possesses Th2-like immunomodulatory activity. Immunization of two strains of rats (Dark Agouti and Albino Oxford) and BALB/c mice with chicken albumin in the presence of T-CHO resulted in selective enhancement of immunoglobulin G1 (IgG1) antibodies, considered to be associated with Th2 responses in both rats and mice. Interleukin-6 (IL-6) followed by IL-10 were the dominant cytokines detected in in vitro cultures of mouse spleen cells stimulated with T-CHO. IL-4 and IL-5 were not detected in these culture supernates. Furthermore, Taenia carbohydrates were mitogenic to spleen cells, activated serine phosphorylation of proteins and up-regulated the expression of the anti-apoptotic protein, Bcl-2. When mouse spleen cells were cultured in the presence of Taenia carbohydrates, a concentration-dependent down-regulation of IL-2 and an overlapping up-regulation of IL-6 secretion were seen. PMID:12460185

Neural metabolite changes in corpus striatum after rat multipotent mesenchymal stem cells transplanted in hemiparkinsonian rats by magnetic resonance spectroscopy.

PubMed

Fu, Wenyu; Zheng, Zhijuan; Zhuang, Wenxin; Chen, Dandan; Wang, Xiaocui; Sun, Xihe; Wang, Xin

2013-12-01

To investigate the biochemical changes in striatum after rat bone marrow mesenchymal stem cells (MSCs) were transplanted into hemiparkinsonian rats and to further confirm the therapeutic effects of rat MSCs for Parkinson's disease (PD). 5-bromo-2-deoxyuridine (BrdU)-labeled MSCs were transplanted into the corpus striatum of the 6-hydroxydopamine (6-OHDA)-injected side of six PD model rats. Before and 8 weeks after MSC transplantation, ethological changes in PD rats were assessed. The expression of tyrosine hydroxylase (TH) in substantia nigra (SN) and striatum were measured using immunohistochemical methods. The differentiation of MSCs was detected by double immunofluorescence techniques. The concentrations of neural metabolites of N-acetylaspartate (NAA), choline (Cho) and creatine (Cr) were measured by ¹H-magnetic resonance spectroscopy (MRS). Relative concentrations of NAA/Cr and Cho/Cr were calculated. The behavior of PD rats in rotarod tests improved, and there were statistical differences in TH-positive cells in SN and TH-positive terminals in striatum after the transplantation of BrdU-labeled MSCs. Transplanted MSCs differentiated into MAP-2-positive neurons. Especially compared with pre-MSC transplantation, the neural metabolite NAA/Cr ratio of the 6-OHDA-injected side of the striatum increased (P < 0.05) and the Cho/Cr ratio decreased (P < 0.05). MSCs transplantation apparently improves neuronal function in the striatum of PD rats.

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.

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.

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.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

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

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

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

Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram

2010-07-06

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-bindingmore » pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.« less

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-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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 ...

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.

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.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

PubMed

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Favourable metabolic effects of a eucaloric lower-carbohydrate diet in women with PCOS.

PubMed

Gower, Barbara A; Chandler-Laney, Paula C; Ovalle, Fernando; Goree, Laura Lee; Azziz, Ricardo; Desmond, Renee A; Granger, Wesley M; Goss, Amy M; Bates, G Wright

2013-10-01

Diet-induced reduction in circulating insulin may be an attractive nonpharmacological treatment for women with polycystic ovary syndrome (PCOS) among whom elevated insulin may exacerbate symptoms by stimulating testosterone synthesis. This study was designed to determine whether a modest reduction in dietary carbohydrate (CHO) content affects β-cell responsiveness, serum testosterone concentration and insulin sensitivity in women with PCOS. In a crossover design, two diets ('Standard,' STD, 55:18:27% energy from carbohydrate/protein/fat; lower-carbohydrate, 41:19:40) were provided for 8 weeks in random order with a 4-week washout between. Thirty women with PCOS. β-cell responsiveness assessed as the C-peptide response to glucose during a liquid meal test; insulin sensitivity from insulin and glucose values throughout the test; insulin resistance (HOMA-IR); and total testosterone by immunoassay. Paired t-test indicated that the lower-CHO diet induced significant decreases in basal β-cell response (PhiB), fasting insulin, fasting glucose, HOMA-IR, total testosterone and all cholesterol measures, and significant increases in insulin sensitivity and dynamic ('first-phase') β-cell response. The STD diet induced a decrease in HDL-C and an increase in the total cholesterol-to-HDL-C ratio. Across all data combined, the change in testosterone was positively associated with the changes in fasting insulin, PhiB and insulin AUC (P < 0·05). In women with PCOS, modest reduction in dietary CHO in the context of a weight-maintaining diet has numerous beneficial effects on the metabolic profile that may lead to a decrease in circulating testosterone. © 2013 John Wiley & Sons Ltd.

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

PubMed

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

2016-03-31

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

ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

PubMed

Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-06-01

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN).

PubMed

Reddy, Gunda; Song, Jian; Kirby, Paul; Johnson, Mark S

2011-12-24

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

PubMed Central

Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

2006-01-01

Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure.

PubMed

Thompson, Gary L; Roth, Caleb C; Kuipers, Marjorie A; Tolstykh, Gleb P; Beier, Hope T; Ibey, Bennett L

2016-01-29

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus. Copyright © 2015 Elsevier Inc. All rights reserved.

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

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-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed. Images PMID:2725506

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes.

PubMed

Xiong, Liang-Bin; Liu, Hao-Hao; Xu, Li-Qin; Sun, Wan-Ju; Wang, Feng-Qing; Wei, Dong-Zhi

2017-05-22

The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC. MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system. Increasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

PubMed

Shahmohammadi-Farid, Sima; Ghods, Roya; Jeddi-Tehrani, Mahmood; Bayat, Ali-Ahmad; Mojtabavi, Nazanin; Razavi, Alireza; Zarnani, Amir-Hassan

2017-12-01

Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.

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

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.

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.

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 encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

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-directed mutant encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. Conclusion The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction. PMID:25056022

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

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.

Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations.

PubMed

Xu, Sen; Hoshan, Linda; Chen, Hao

2016-11-01

In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10(6) cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

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

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-06-06

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. Wemore » propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.« less

Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species.

PubMed

Shen, Yajing; Zhang, Ye; Zhang, Xiang; Zhou, Xiuhong; Teng, Xiyao; Yan, Manqing; Bi, Hong

2015-02-21

Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles (MMSNs-HRP) have been synthesized by a NHS/EDC coupling between the amino groups of horseradish peroxidase (HRP) and the carboxyl groups on the MMSNs surface. It is found that the immobilized HRP on MMSNs still retain high activity and the MMSNs-HRP can eliminate the reactive oxygen species (ROS) in Chinese hamster ovary (CHO) cells induced by the addition of H2O2 aqueous solution. Further, the fluorescent MMSN-HRP-CD nanoparticles have been prepared by attaching biocompatible, fluorescent carbon dots (CDs) to MMSNs-HRP. We have also investigated the effect of an applied magnetic field on cellular uptake of MMSNs-HRP-CDs and found that the internalization of MMSNs-HRP-CDs by CHO cells could be enhanced within 2 hours under the magnetic field. This work provides us with a novel and efficient method to eliminate ROS in living cells by using HRP-immobilized nanoparticles.

High frequency electroporation efficiency is under control of membrane capacitive charging and voltage potential relaxation.

PubMed

Novickij, Vitalij; Ruzgys, Paulius; Grainys, Audrius; Šatkauskas, Saulius

2018-02-01

The study presents the proof of concept for a possibility to achieve a better electroporation in the MHz pulse repetition frequency (PRF) region compared to the conventional low frequency protocols. The 200ns×10 pulses bursts of 10-14kV/cm have been used to permeabilize Chinese hamster ovary (CHO) cells in a wide range (1Hz-1MHz) of PRF. The permeabilization efficiency was evaluated using fluorescent dye assay (propidium iodide) and flow cytometry. It was determined that a threshold PRF exists when the relaxation of the cell transmembrane potential is longer than the delay between the consequent pulses, which results in accumulation of the charge on the membrane. For the CHO cells and 0.1S/m electroporation medium, this phenomenon is detectable in the 0.5-1MHz range. It was shown that the PRF is an important parameter that could be used for flexible control of electroporation efficiency in the high frequency range. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-09-01

Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application. Copyright © 2016 Elsevier Ltd. All rights reserved.

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 cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process. © 2014 American Institute of Chemical Engineers.

Mitochondrial dependent oxidative stress in cell culture induced by laser radiation at 1265 nm.

PubMed

Saenko, Yury V; Glushchenko, Eugenia S; Zolotovskii, Igor O; Sholokhov, Evgeny; Kurkov, Andrey

2016-04-01

Photodynamic therapy is the main technique applied for surface carcinoma treatment. This technique employs singlet oxygen generated via a laser excited photosensitizer as a main damaging agent. However, prolonged sensitivity to intensive light, relatively low tissue penetration by activating light the cost of photosensitizer (PS) administration can limit photodynamic therapy applications. Early was reported singlet oxygen generation without photosensitizer induced by a laser irradiation at the wavelength of 1250-1270 nm. Here, we study the dynamics of oxidative stress, DNA damage, changes of mitochondrial potential, and mitochondrial mass induced by a laser at 1265 nm have been studied in HCT-116 and CHO-K cells. Laser irradiation of HCT-116 and CHO-K cells has induced a dose-dependent cell death via increasing intracellular reactive oxygen species (ROS) concentration, increase of DNA damage, decrease of mitochondrial potential, and reduced glutathione. It has been shown that, along with singlet oxygen generation, the increase of the intracellular ROS concentration induced by mitochondrial damage contributes to the damaging effect of the laser irradiation at 1265 nm.

Water and solute absorption from carbohydrate-electrolyte solutions in the human proximal small intestine: a review and statistical analysis.

PubMed

Shi, Xiaocai; Passe, Dennis H

2010-10-01

The purpose of this study is to summarize water, carbohydrate (CHO), and electrolyte absorption from carbohydrate-electrolyte (CHO-E) solutions based on all of the triple-lumen-perfusion studies in humans since the early 1960s. The current statistical analysis included 30 reports from which were obtained information on water absorption, CHO absorption, total solute absorption, CHO concentration, CHO type, osmolality, sodium concentration, and sodium absorption in the different gut segments during exercise and at rest. Mean differences were assessed using independent-samples t tests. Exploratory multiple-regression analyses were conducted to create prediction models for intestinal water absorption. The factors influencing water and solute absorption are carefully evaluated and extensively discussed. The authors suggest that in the human proximal small intestine, water absorption is related to both total solute and CHO absorption; osmolality exerts various impacts on water absorption in the different segments; the multiple types of CHO in the ingested CHO-E solutions play a critical role in stimulating CHO, sodium, total solute, and water absorption; CHO concentration is negatively related to water absorption; and exercise may result in greater water absorption than rest. A potential regression model for predicting water absorption is also proposed for future research and practical application. In conclusion, water absorption in the human small intestine is influenced by osmolality, solute absorption, and the anatomical structures of gut segments. Multiple types of CHO in a CHO-E solution facilitate water absorption by stimulating CHO and solute absorption and lowering osmolality in the intestinal lumen.

Role of cytoskeleton and elastic moduli in cellular response to nanosecond pulsed electric fields

NASA Astrophysics Data System (ADS)

Thompson, Gary L.; Roth, Caleb; Tolstykh, Gleb; Kuipers, Marjorie; Ibey, Bennett L.

2013-02-01

Nanosecond pulsed electric fields (nsPEFs) are known to increase cell membrane permeability to small molecules in accordance with dosages. As previous work has focused on nsPEF exposures in whole cells, electrodeformation may contribute to this induced-permeabilization in addition to other biological mechanisms. Here, we hypothesize that cellular elasticity, based upon the cytoskeleton, affects nsPEF-induced decrease in cellular viability. Young's moduli of various types of cells have been calculated from atomic force microscopy (AFM) force curve data, showing that CHO cells are stiffer than non-adherent U937 and Jurkat cells, which are more susceptible to nsPEF exposure. To distinguish any cytoskeletal foundation for these observations, various cytoskeletal reagents were applied. Inhibiting actin polymerization significantly decreased membrane integrity, as determined by relative propidium uptake and phosphatidylserine externalization, upon exposure at 150 kV/cm with 100 pulses of 10 ns pulse width. Exposure in the presence of other drugs resulted in insignificant changes in membrane integrity and 24-hour viability. However, Jurkat cells showed greater lethality than latrunculin-treated CHO cells of comparable elasticity. From these results, it is postulated that cellular elasticity rooted in actin-membrane interaction is only a minor contributor to the differing responses of adherent and non-adherent cells to nsPEF insults.

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

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

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Singh, Surender; Kumar, Sudarshan; Kaushik, Jai K; Mohanty, Ashok K; Malakar, Dhruba

2015-12-01

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

Comparison of [¹¹C]choline ([¹¹C]CHO) and S(+)-β-methyl-[¹¹C]choline ([¹¹C]SMC) as imaging probes for prostate cancer in a PC-3 prostate cancer xenograft model.

PubMed

Schwarzenböck, Sarah Marie; Gertz, Jana; Souvatzoglou, Michael; Kurth, Jens; Sachs, David; Nawroth, Roman; Treiber, Uwe; Schuster, Tibor; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Ziegler, Sibylle Ilse; Henriksen, Gjermund; Wester, Hans-Jürgen; Krause, Bernd Joachim

2015-04-01

Carbon-11- and fluorine-18-labeled choline derivatives have been introduced as promising tracers for prostate cancer imaging. However, due to limited specificity and sensitivity, there is a need for new tracers with higher sensitivity and specificity for diagnosing prostate cancer to improve tracer uptake and enhance imaging contrast. The aim of this study was to compare the properties of [(11)C]choline ([(11)C]CHO) with S(+)-β-methyl-[(11)C]choline ([(11)C]SMC) as tracer for prostate cancer imaging in a human prostate tumor mouse xenograft model by small-animal positron emission tomography/X-ray computed tomography (PET/CT). We carried out a dual-tracer small-animal PET/CT study comparing [(11)C]CHO and [(11)C]SMC. The androgen-independent human prostate tumor cell line PC3 was implanted subcutaneously in the flanks of Naval Medical Research Institute (NMRI) (nu/nu) mice (n = 11). Mice-6 weeks post-xenograft implantation-were injected with 37 MBq [(11)C]CHO via the tail vein. On a separate day, the mice were injected with 37 MBq [(11)C]SMC. Dynamic imaging was performed for 60 min with the Inveon animal PET/CT scanner (Siemens Medical Solutions) on two separate days (randomizing the sequence of the tracers). The dynamic PET images were acquired in list mode. Regions of interest (5 × 5 × 5 mm) were placed in transaxial slices in tumor, muscle (thigh), liver, kidney, and blood. Image analysis was performed calculating tumor to muscle (T/M) ratios based on summed images as well as dynamic data. For [(11)C]SMC, the mean T/M ratio was 2.24 ± 0.56 while the corresponding mean [(11)C]CHO T/M ratio was 1.35 ± 0.28. The T/M ratio for [(11)C]SMC was significant higher compared to [(11)C]CHO (p < 0.001). The time course of T/M ratio (T/Mdyn ratio) of [(11)C]SMC was higher compared to [(11)C]CHO with a statistically significant difference between the magnitudes of the T/M ratios and a significant different change of the T/M ratios over time between [(11)C]CHO and [(11)C]SMC. Our results demonstrate that [(11)C]SMC is taken up by the tumor in the PC-3 prostate cancer xenograft model. [(11)C]SMC uptake was significantly higher compared to the clinically utilized [(11)C]CHO tracer with a higher contrast allowing imaging of a prostate cancer xenograft.

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-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments. PMID:1846001

Effects of carbohydrate ingestion during exercise on substrate oxidation in physically active women with different body compositions.

PubMed

Mitchell, Nicole M; Potteiger, Jeffery A; Bernardoni, Brittney; Claytor, Randal P

2013-03-01

We examined the effects of consuming a 6% carbohydrate (CHO) beverage during exercise on substrate oxidation in active, young Caucasian women with higher body fat (HF) (>25%) and lower body fat (LF) (<25%). The HF group (n = 9) had 32.4% ± 5.6% fat and the LF group (n = 8) had 20.0% ± 3.0% fat. Subjects completed 2 sessions of 45 min of treadmill exercise at 55% of maximal oxygen consumption. Immediately prior to and at 15-min intervals during exercise, subjects consumed 25% of a total volume of a CHO beverage (1 g CHO·kg(-1)) or a placebo (PLC). During exercise and for 2 h after exercise, expired gases were analyzed to determine oxidation rates for CHO (CHO-OX) and fat (FAT-OX). During exercise, significant differences (p < 0.05) in CHO-OX (mg·kg fat-free mass(-1)·min(-1)) were found between groups for the CHO trial (LF, 35.4 ± 4.7 vs. HF, 29.8 ± 3.6) and the PLC trial (LF, 33.7 ± 6.4 vs. HF, 26.3 ± 4.3). CHO-OX was significantly higher during the first hour of recovery in both the LF group (CHO, 9.3 ± 2.1 vs. PLC, 5.3 ± 2.4) and the HF group (CHO, 8.7 ± 2.0 vs. PLC, 4.2 ± 1.0), but during the second hour of recovery, only the HF group had a higher CHO-OX (CHO, 5.3 ± 1.8 vs. PLC, 3.9 ± 1.1). FAT-OX was significantly lower during the first hour of recovery in both the LF group (CHO, 0.6 ± 0.4 vs. PLC, 1.0 ± 0.4) and the HF group (CHO, 0.4 ± 0.4 vs. PLC, 1.4 ± 0.4), as well as during the second hour of recovery in both the LF group (CHO, 0.8 ± 0.4 vs. PLC, 1.3 ± 0.5) and the HF group (CHO, 0.9 ± 0.6 vs. PLC, 1.6 ± 0). CHO ingestion promotes CHO-OX and suppresses FAT-OX during and following exercise in physically active women with low and high levels of body fat.

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.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

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.

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

PubMed

Sou, Si Nga; Jedrzejewski, Philip M; Lee, Ken; Sellick, Christopher; Polizzi, Karen M; Kontoravdi, Cleo

2017-07-01

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb ) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc-glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N-linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N-linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc-glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570-1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

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

Cytotoxic, antioxidative, genotoxic and antigenotoxic effects of Horchata, beverage of South Ecuador.

PubMed

Bailon-Moscoso, Natalia; Tinitana, Fani; Martínez-Espinosa, Ruth; Jaramillo-Velez, Andrea; Palacio-Arpi, Alejandra; Aguilar-Hernandez, Jessica; Romero-Benavides, Juan Carlos

2017-12-19

"Horchata" is an herbal mixture infusion consumed in Southern Ecuador; 66% of its plants are anti-inflammatory medicinal plant, and 51% are analgesics. Anti-inflammatory substances can prevent carcinogenesis mediated by cytotoxic effects and can prevent DNA damage. The aim of this study was to evaluate the cytotoxicity and apoptotic/antigenotoxic effects of horchata as well as its mechanism. Nine different varieties of horchata were prepared in the traditional way and then freeze-dried. Phytochemical screening tested for the presence of secondary metabolites using standard procedures and antioxidant activities. The cytotoxic activity was evaluated on cerebral astrocytoma (D-384), prostate cancer (PC-3), breast cancer (MCF-7), colon cancer (RKO), lung cancer (A-549), immortalized Chinese hamster ovary cells (CHO-K1), and human peripheral blood lymphocytes via a MTS assay. The pro-apoptotic effects were evaluated with Anexin V/Propidium Iodide and western blot of Bax, Bcl-2, TP53, and TP73. Induction and reduction of ROS were assessed by fluorimetry. Genotoxic and antigenotoxic effects were evaluated with a comet assay and micronuclei on binucleated cells. Five of nine horchatas had cytotoxic effects against D-384 while not affecting normal cells. These horchatas induce cell death by apoptosis modulated by p53/p73. In CHO-K1 cells, the horchatas decrease the damage induced by hydrogen peroxide and Mitomycin C measured in the comet and micronucleus assay respectively. The IC 50 range of effective horchatas in D-384 was 41 to 122 μg·mL -1 . This effect may be related to its use in traditional medicine (brain tonic). On the other hand, immortalized Chinese hamster ovary cells (CHO-K1) and lymphocytes did not show a cytotoxic effect. The most potent horchata induced apoptosis via a p53/p73-mediated mechanism. The horchatas present antigenotoxic properties, which may be related to the antioxidant capacity. Future studies on horchata components are necessary to understand the interactions and beneficial properties.

Mismatch repair deficiency associated with overexpression of the MSH3 gene.

PubMed

Marra, G; Iaccarino, I; Lettieri, T; Roscilli, G; Delmastro, P; Jiricny, J

1998-07-21

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

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.

Unprecedented Cell-Selection Using Ultra-Quick Freezing Combined with Aquaporin Expression

PubMed Central

Kato, Yasuhiro; Miyauchi, Takayuki; Abe, Youichiro; Kojić, Dušan; Tanaka, Manami; Chikazawa, Nana; Nakatake, Yuhki; Ko, Shigeru B. H.; Kobayashi, Daisuke; Hazama, Akihiro; Fujiwara, Shoko; Uchida, Tatsuya; Yasui, Masato

2014-01-01

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols. PMID:24558371

Effect of carbohydrate composition on fluid balance, gastric emptying, and exercise performance.

PubMed

Cole, K J; Grandjean, P W; Sobszak, R J; Mitchell, J B

1993-12-01

This study examined the effects of serial feedings of different carbohydrate (CHO) solutions on plasma volume, gastric emptying (GE), and performance during prolonged cycling exercise. Solutions containing 6 g% glucose-sucrose (CHO-6GS), 8.3 g% high fructose corn syrup (CHO-8HF), 6.3 g% high fructose corn syrup + 2 g% glucose polymer (CHO-8HP), and a water placebo (WP) were compared. Ten trained male cyclists performed four cycling trials consisting of 105 min at 70% VO2max followed by a 15-min all-out, self-paced performance ride. Every 15 min the men consumed one of the four test solutions. Blood samples were taken before, during, and after exercise to determine blood glucose and plasma volume changes. There were no significant differences in performance, GE, or plasma volume changes between trials. Blood glucose was significantly elevated at the 105-min time-point in all CHO trials when compared to WP. The CHO-8HF and CHO-8HP drinks resulted in a significantly higher delivery of CHO to the intestine. Higher rates of CHO oxidation during the steady-state ride were observed only with the CHO-6GS drink.

Using vibrational molecular spectroscopy to reveal association of steam-flaking induced carbohydrates molecular structural changes with grain fractionation, biodigestion and biodegradation

NASA Astrophysics Data System (ADS)

Xu, Ningning; Liu, Jianxin; Yu, Peiqiang

2018-04-01

Advanced vibrational molecular spectroscopy has been developed as a rapid and non-destructive tool to reveal intrinsic molecular structure conformation of biological tissues. However, this technique has not been used to systematically study flaking induced structure changes at a molecular level. The objective of this study was to use vibrational molecular spectroscopy to reveal association between steam flaking induced CHO molecular structural changes in relation to grain CHO fractionation, predicted CHO biodegradation and biodigestion in ruminant system. The Attenuate Total Reflectance Fourier-transform Vibrational Molecular Spectroscopy (ATR-Ft/VMS) at SRP Key Lab of Molecular Structure and Molecular Nutrition, Ministry of Agriculture Strategic Research Chair Program (SRP, University of Saskatchewan) was applied in this study. The fractionation, predicted biodegradation and biodigestion were evaluated using the Cornell Net Carbohydrate Protein System. The results show that: (1) The steam flaking induced significant changes in CHO subfractions, CHO biodegradation and biodigestion in ruminant system. There were significant differences between non-processed (raw) and steam flaked grain corn (P < .01); (2) The ATR-Ft/VMS molecular technique was able to detect the processing induced CHO molecular structure changes; (3) Induced CHO molecular structure spectral features are significantly correlated (P < .05) to CHO subfractions, CHO biodegradation and biodigestion and could be applied to potentially predict CHO biodegradation (R2 = 0.87, RSD = 0.74, P < .01) and intestinal digestible undegraded CHO (R2 = 0.87, RSD = 0.24, P < .01). In summary, the processing induced molecular CHO structure changes in grain corn could be revealed by the ATR-Ft/VMS vibrational molecular spectroscopy. These molecular structure changes in grain were potentially associated with CHO biodegradation and biodigestion.

Identification and functional characterization of hemorphins VV-H-7 and LVV-H-7 as low-affinity agonists for the orphan bombesin receptor subtype 3

PubMed Central

Lammerich, Hans-Peter; Busmann, Annette; Kutzleb, Christian; Wendland, Martin; Seiler, Petra; Berger, Claudia; Eickelmann, Peter; Meyer, Markus; Forssmann, Wolf-Georg; Maronde, Erik

2003-01-01

The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines. Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed. PMID:12721098

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.

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.

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

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.

Determination of cellular injury and death thresholds following exposure to high voltage 10ns electrical pulses

NASA Astrophysics Data System (ADS)

Ibey, Bennett L.; Roth, Caleb C.; Bernhard, Joshua A.; Pakhomov, Andrei G.; Wilmink, Gerald J.; Pakhomova, Olga

2011-03-01

Intense, nanosecond-duration electric pulses (nsEP) have been introduced as a novel modality to alter cellular function, with a mechanism of action qualitatively different from micro- and millisecond duration pulses used in electroporation. In this study, we determined the thresholds for plasma membrane injury (within 15 minutes) and cell death (at 24 hours) for 4 different cell types (CHO-K1, HeLa, Jurkat and U937). Plasma membrane injury was measured by flow cytometry using two fluorescent dyes, namely Annexin V-FITC, which binds to phosphatidylserine (PS) upon its externalization (subtle membrane injury), and propidium iodide (PI), which is typically impermeable to the cell, but enters when large pores are formed in the plasma membrane. In all cell types, 10-ns pulses caused phosphatidylserine (PS) externalization at low doses (<150kV/cm and 100 pulses for each cell type) and no PI uptake. Jurkat and U937 cell lines showed substantial cell death without uptake of PI (15 minutes post exposure) suggesting either delayed permeabilization due to swelling, or damage to intracellular components. In CHO-K1 and HeLa cell lines, PI uptake occurred at low doses relative to that necessary to cause cell death suggesting a necrotic death similar to longer pulse exposures. These findings suggest that nanosecond pulses may be beneficial in applications that require selective elimination of specific cell types.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

PubMed Central

Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

1996-01-01

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

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.

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.

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.

Fuel for the Work Required: A Theoretical Framework for Carbohydrate Periodization and the Glycogen Threshold Hypothesis.

PubMed

Impey, Samuel G; Hearris, Mark A; Hammond, Kelly M; Bartlett, Jonathan D; Louis, Julien; Close, Graeme L; Morton, James P

2018-05-01

Deliberately training with reduced carbohydrate (CHO) availability to enhance endurance-training-induced metabolic adaptations of skeletal muscle (i.e. the 'train low, compete high' paradigm) is a hot topic within sport nutrition. Train-low studies involve periodically training (e.g., 30-50% of training sessions) with reduced CHO availability, where train-low models include twice per day training, fasted training, post-exercise CHO restriction and 'sleep low, train low'. When compared with high CHO availability, data suggest that augmented cell signalling (73% of 11 studies), gene expression (75% of 12 studies) and training-induced increases in oxidative enzyme activity/protein content (78% of 9 studies) associated with 'train low' are especially apparent when training sessions are commenced within a specific range of muscle glycogen concentrations. Nonetheless, such muscle adaptations do not always translate to improved exercise performance (e.g. 37 and 63% of 11 studies show improvements or no change, respectively). Herein, we present our rationale for the glycogen threshold hypothesis, a window of muscle glycogen concentrations that simultaneously permits completion of required training workloads and activation of the molecular machinery regulating training adaptations. We also present the 'fuel for the work required' paradigm (representative of an amalgamation of train-low models) whereby CHO availability is adjusted in accordance with the demands of the upcoming training session(s). In order to strategically implement train-low sessions, our challenge now is to quantify the glycogen cost of habitual training sessions (so as to inform the attainment of any potential threshold) and ensure absolute training intensity is not compromised, while also creating a metabolic milieu conducive to facilitating the endurance phenotype.

Effects of preoperative oral carbohydrates and trace elements on perioperative nutritional status in elective surgery patients.

PubMed

Oyama, Yoshimasa; Iwasaka, Hideo; Shiihara, Keisuke; Hagiwara, Satoshi; Kubo, Nobuhiro; Fujitomi, Yutaka; Noguchi, Takayuki

2011-10-01

In order to enhance postoperative recovery, preoperative consumption of carbohydrate (CHO) drinks has been used to suppress metabolic fluctuations. Trace elements such as zinc and copper are known to play an important role in postoperative recovery. Here, we examined the effects of preoperatively consuming a CHO drink containing zinc and copper. Subjects were 122 elective surgery patients divided into two groups (overnight fasting and CHO groups); each group was further divided into morning or afternoon surgery groups. Subjects in the CHO group consumed 300 mL of a CHO drink the night before surgery, followed by 200 ml before morning surgery or 700 ml before afternoon surgery (> or =2 hours before anesthesia induction). Blood levels of glucose, nonesterified fatty acids (NEFA), retinol-binding protein, zinc, and copper were determined. One subject in the CHO group was excluded after refusing the drink. There were no adverse effects from the CHO drink. NEFA levels increased in the fasting groups. Although zinc levels increased in the CHO group immediately after anesthesia induction, no group differences were observed the day after surgery. Preoperative consumption of a CHO drink containing trace elements suppressed preoperative metabolic fluctuations without complications and prevented trace element deficiency. Further beneficial effects during the perioperative period can be expected by adding trace elements to CHO supplements.

Myo-inositol metabolism in appropriately grown and growth-restricted fetuses: a proton magnetic resonance spectroscopy study.

PubMed

Story, Lisa; Damodaram, Mellisa S; Supramaniam, Veena; Allsop, Joanna M; Mcguinness, Amy; Patel, Abhilasha; Wylezinska, Marzena; Kumar, Sailesh; Rutherford, Mary A

2013-09-01

Myo-inositol (Myo-ins) is a marker of neuroglial cells, being present in the astrocytes of brain tissue, but also functions as an osmolyte. Numbers of astrocytes are known to increase following injury to the brain. Growth-restricted fetuses are at increased risk of later neurodevelopmental impairments even in the absence of overt lesions and despite preserved/increased cerebral blood flow. This study aims to investigate brain Myo-ins metabolism in fetuses with intrauterine growth restriction (IUGR) and evidence of cerebral redistribution using magnetic resonance spectroscopy (MRS) at a short echo time. Biometry and Doppler assessment of blood flow was assessed using ultrasound in 28 fetuses with IUGR and 47 appropriately grown control subjects. MRI was used to exclude overt brain injury. Proton magnetic resonance spectroscopy of the fetal brain was then performed at an echo time of 42 ms to examine the Myo-ins:Choline (Cho), Myo-ins:Creatine (Cr) and Cho:Cr ratios. No alterations in brain Myo-ins:Cho, Myo-ins:Cr or Cho:Cr ratios were detected between appropriately grown and growth restricted fetuses. IUGR is not associated with a measureable difference in brain myo-inositol ratios. This may be due to the protective effects of preserved cerebral blood flow in growth restriction and comparable astrocyte numbers when compared to controls. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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.

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 process, showing that protein-specific cell/process engineering can provide a solution that exceeds the limits of genetic/functional diversity within heterogeneous host cell populations. . © 2015 Wiley Periodicals, Inc.

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

PubMed

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

2016-09-01

In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. This requires applying appropriate methods during bioprocess development to enable meaningful characterization of CHO clones and processes. Here, we present a novel hybrid approach for supporting comprehensive characterization of metabolic clone performance. The approach combines metabolite profiling with multivariate data analysis and fluxomics to enable a data-driven mechanistic analysis of key metabolic traits associated with desired cell phenotypes. We applied the methodology to quantify and compare metabolic performance in a set of 10 recombinant CHO-K1 producer clones and a host cell line. The comprehensive characterization enabled us to derive an extended set of clone performance criteria that not only captured growth and product formation, but also incorporated information on intracellular clone physiology and on metabolic changes during the process. These criteria served to establish a quantitative clone ranking and allowed us to identify metabolic differences between high-producing CHO-K1 clones yielding comparably high product titers. Through multivariate data analysis of the combined metabolite and flux data we uncovered common metabolic traits characteristic of high-producer clones in the screening setup. This included high intracellular rates of glutamine synthesis, low cysteine uptake, reduced excretion of aspartate and glutamate, and low intracellular degradation rates of branched-chain amino acids and of histidine. Finally, the above approach was integrated into a workflow that enables standardized high-content selection of CHO producer clones in a high-throughput fashion. In conclusion, the combination of quantitative metabolite profiling, multivariate data analysis, and mechanistic network model simulations can identify metabolic traits characteristic of high-performance clones and enables informed decisions on which clones provide a good match for a particular process platform. The proposed approach also provides a mechanistic link between observed clone phenotype, process setup, and feeding regimes, and thereby offers concrete starting points for subsequent process optimization. Biotechnol. Bioeng. 2016;113: 2005-2019. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

PubMed

Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

2017-03-01

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficacy and safety of two new formulations of artificial tears in subjects with dry eye disease: a 3-month, multicenter, active-controlled, randomized trial

PubMed Central

Simmons, Peter A; Liu, Haixia; Carlisle-Wilcox, Cindy; Vehige, Joseph G

2015-01-01

Purpose To evaluate and compare the efficacy and safety of two investigational artificial tear formulations (CHO-1 and CHO-2) containing carmellose sodium, hyaluronic acid at different concentrations, and osmoprotectants, with a standard carmellose sodium-containing formulation (Refresh Tears [RT]) in the treatment of dry eye disease. Subjects and methods In this 3-month, double-masked, multicenter study, subjects (n=305) were randomized 1:1:1 to receive CHO-1, CHO-2, or RT, used as needed but at least twice daily. The primary endpoint was change in ocular surface disease index (OSDI) score from baseline to day 90. Other key outcomes included symptoms evaluated on a visual analog scale, corneal and conjunctival staining, and adverse events. Results OSDI scores and dry eye symptoms showed a rapid and sustained reduction from baseline in each group. Both CHO-1 and CHO-2 met the primary efficacy endpoint of noninferiority to RT in day 90 OSDI score change from baseline. OSDI ocular symptoms subscale improved more with CHO-1 than CHO-2 (P=0.048). In subjects with clinically relevant baseline ocular surface staining (>14 total score of a maximum of 55), day 90 improvements were greater with CHO-1 and CHO-2 than RT (P≤0.044). Day 90 improvements in OSDI ocular symptoms subscale scores were also greater with CHO-1 than RT (P<0.007) in subjects with clinically relevant ocular staining. All treatments were well tolerated. Conclusion Both combination artificial tear formulations were efficacious and well tolerated in subjects with dry eye. CHO-1 demonstrated the best performance in improving ocular symptoms and reducing ocular staining in this heterogeneous study population. PMID:25931807

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

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.

Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D.

PubMed

Van de Walle, Gerlinde R; Peters, Sarah T; VanderVen, Brian C; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2008-12-01

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

Gut-training: the impact of two weeks repetitive gut-challenge during exercise on gastrointestinal status, glucose availability, fuel kinetics, and running performance.

PubMed

Costa, Ricardo J S; Miall, Atlanta; Khoo, Anthony; Rauch, Christopher; Snipe, Rhiannon; Camões-Costa, Vera; Gibson, Peter

2017-05-01

Due to gastrointestinal tract adaptability, the study aimed to determine the impact of gut-training protocol over 2 weeks on gastrointestinal status, blood glucose availability, fuel kinetics, and running performance. Endurance runners (n = 25) performed a gut-challenge trial (GC1), consisting of 2 h running exercise at 60% V̇O 2max whilst consuming gel-discs containing 30 g carbohydrates (2:1 glucose/fructose, 10% w/v) every 20 min and a 1 h distance test. Participants were then randomly assigned to a carbohydrate gel-disc (CHO-S), carbohydrate food (CHO-F), or placebo (PLA) gut-training group for 2 weeks of repetitive gut-challenge intervention. Participants then repeated a second gut-challenge trial (GC2). Gastrointestinal symptoms reduced in GC2 on CHO-S (60%; p = 0.008) and CHO-F (63%; p = 0.046); reductions were greater than PLA (p < 0.05). H 2 peak was lower in GC2 on CHO-S (mean (CI): 6 (4-8) ppm) compared with CHO-F (9 (6-12) ppm) and PLA (12 (2-21) ppm) (trial × time: p < 0.001). Blood glucose concentration was higher in GC2 on CHO-S (7.2 (6.3-8.1) mmol·L -1 ) compared with CHO-F (6.1 (5.7-6.5) mmol·L -1 ) and PLA (6.2 (4.9-7.5) mmol·L -1 ) (trial × time: p = 0.015). No difference in oxidation rates, plasma I-FABP, and cortisol concentrations were observed between groups and trials. Distance test improved on CHO-S (5.2%) and CHO-F (4.3%) in GC2, but not on PLA (-2.1%) (trial × time: p = 0.009). Two weeks of gut-training with CHO-S and CHO-F improved gastrointestinal symptoms and running performance compared with PLA. CHO-S also reduced malabsorption and increased blood glucose availability during endurance running compared with PLA.

Caffeine and carbohydrate supplementation during exercise when in negative energy balance: effects on performance, metabolism, and salivary cortisol.

PubMed

Slivka, Dustin; Hailes, Walter; Cuddy, John; Ruby, Brent

2008-12-01

The ingestion of carbohydrate (+CHO) and caffeine (+CAF) during exercise is a commonly used ergogenic practice. Investigations are typically conducted with subjects who are in a rested state after an overnight fast. However, this state of positive energy balance is not achieved during many work and exercise circumstances. The aim of this study was to evaluate the substrate use and performance effects of caffeine and carbohydrate consumed alone and in combination while participants were in negative energy balance. Male participants (n = 9; 23 +/- 3 years; 74.1 +/- 10.6 kg) completed 4 trials in random order: -CAF/-CHO, -CAF/+CHO, +CAF/-CHO, and +CAF/+CHO. Diet and exercise were prescribed for 2 days before each trial to ensure negative energy balance. For each trial, before and after 2 h of cycling at 50% of maximal watts, a saliva sample and a muscle biopsy (vastus lateralis) were obtained. A simulated 20 km time trial was then performed. The respiratory exchange ratio was higher (p < 0.05) in +CHO trials and lower (p < 0.05) in the +CAF/+CHO trial than in the -CAF/+CHO trial. Salivary cortisol response was higher (p < 0.05) in the +CAF/-CHO trial than in any of the other trials. Muscle glycogen and heart rates were similar in all trials. Performance in the 20 km time trial was better in the -CAF/+CHO trial than in the -CAF/-CHO trial (p < 0.05), but the +CAF/+CHO trial was no better than the +CAF/-CHO trial (p > 0.05), or any of the other trials. When co-ingested with carbohydrate, caffeine increased fat use and decreased nonmuscle glycogen carbohydrate use over carbohydrate alone when participants are in negative energy balance; however, caffeine had no effect on the 20 km cycling time trial performance.

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 than 32 weeks gestation. RSNA, 2013

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), never producing values lower than baseline. Earlier use of CB and CR in the life cycle appears more effective in overcoming CHO-induced perturbations than later use.

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

Weber, Thomas J.; Markillie, Lye MENG.

The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cellsmore » transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.« less

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

Su, Cathy; Allum, Allison J.; Aizawa, Yasushi

Glyceryl glucoside (GG, α-D-glucosyglycerol) is a natural glycerol derivative found in alcoholic drinks. Recently GG has been used as an alternative for glycerol in cosmetic products. However, the safety of using GG is still unclear. Currently, dimethyl sulfoxide (DMSO) and glycerol are wildly used in cryopreservation. Despite GG being a derivative of glycerol, the ability of GG in cryopreservation is still unknown. By using a system of Chinese Hamster Ovary cells (CHO), A549 cells and AG1522 cells, the study examined the cryoprotective effects of DMSO, glycerol and GG. Cytotoxic and genotoxic responses induced by the three chemicals were also investigated with CHOmore » to determine the safety of GG for cosmetic products. Our data suggests that GG has great cryopresearvation ability in the concentration of 30%–40% (v/v). For cytotoxic studies, DMSO showed the highest cytotoxicity above 3% (v/v) in cell doubling time delay among three chemicals. For the acute cytotoxicity with trypan blue dye exclusion assay, GG showed stronger cell killing effect within 24 h above 4% (v/v). For the continuous cytotoxicity with colony formation assay for 7 days, DMSO showed significantly reduced clonogenic ability above 2%. In genotoxicity studies, CHO treated with glycerol at 2% concentration induced three times higher frequencies of sister chromatid exchange (SCE) than background levels. GG did not induce significant amounts of SCE compared to background. Micronuclei formation was equally observed in the 2% and above concentrations of glycerol and GG. Our data showed that GG has significant effects on cryopreservation compared to DMSO. Glycerol and GG have similar cytotoxicity effects to CHO, but glycerol induced genotoxic responses in the same concentration. Therefore, we conclude that GG may be a safer alternative compound to glycerol in cosmetic products and safer alternative to DMSO in cryopreservation. -- Highlights: •Glyceryl Glucoside is low cytotoxicity and genotoxicity. •Glyceryl Glucoside is better cyroprotective agent than glycerol. •Glycerol has higher genotoxicity than Glyceryl Glucoside. •DMSO has higher cytotoxicity than Glyceryl Glucoside.« less

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.

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.

Microbial modulation of host immunity with the small molecule phosphorylcholine.

PubMed

Clark, Sarah E; Weiser, Jeffrey N

2013-02-01

All microorganisms dependent on persistence in a host for survival rely on either hiding from or modulating host responses to infection. The small molecule phosphorylcholine, or choline phosphate (ChoP), is used for both of these purposes by a wide array of bacterial and parasitic microbes. While the mechanisms underlying ChoP acquisition and expression are diverse, a unifying theme is the use of ChoP to reduce the immune response to infection, creating an advantage for ChoP-expressing microorganisms. In this minireview, we discuss several benefits of ChoP expression during infection as well as how the immune system fights back against ChoP-expressing pathogens.

Effects of Three Commercially Available Sports Drinks on Substrate Metabolism and Subsequent Endurance Performance in a Postprandial State.

PubMed

Qin, Lu; Wang, Qi-Rong; Fang, Zi-Long; Wang, Ting; Yu, Ai-Qi; Zhou, Yu-Jie; Zheng, Yi; Yi, Mu-Qing

2017-04-12

Purpose: To examine the effects of commercially available sports beverages with various components on substrate metabolism and subsequent performance. Methods: Two studies were conducted in a double-blinded, counterbalanced manner. Study I was designed to determine the glycemic index, while study II determined the utilization of substrates and subsequent exercise performance. Ten healthy male participants (age 21.70 ± 2.41 years, height 176.60 ± 5.23 cm, weight 66.58 ± 5.38 kg, V̇O 2max 48.1 ± 8.4 mL/kg/min) participated in both study I and study II. Three types of commercially available sports beverage powders were used. The powders consisted primarily of oligosaccharides (low molecular weight carbohydrates, L-CHO), hydrolyzed starch (high molecular weight CHO, H-CHO), and whey protein powder with carbohydrate (CHO-PRO). They were dissolved in purified water with identical CHO concentration of 8% ( w / v ). In study I, each participant underwent two oral glucose tolerance tests (OGTT) and one glycemic response test for each sports drink. In study II, participants cycled for 60 min at 70% V̇O 2max , one hour after consuming a standardized breakfast. One of four prescribed beverages (L-CHO, H-CHO, CHO-PRO, and Placebo control, PLA) was served at 0, 15, 30, 45 min during the exercise. Six hours after the first exercise session, participants came back for a "time to exhaustion test" (TTE). Blood samples were drawn at 0, 30, and 60 min in the first exercise session, while arterial blood gas analysis was conducted at 0, 30, and 60 min in both sessions. Subjective feelings (rating of perceived exertion and abdominal discomfort) were also evaluated every 30 min during exercise. Results: Compared to the reference standardized glucose solution, the glycemic index of the L-CHO beverage was 117.70 ± 14.25, while H-CHO was 105.50 ± 12.82, and CHO-PRO was 67.23 ± 5.88. During the exercise test, the insulin level at 30 and 60 min was significantly lower than baseline following the treatment of L-CHO, H-CHO, and PLA ( p < 0.05). The CHO oxidation rate at 60 min in the first exercise session was significantly higher than that at 60 min in the second exercise session following the L-CHO treatment ( p < 0.05). Time to exhaustion was not significantly different ( p > 0.05). Conclusion: The CHO sports beverage with additional PRO maintains insulin production during endurance cycling at 70% V̇O 2max in the postprandial state. L-CHO sports beverage suppresses fat utilization during the subsequent exercise performance test. The subsequent exercise performance (as evaluated by TTE) was not influenced by the type of CHO or the addition of PRO in the commercially available sports beverages used in the present study.

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...

Relationship of carbohydrate molecular spectroscopic features in combined feeds to carbohydrate utilization and availability in ruminants

NASA Astrophysics Data System (ADS)

Zhang, Xuewei; Yu, Peiqiang

To date, there is no study on the relationship between carbohydrate (CHO) molecular structures and nutrient availability of combined feeds in ruminants. The objective of this study was to use molecular spectroscopy to reveal the relationship between CHO molecular spectral profiles (in terms of functional groups (biomolecular, biopolymer) spectral peak area and height intensity) and CHO chemical profiles, CHO subfractions, energy values, and CHO rumen degradation kinetics of combined feeds of hulless barley with pure wheat dried distillers grains with solubles (DDGS) at five different combination ratios (hulless barley to pure wheat DDGS: 100:0, 75:25, 50:50, 25:75, 0:100). The molecular spectroscopic parameters assessed included: lignin biopolymer molecular spectra profile (peak area and height, region and baseline: ca. 1539-1504 cm-1); structural carbohydrate (STCHO, peaks area region and baseline: ca. 1485-1186 cm-1) mainly associated with hemi- and cellulosic compounds; cellulosic materials peak area (centered at ca. 1240 cm-1 with region and baseline: ca. 1272-1186 cm-1); total carbohydrate (CHO, peaks area region and baseline: ca. 1186-946 cm-1). The results showed that the functional groups (biomolecular, biopolymer) in the combined feeds are sensitive to the changes of carbohydrate chemical and nutrient profiles. The changes of the CHO molecular spectroscopic features in the combined feeds were highly correlated with CHO chemical profiles, CHO subfractions, in situ CHO rumen degradation kinetics and fermentable organic matter supply. Further study is needed to investigate possibility of using CHO molecular spectral features as a predictor to estimate nutrient availability in combined feeds for animals and quantify their relationship.

Using channelized Hotelling observers to quantify temporal effects of medical liquid crystal displays on detection performance

NASA Astrophysics Data System (ADS)

Platiša, Ljiljana; Goossens, Bart; Vansteenkiste, Ewout; Badano, Aldo; Philips, Wilfried

2010-02-01

Clinical practice is rapidly moving in the direction of volumetric imaging. Often, radiologists interpret these images in liquid crystal displays at browsing rates of 30 frames per second or higher. However, recent studies suggest that the slow response of the display can compromise image quality. In order to quantify the temporal effect of medical displays on detection performance, we investigate two designs of a multi-slice channelized Hotelling observer (msCHO) model in the task of detecting a single-slice signal in multi-slice simulated images. The design of msCHO models is inspired by simplifying assumptions about how humans observe while viewing in the stack-browsing mode. For comparison, we consider a standard CHO applied only on the slice where the signal is located, recently used in a similar study. We refer to it as a single-slice CHO (ssCHO). Overall, our results confirm previous findings that the slow response of displays degrades the detection performance of the observers. More specifically, the observed performance range of msCHO designs is higher compared to the ssCHO suggesting that the extent and rate of degradation, though significant, may be less drastic than previously estimated by the ssCHO. Especially, the difference between msCHO and ssCHO is more significant for higher browsing speeds than for slow image sequences or static images. This, together with their design criteria driven by the assumptions about humans, makes the msCHO models promising candidates for further studies aimed at building anthropomorphic observer models for the stack-mode image presentation.

Development of an Improved Mammalian Overexpression Method for Human CD62L

PubMed Central

Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

2014-01-01

We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

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.

Ethanol precipitation for purification of recombinant antibodies.

PubMed

Tscheliessnig, Anne; Satzer, Peter; Hammerschmidt, Nikolaus; Schulz, Henk; Helk, Bernhard; Jungbauer, Alois

2014-10-20

Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

PubMed

Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

2016-05-01

Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aromatic compounds produced by Periconia atropurpurea, an endophytic fungus associated with Xylopia aromatica.

PubMed

Teles, Helder Lopes; Sordi, Renata; Silva, Geraldo Humberto; Castro-Gamboa, Ian; Bolzani, Vanderlan da Silva; Pfenning, Ludwig Heinrich; de Abreu, Lucas Magalhães; Costa-Neto, Claudio Miguel; Young, Maria Claudia Marx; Araújo, Angela Regina

2006-12-01

6,8-Dimethoxy-3-(2'-oxo-propyl)-coumarin (1) and 2,4-dihydroxy-6-[(1'E,3'E)-penta-1',3'-dienyl]-benzaldehyde (2), in addition to the known compound periconicin B (3), were isolated from the ethyl acetate extract of Periconia atropurpurea, an endophytic fungus obtained from the leaves of Xylopia aromatica, a native plant of the Brazilian Cerrado. Their chemical structures were assigned based on analyses of MS, 1D and 2D-NMR spectroscopic experiments. Biological analyses were performed using two mammalian cell lines, human cervix carcinoma (HeLa) and Chinese hamster ovary (CHO). The results showed that compound 1 had no effect when compared to the control group, which was treated with the vehicle (DMSO). Compound 2 was able to induce a slight increase in cell proliferation of HeLa (37% of increase) and CHO (38% of increase) cell lines. Analysis of compound 3 showed that it has potent cytotoxic activity against both cell lines, with an IC50 of 8.0 microM. Biological analyses using the phytopathogenic fungi Cladosporium sphaerospermum and C. cladosporioides revealed that also 2 showed potent antifungal activity compared to nystatin.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

PubMed

Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

1998-06-01

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

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.

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.

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.

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase

PubMed Central

McNeil, Scott D.; Nuccio, Michael L.; Ziemak, Michael J.; Hanson, Andrew D.

2001-01-01

Choline (Cho) is the precursor of the osmoprotectant glycine betaine and is itself an essential nutrient for humans. Metabolic engineering of Cho biosynthesis in plants could therefore enhance both their resistance to osmotic stresses (drought and salinity) and their nutritional value. The key enzyme of the plant Cho-synthesis pathway is phosphoethanolamine N-methyltransferase, which catalyzes all three of the methylations required to convert phosphoethanolamine to phosphocholine. We show here that overexpressing this enzyme in transgenic tobacco increased the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting phosphatidylcholine content or growth. Moreover, the expanded Cho pool led to a 30-fold increase in synthesis of glycine betaine via an engineered glycine betaine pathway. Supplying the transgenics with the Cho precursor ethanolamine (EA) further enhanced Cho levels even though the supplied EA was extensively catabolized. These latter results establish that there is further scope for improving Cho synthesis by engineering an increased endogenous supply of EA and suggest that this could be achieved by enhancing EA synthesis and/or by suppressing its degradation. PMID:11481443

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.

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

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.

Comparison of flow cytometry, fluorescence microscopy and spectrofluorometry for analysis of gene electrotransfer efficiency.

PubMed

Marjanovič, Igor; Kandušer, Maša; Miklavčič, Damijan; Keber, Mateja Manček; Pavlin, Mojca

2014-12-01

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.

Low-Carbohydrate Diet for the Treatment of Gestational Diabetes Mellitus

PubMed Central

Moreno-Castilla, Cristina; Hernandez, Marta; Bergua, Merce; Alvarez, Maria C.; Arce, Maria A.; Rodriguez, Karen; Martinez-Alonso, Montserrat; Iglesias, Montserrat; Mateu, Magdalena; Santos, Maria D.; Pacheco, Linda R.; Blasco, Yolanda; Martin, Eva; Balsells, Nuria; Aranda, Nuria; Mauricio, Didac

2013-01-01

OBJECTIVE Medical nutrition therapy based on the control of the amount and distribution of carbohydrates (CHO) is the initial treatment for gestational diabetes mellitus (GDM), but there is a need for randomized controlled trials comparing different dietary strategies. The purpose of this study was to test the hypothesis that a low-CHO diet for the treatment of GDM would lead to a lower rate of insulin treatment with similar pregnancy outcomes compared with a control diet. RESEARCH DESIGN AND METHODS A total of 152 women with GDM were included in this open, randomized controlled trial and assigned to follow either a diet with low-CHO content (40% of the total diet energy content as CHO) or a control diet (55% of the total diet energy content as CHO). CHO intake was assessed by 3-day food records. The main pregnancy outcomes were also assessed. RESULTS The rate of women requiring insulin was not significantly different between the treatment groups (low CHO 54.7% vs. control 54.7%; P = 1). Daily food records confirmed a difference in the amount of CHO consumed between the groups (P = 0.0001). No differences were found in the obstetric and perinatal outcomes between the treatment groups. CONCLUSIONS Treatment of women with GDM using a low-CHO diet did not reduce the number of women needing insulin and produced similar pregnancy outcomes. In GDM, CHO amount (40 vs. 55% of calories) did not influence insulin need or pregnancy outcomes. PMID:23564917

Preservation of the gut by preoperative carbohydrate loading improves postoperative food intake.

PubMed

Luttikhold, Joanna; Oosting, Annemarie; van den Braak, Claudia C M; van Norren, Klaske; Rijna, Herman; van Leeuwen, Paul A M; Bouritius, Hetty

2013-08-01

A carbohydrate (CHO) drink given preoperatively changes the fasted state into a fed state. The ESPEN guidelines for perioperative care include preoperative CHO loading and re-establishment of oral feeding as early as possible after surgery. An intestinal ischaemia reperfusion (IR) animal model was used to investigate whether preoperative CHO loading increases spontaneous postoperative food intake, intestinal barrier function and the catabolic response. Male Wistar rats (n = 65) were subjected to 16 h fasting with ad libitum water and: A) sham laparotomy (Sham fasted, n = 24); B) intestinal ischaemia (IR fasted, n = 27); and C) intestinal ischaemia with preoperatively access to a CHO drink (IR CHO, n = 14). Spontaneous food intake, intestinal barrier function, insulin sensitivity, intestinal motility and plasma amino acids were measured after surgery. The IR CHO animals started eating significantly earlier and also ate significantly more than the IR fasted animals. Furthermore, preoperative CHO loading improved the intestinal barrier function, functional enterocyte metabolic mass measured by citrulline and reduced muscle protein catabolism, as indicated by normalization of the biomarker 3-methylhistidine. Preoperative CHO loading improves food intake, preserves the GI function and reduces the catabolic response in an IR animal model. These findings suggest that preoperative CHO loading preserves the intestinal function in order to accelerate recovery and food intake. If this effect is caused by overcoming the fasted state or CHO loading remains unclear. Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

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.

Effects of a low- or a high-carbohydrate diet on performance, energy system contribution, and metabolic responses during supramaximal exercise.

PubMed

Lima-Silva, Adriano E; Pires, Flavio O; Bertuzzi, Romulo; Silva-Cavalcante, Marcos D; Oliveira, Rodrigo S F; Kiss, Maria Augusta; Bishop, David

2013-09-01

The purpose of the present study was to examine the effects of a high- or low-carbohydrate (CHO) diet on performance, aerobic and anaerobic contribution, and metabolic responses during supramaximal exercise. Six physically-active men first performed a cycling exercise bout at 115% maximal oxygen uptake to exhaustion after following their normal diet for 48 h (∼50% of CHO, control test). Seventy-two hours after, participants performed a muscle glycogen depletion exercise protocol, followed by either a high- or low-CHO diet (∼70 and 25% of CHO, respectively) for 48 h, in a random, counterbalanced order. After the assigned diet period (48 h), the supramaximal cycling exercise bout (115% maximal oxygen consumption) to exhaustion was repeated. The low-CHO diet reduced time to exhaustion when compared with both the control and the high-CHO diet (-19 and -32%, respectively, p < 0.05). The reduced time to exhaustion following the low-CHO diet was accompanied by a lower total aerobic energy contribution (-39%) compared with the high-CHO diet (p < 0.05). However, the aerobic and anaerobic energy contribution at the shortest time to exhaustion (isotime) was similar among conditions (p > 0.05). The low-CHO diet was associated with a lower blood lactate concentration (p < 0.05), with no effect on the plasma concentration of insulin, glucose and K(+) (p > 0.05). In conclusion, a low-CHO diet reduces both performance and total aerobic energy provision during supramaximal exercise. As peak K(+) concentration was similar, but time to exhaustion shorter, the low-CHO diet was associated with an earlier attainment of peak plasma K(+) concentration.

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.

Metabolic and sarcoplasmic reticulum Ca2+ cycling responses in human muscle 4 days following prolonged exercise.

PubMed

Duhamel, T A; Green, H J; Perco, J G; Ouyang, J

2005-07-01

This study investigated the effects of prolonged exercise on muscle sarcoplasmic reticulum (SR) Ca2+ cycling properties and the metabolic responses with and without a session of exercise designed to reduce muscle glycogen reserves while on a normal carbohydrate (CHO) diet. Eight untrained males (VO(2peak) = 3.81 +/- 0.12 L/min, mean +/- SE) performed a standardized cycle-to-fatigue at 55% VO(2peak) while on a normal CHO diet (Norm CHO) and 4 days following prolonged exercise while on a normal CHO diet (Ex+Norm CHO). Compared to rest, exercise in Norm CHO to fatigue resulted in significant reductions (p < 0.05) in Ca2+ uptake (3.17 +/- 0.21 vs. 2.47 +/- 0.12 micromol.(g protein)-1.min-1), maximal Ca2+ ATPase activity (Vmax, 152 +/- 12 vs. 119 +/- 9 micromol.(g protein)-1.min-1) and both phase 1 (15.1 +/- 0.98 vs. 13.1 +/- 0.28 micromol.(g protein)-1.min-1) and phase 2 (6.56 +/- 0.33 vs. 4.91 +/- 0.28 micromol.(g protein)-1.min-1) Ca2+ release in vastus lateralis muscle. No differences were observed between Norm CHO and Ex-Norm CHO in the response of these properties to exercise. Compared with Norm CHO, Ex+Norm CHO resulted in higher (p < 0.05) resting Ca2+ uptake (3.17 +/- 0.21 vs. 3.49 +/- 0.24 micromol.(g protein).min-1 and higher ionophore ratio, defined as the ratio of Vmax measured with and without the Ca2+-ionophore A23187, (2.3 +/- 0.3 vs. 4.4 +/- 0.3 micromol.(g protein).min-1) at fatigue. No differences were observed between conditions in the concentration of muscle glycogen, the high-energy phosphates (ATP and PCr), or metabolites (Pi, Cr, and lactate). Ex+Norm CHO also failed to modify the exercise-induced changes in CHO and fat oxidation. We conclude that prolonged exercise to fatigue performed 4 days following glycogen-depleting exercise while on a normal CHO diet elevates resting Ca2+ uptake and prevents increases in SR membrane permeability to Ca2+ as measured by the ionophore ratio.

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

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 calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Characterization and analyses of multidrug resistance-associated protein 1 (MRP1/ABCC1) polymorphisms in Chinese population

PubMed Central

Yin, Ji-Ye; Huang, Qiong; Yang, Youyun; Zhang, Jian-Ting; Zhong, Mei-Zuo; Zhou, Hong-Hao; Liu, Zhao-Qian

2009-01-01

Multidrug resistance (MDR) is one of the major obstacles for successful cancer chemotherapy. Over-expression of ATP-binding cassette (ABC) transporters such as MRP1/ABCC1 has been suggested to cause MDR. In this study, we explored the distribution frequencies of four common single nucleotide polymorphisms (SNPs) of MRP1/ABCC1 in a mainland Chinese population and investigated whether these SNPs affect the expression and function of the MRP1/ABCC1. We found that the allelic frequencies of Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A) and Arg1058Gln (3173G>A) in mainland Chinese were 0.5%, 1.4%, 5.8% and 0.5%, respectively. These four SNPs were recreated by site-directed mutagenesis and tested for their effect on MRP1/ABCC1 expression and MDR function in HEK293 and CHO-K1 cells lines. We found that none of these mutations had any effect on MRP1/ABCC1 expression and trafficking, but that Arg723Gln mutation significantly reduced MRP1/ABCC1-mediated resistance to daunorubicin, doxorubicin, etoposide, vinblastine and vincristine. The Cys43Ser mutation did not affect all tested drugs resistance. On the other hand, the Thr73Ile mutation reduced resistance to methotrexate and etoposide while the Arg1058Gln mutation increased the response of two anthracycline drugs and etoposide in HEK293 and CHO-K1 cells as well as vinblastine and methotrexate in CHO-K1 cells. We conclude that the allelic frequency of the Arg723Gln mutation is relatively higher than other SNPs in mainland Chinese population and therefore this mutation significantly reduces MRP1/ABCC1 activity in MDR. PMID:19214144

Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands.

PubMed

Kish, William S; Roach, Matthew K; Sachi, Hiroyuki; Naik, Amith D; Menegatti, Stefano; Carbonell, Ruben G

2018-05-15

Prior work described the identification and characterization of erythropoietin-binding cyclic peptides SLFFLH, VVFFVH, FSLLHH and FSLLSH (all of the form cyclo[(N α -Ac)Dap(A)-X 1 -X 6 -AE], wherein X 1 -X 6 is the listed sequences). In this work, the peptide ligands were synthesized on Toyopearl chromatographic resins and utilized for purifying recombinant human erythropoietin (rHuEPO) from complex sources. Elution buffer pH and composition were optimized to maximize the recovery of standard rHuEPO from the peptide resins. The peptide-based adsorbents were employed for separating rHuEPO from a mixture of albumin, myoglobin, and IgG to examine their selectivity. When using FSLLHH, the inclusion of low amounts of surfactants in the wash and elution buffers facilitated the recovery of rHuEPO with high yield and purity. Specifically, FSLLSH and VVFFVH afforded the most efficient separation of rHuEPO, with yield and purity of 85% and 95-97%, respectively. The affinity resins were also utilized to purify rHuEPO from spiked CHO cell culture fluid. In particular, FSLLSH provided the most successful separation from CHO, with yield and purity above 90%, and 1.0 log 10 reduction of host cell proteins. The influence of conductivity and pH in the CHO-rHuEPO load was investigated. Finally, FSLLSH-based resins were used to purify rHuEPO spiked into a Pichia pastoris cell culture fluid, resulting in product yield and purity of 96% and 84%, respectively, and 1.3 log 10 reduction of host DNA. These results compare well with values obtained using wheat germ agglutinin agarose and clearly indicate the potential of the cyclic peptide resins as a viable tool for rHuEPO purification. Copyright © 2018 Elsevier B.V. All rights reserved.

De novo amplification within a silent human cholinesterase gene in a family subjected to prolonged exposure to organophosphorus insecticides

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

Prody, C.A.; Dreyfus, P.; Soreq, H.

1989-01-01

A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing silent CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorus insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the onion skin model, based on amplification of genesmore » in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where the authors previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. These findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor.« less

Human Metapneumovirus (HMPV) Binding and Infection Are Mediated by Interactions between the HMPV Fusion Protein and Heparan Sulfate

PubMed Central

Chang, Andres; Masante, Cyril; Buchholz, Ursula J.

2012-01-01

Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require β1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans. PMID:22238303

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. Based on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

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

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.

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

NASA Astrophysics Data System (ADS)

Saren, Gaowa; Zhao, Yonggang

2009-05-01

A full length amphioxus cDNA, encoding a novel phosducin-like protein ( Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/ Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

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

Fang Qing; Yang Lin; Zhu Weijun

2005-05-10

Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene.more » Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.« less

Cytotoxicities and genotoxicities of cements based on calcium silicate and of dental formocresol.

PubMed

Ko, Hyunjung; Jeong, Youngdan; Kim, Miri

2017-03-01

Increasing interest is being paid to the toxicities of dental materials. The purpose of this study was to determine the cytotoxicities and genotoxicities of endodontic compounds to Chinese hamster ovary (CHO-K1) reproductive cells. Cultured CHO-K1 cells were treated with dental formocresol, two types of calcium hydroxide paste, and two types of mineral trioxide aggregate cement for 24h. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed on each culture, and the micronucleus frequency was determined by performing a micronucleus assay. Alkaline comet assay and γ-H2AX immunofluorescence assay were used to detect DNA damage. Out of the five materials tested, only dental formocresol significantly increased DNA damage. The mineral trioxide aggregate cements based on calcium silicate were not found to be potentially genotoxic. The data suggest that dental formocresol should not be recommended for use in vital pulp therapy on young teeth. Copyright © 2017 Elsevier B.V. All rights reserved.

Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

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

Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel

2007-09-21

The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount ofmore » AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.« less

Hippocampal metabolic dysfunction in juvenile myoclonic epilepsy: 3D multivoxel spectroscopy study.

PubMed

Ristić, Aleksandar J; Ostojić, Jelena; Kozić, Duško; Vojvodić, Nikola M; Popović, Ljubica M; Janković, Slavko; Baščarević, Vladimir; Sokić, Dragoslav V

2011-06-15

To investigate the metabolic differences in hippocampi of patients with juvenile myoclonic epilepsy (JME) and healthy controls using magnetic resonance spectroscopy (MRS). A 3D multivoxel SE 135 MRS study on 1.5 T scanner of both hippocampi was performed in 17 patients with JME and normal brain MRI and in 19 age and sex matched controls. Three dominant signals were measured: Choline (Cho), Creatine (tCr) and N-Acetylaspartate (NAA) and expressed as ratios of Cho:tCr, NAA:tCr, NAA:Cho and NAA:(Cho+tCr). Metabolite ratios in head, body and tail of each hippocampus in the JME group of patients were compared with ratios from corresponding structures in the control group. We found a significant difference in metabolite ratios of both hippocampi between the JME and the control groups. We detected significant differences of Cho:tCr in the head, NAA:tCr in the head, body and tail, NAA:Cho and NAA:(Cho+tCr) in the body and tail of the left hippocampus, and NAA:Cho and NAA:(Cho+tCr) in the body and tail of the right hippocampus. Although not previously recognized as a part of the epileptogenic network, our results suggest that the hippocampus, well recognized as a key player in focal epilepsies, may have a certain role in the pathogenesis of JME. Copyright © 2010. Published by Elsevier B.V.

Randomized clinical trial of the effect of preoperative oral carbohydrate treatment on postoperative whole-body protein and glucose kinetics.

PubMed

Svanfeldt, M; Thorell, A; Hausel, J; Soop, M; Rooyackers, O; Nygren, J; Ljungqvist, O

2007-11-01

Preoperative oral carbohydrate (CHO) reduces postoperative insulin resistance. In this randomized trial, the effect of CHO on postoperative whole-body protein turnover was studied. Glucose and protein kinetics ([6,6(2)H(2)]D-glucose, [(2)H(5)]phenylalanine, [(2)H(2)]tyrosine and [(2)H(4)]tyrosine) and substrate oxidation (indirect calorimetry) were studied at baseline and during hyperinsulinaemic normoglycaemic clamping before and on the first day after colorectal resection. Fifteen patients were randomized to receive a preoperative beverage with high (125 mg/ml) or low (25 mg/ml) CHO content. Three patients were excluded after the intervention, leaving six patients in each group. After surgery whole-body protein balance did not change in the high oral CHO group, whereas it was more negative in the low oral CHO group after surgery at baseline (P = 0.003) and during insulin stimulation (P = 0.005). Insulin-stimulated endogenous glucose release was similar before and after surgery in the high oral CHO group, but was higher after surgery in the low oral CHO group (P = 0.013) and compared with the high oral CHO group (P = 0.044). Whole-body protein balance and the suppressive effect of insulin on endogenous glucose release are better maintained when patients receive a CHO-rich beverage before surgery. Copyright (c) 2007 British Journal of Surgery Society Ltd.

The factors affecting on estimation of carbohydrate content of meals in carbohydrate counting

PubMed Central

Kawamura, Tomoyuki; Takamura, Chihiro; Hirose, Masakazu; Hashimoto, Tomomi; Higashide, Takashi; Kashihara, Yoneo; Hashimura, Kayako; Shintaku, Haruo

2015-01-01

Abstract. The objective of this study was to identify factors affecting on errors in carbohydrate (CHO) content estimation during CHO counting. Thirty-seven type 1 diabetes patients and 22 of their parents and 28 physicians/dieticians were enrolled in this study. CHO counting was counted in “Carb”, with 1 Carb defined as 10 g of CHO. To evaluate the accuracy of CHO counting, 80 real-size photographs of cooked meals were presented to the subjects for Carb estimation. Carbs tended to be overestimated for foods containing relatively small amounts of Carbs. On the other hands, Carbs tended to be underestimated for foods with higher than 6 Carbs. Accurate estimation of the Carbs in food containing a large amount of rice was particularly difficult even in the subjects having the CHO counting experience. The Carb contents of high-calorie foods such as meats, fried foods, and desserts tended to be overestimated. This error was smaller in subjects having the CHO counting experience. In conclusion, misunderstanding of high-calorie dishes containing high amounts of CHO was observed in inexperienced subjects, indicating the efficacy of the current methodology of CHO counting. On the other hand it was difficult even for experienced subjects to assess the amount of seasoned rice, suggesting the need for a new methodology for accurate estimation. PMID:26568656

AFRRI Reports, Fourth Quarter 1991

DTIC Science & Technology

1992-01-01

Gallin, E. K. Heat induces intracellular acidification in human A-431 cells : role of Na’-H’ exchange and metabolism. SR91-49: MacVittie, T. J., Monroy, R... induced release of DNA front agarose plugs treated with Not I restriction enzyme As above, wild-type and strain 112 cells were exposed to 2 kGy and...times. Ager and co-workers (1990) found that following irradiation of CHO-KI cells , replication forks showed reduced PFGE- induced migration out of the

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.

Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner

PubMed Central

Kurita-Ochiai, Tomoko; Ochiai, Kuniyasu; Fukushima, Kazuo

2001-01-01

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis. PMID:11238216

The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity

PubMed Central

Froese, Sean; Dai, Feihan F.; Robitaille, Mélanie; Bhattacharjee, Alpana; Huang, Xinyi; Jia, Weiping; Angers, Stéphane; Wheeler, Michael B.; Wei, Li

2015-01-01

Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D. PMID:26075596

Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres.

PubMed

Morrish, Tammy A; Garcia-Perez, José Luis; Stamato, Thomas D; Taccioli, Guillermo E; Sekiguchi, JoAnn; Moran, John V

2007-03-08

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise approximately 17% of human DNA. The average human genome contains approximately 80-100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (EN(i)) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair. Here we have characterized EN(i) retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, approximately 30% of EN(i) retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among EN(i) retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated EN(i) retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of EN(i) retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends. Thus, we propose that EN(i) retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.

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

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.

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.

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

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.

Developing global regression models for metabolite concentration prediction regardless of cell line.

PubMed

André, Silvère; Lagresle, Sylvain; Da Sliva, Anthony; Heimendinger, Pierre; Hannas, Zahia; Calvosa, Éric; Duponchel, Ludovic

2017-11-01

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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...

Neurometabolite Alterations Associated With Cognitive Performance in Perinatally HIV-Infected Children.

PubMed

Van Dalen, Yvonne W; Blokhuis, Charlotte; Cohen, Sophie; Ter Stege, Jacqueline A; Teunissen, Charlotte E; Kuhle, Jens; Kootstra, Neeltje A; Scherpbier, Henriette J; Kuijpers, Taco W; Reiss, Peter; Majoie, Charles B L M; Caan, Matthan W A; Pajkrt, Dasja

2016-03-01

Despite treatment with combination antiretroviral therapy (cART), cognitive impairment is still observed in perinatally HIV-infected children. We aimed to evaluate potential underlying cerebral injury by comparing neurometabolite levels between perinatally HIV-infected children and healthy controls. This cross-sectional study evaluated neurometabolites, as measured by Magnetic Resonance Spectroscopy (MRS), in perinatally HIV-infected children stable on cART (n = 26) and healthy controls (n = 36).Participants were included from a cohort of perinatally HIV-infected children and healthy controls, matched group-wise for age, gender, ethnicity, and socio-economic status. N-acetylaspartate (NAA), glutamate (Glu), myo-inositol (mI), and choline (Cho) levels were studied as ratios over creatine (Cre). Group differences and associations with HIV-related parameters, cognitive functioning, and neuronal damage markers (neurofilament and total Tau proteins) were determined using age-adjusted linear regression analyses.HIV-infected children had increased Cho:Cre in white matter (HIV-infected = 0.29 ± 0.03; controls = 0.27 ± 0.03; P value = 0.045). Lower nadir CD4+ T-cell Z-scores were associated with reduced neuronal integrity markers NAA:Cre and Glu:Cre. A Centers for Disease Control and Prevention (CDC) stage C diagnosis was associated with higher glial markers Cho:Cre and mI:Cre. Poorer cognitive performance was mainly associated with higher Cho:Cre in HIV-infected children, and with lower NAA:Cre and Glu:Cre in healthy controls. There were no associations between neurometabolites and neuronal damage markers in blood or CSF.Compared to controls, perinatally HIV-infected children had increased Cho:Cre in white matter, suggestive of ongoing glial proliferation. Levels of several neurometabolites were associated with cognitive performance, suggesting that MRS may be a useful method to assess cerebral changes potentially linked to cognitive outcomes.

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

PubMed

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

2016-04-14

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

Energy substrate utilization with and without exogenous carbohydrate intake in boys and men exercising in the heat.

PubMed

Leites, Gabriela T; Cunha, Giovani S; Chu, Lisa; Meyer, Flavia; Timmons, Brian W

2016-11-01

Little is known about energy yield during exercise in the heat in boys compared with men. To investigate substrate utilization with and without exogenous carbohydrate (CHO exo ) intake, seven boys [11.2 ± 0.2 (SE) yr] and nine men (24.0 ± 1.1 yr) cycled (4 × 20-min bouts) at a fixed metabolic heat production (Ḣ p ) per unit body mass (6 W/kg) in a climate chamber (38°C and 50% relative humidity), on two occasions. Participants consumed a 13 C-enriched 8% CHO beverage (CARB) or placebo beverage (CONT) in a double-blinded, counterbalanced manner. Substrate utilization was calculated for the last 60 min of exercise. CHO exo oxidation rate (2.0 ± 0.3 vs. 2.5 ± 0.2 mg·kg fat-free mass -1 ·min -1 , P = 0.02) and CHO exo oxidation efficiency (12.8 ± 0.6 vs. 16.0 ± 0.9%, P = 0.01) were lower in boys compared with men exercising in the heat. Total carbohydrate (CHO total ), endogenous CHO (CHO endo ), and total fat (Fat total ) remained stable in boys and men (P > 0.05) during CARB, whereas CHO total oxidation rate decreased (P < 0.001) and Fat total oxidation rate increased over time similarly in boys and men during CONT (P < 0.001). The relative contribution of CHO exo to total energy yield increased over time in both groups (P < 0.001). In conclusion, endogenous substrate metabolism and the relative contribution of fuels to total energy yield were not different between groups. The ingestion of a CHO beverage during exercise in the heat may be as beneficial for boys as men to spare endogenous substrate. Copyright © 2016 the American Physiological Society.

Combined carbohydrate-protein supplementation improves competitive endurance exercise performance in the heat.

PubMed

Cathcart, Andrew J; Murgatroyd, Scott R; McNab, Alison; Whyte, Laura J; Easton, Chris

2011-09-01

Laboratory-based studies have demonstrated that adding protein (PRO) to a carbohydrate (CHO) supplement can improve thermoregulatory capacity, exercise performance and recovery. However, no study has investigated these effects in a competitive sporting context. This study assessed the effects of combined CHO-PRO supplementation on physiological responses and exercise performance during 8 days of strenuous competition in a hot environment. Twenty-eight cyclists participating in the TransAlp mountain bike race were randomly assigned to fitness-matched placebo (PLA 76 g L(-1) CHO) or CHO-PRO (18 g L(-1) PRO, 72 g L(-1) CHO) groups. Participants were given enough supplements to allow ad libitum consumption. Physiological and anthropometric variables were recorded pre- and post-exercise. Body mass decreased significantly from race stage 1 to 8 in the PLA group (-0.75 ± 0.22 kg, P = 0.01) but did not change in the CHO-PRO group (0.42 ± 0.42 kg, P = 0.35). Creatine kinase concentration and muscle soreness were substantially elevated during the race, but were not different between groups (P = 0.82, P = 0.44, respectively). Urine osmolality was significantly higher in the CHO-PRO versus the PLA group (P = 0.04) and the rise in tympanic temperature from pre- to post-exercise was significantly less in CHO-PRO versus PLA (P = 0.01). The CHO-PRO group also completed the 8 stages significantly quicker than the PLA group (2,277 ± 127 vs. 2,592 ± 68 min, respectively, P = 0.02). CHO-PRO supplementation therefore appears to prevent body mass loss, enhance thermoregulatory capacity and improve competitive exercise performance despite no effect on muscle damage.

The effect of carbohydrate ingestion on performance during a simulated soccer match.

PubMed

Goedecke, Julia H; White, Nicholas J; Chicktay, Waheed; Mahomed, Hafsa; Durandt, Justin; Lambert, Michael I

2013-12-16

This study investigated how performance was affected after soccer players, in a postprandial state, ingested a 7% carbohydrate (CHO) solution compared to a placebo (0% CHO) during a simulated soccer match. Using a double-blind placebo-controlled design, 22 trained male league soccer players (age: 24 ± 7 years, wt: 73.4 ± 12.0 kg, VO2max: 51.8 ± 4.3 mL O2/kg/min) completed two trials, separated by 7 days, during which they ingested, in random order, 700 mL of either a 7% CHO or placebo drink during a simulated soccer match. Ratings of perceived exertion (RPE), agility, timed and run to fatigue were measured during the trials. Change in agility times was not altered by CHO vs. placebo ingestion (0.57 ± 1.48 vs. 0.66 ± 1.00, p = 0.81). Timed runs to fatigue were 381 ± 267 s vs. 294 ± 159 s for the CHO and placebo drinks, respectively (p = 0.11). Body mass modified the relationship between time to fatigue and drink ingestion (p = 0.02 for drink × body mass), such that lower body mass was associated with increased time to fatigue when the players ingested CHO, but not placebo. RPE values for the final stage of the simulated soccer match were 8.5 ± 1.7 and 8.6 ± 1.5 for the CHO and placebo drinks respectively (p = 0.87). The group data showed that the 7% CHO solution (49 g CHO) did not significantly improve performance during a simulated soccer match in league soccer players who had normal pre-match nutrition. However, when adjusting for body mass, increasing CHO intake was associated with improved time to fatigue during the simulated soccer match.

The effect of high vs. low carbohydrate diets on distances covered in soccer.

PubMed

Souglis, Athanasios G; Chryssanthopoulos, Costas I; Travlos, Antonios K; Zorzou, Amalia E; Gissis, Ioannis T; Papadopoulos, Christos N; Sotiropoulos, Aristomenis A

2013-08-01

The purpose of this study was to compare the distances covered during a 11-a-side soccer match after players had consumed either a high carbohydrate (CHO) or a low CHO diet. Twenty-two male professional soccer players formed 2 teams (A and B), of similar age, body characteristics, and training experience. The 2 teams played against each other twice with a week interval between. For 3.5 days before the first match, the players of team A followed a high CHO diet that provided 8 g CHO per kg body mass (BM) (HC), whereas team B players followed a low CHO diet that provided 3 g CHO per kg BM (LC) for the same time period. Before the second match the dietary treatment was reversed and followed for the same time period. Training during the study was controlled, and distances covered were measured using global positioning system technology. Every player covered a greater total distance in HC compared with the distance covered in LC (HC: 9,380 ± 98 m vs. LC: 8,077 ± 109 m; p < 0.01). All distances covered from easy jogging (7.15 km·h-1) to sprinting (24.15 km·h-1) were also higher in HC compared with LC (p < 0.01). When players followed the HC treatment, they won the match (team A vs. team B: 3-1 for the first game and 1-2 for the second game). The HC diet probably helped players to cover a greater distance compared with LC. Soccer players should avoid eating a low (3 g CHO per kg BM) CHO diet 3-4 days before an important soccer match and have a high CHO intake that provides at least 8 g CHO per kg BM.

Intake of Protein Plus Carbohydrate during the First Two Hours after Exhaustive Cycling Improves Performance the following Day.

PubMed

Rustad, Per I; Sailer, Manuela; Cumming, Kristoffer T; Jeppesen, Per B; Kolnes, Kristoffer J; Sollie, Ove; Franch, Jesper; Ivy, John L; Daniel, Hannelore; Jensen, Jørgen

2016-01-01

Intake of protein immediately after exercise stimulates protein synthesis but improved recovery of performance is not consistently observed. The primary aim of the present study was to compare performance 18 h after exhaustive cycling in a randomized diet-controlled study (175 kJ·kg(-1) during 18 h) when subjects were supplemented with protein plus carbohydrate or carbohydrate only in a 2-h window starting immediately after exhaustive cycling. The second aim was to investigate the effect of no nutrition during the first 2 h and low total energy intake (113 kJ·kg(-1) during 18 h) on performance when protein intake was similar. Eight endurance-trained subjects cycled at 237±6 Watt (~72% VO2max) until exhaustion (TTE) on three occasions, and supplemented with 1.2 g carbohydrate·kg(-1)·h(-1) (CHO), 0.8 g carbohydrate + 0.4 g protein·kg(-1)·h(-1) (CHO+PRO) or placebo without energy (PLA). Intake of CHO+PROT increased plasma glucose, insulin, and branch chained amino acids, whereas CHO only increased glucose and insulin. Eighteen hours later, subjects performed another TTE at 237±6 Watt. TTE was increased after intake of CHO+PROT compared to CHO (63.5±4.4 vs 49.8±5.4 min; p<0.05). PLA reduced TTE to 42.8±5.1 min (p<0.05 vs CHO). Nitrogen balance was positive in CHO+PROT, and negative in CHO and PLA. In conclusion, performance was higher 18 h after exhaustive cycling with intake of CHO+PROT compared to an isocaloric amount of carbohydrate during the first 2 h post exercise. Intake of a similar amount of protein but less carbohydrate during the 18 h recovery period reduced performance.

Can children with Type 1 diabetes and their caregivers estimate the carbohydrate content of meals and snacks?

PubMed

Smart, C E; Ross, K; Edge, J A; King, B R; McElduff, P; Collins, C E

2010-03-01

Carbohydrate (CHO) counting allows children with Type 1 diabetes to adjust mealtime insulin dose to carbohydrate intake. Little is known about the ability of children to count CHO and whether a particular method for assessing CHO quantity is better than others. We investigated how accurately children and their caregivers estimate carbohydrate, and whether counting in gram increments improves accuracy compared with CHO portions or exchanges. One hundred and two children and adolescents (age range 8.3-18.1 years) on intensive insulin therapy and 110 caregivers independently estimated the CHO content of 17 standardized meals (containing 8-90 g CHO), using whichever method of carbohydrate quantification they had been taught (gram increments, 10-g portions or 15-g exchanges). Seventy-three per cent (n = 2530) of all estimates were within 10-15 g of actual CHO content. There was no relationship between the mean percentage error and method of carbohydrate counting or glycated haemoglobin (HbA(1c)) (P > 0.05). Mean gram error and meal size were negatively correlated (r = -0.70, P < 0.0001). The longer children had been CHO counting the greater the mean percentage error (r = 0.173, P = 0.014). Core foods in non-standard quantities were most frequently inaccurately estimated, while individually labelled foods were most often accurately estimated. Children with Type 1 diabetes and their caregivers can estimate the carbohydrate content of meals with reasonable accuracy. Teaching CHO counting in gram increments did not improve accuracy compared with CHO portions or exchanges. Large meals tended to be underestimated and snacks overestimated. Repeated age-appropriate education appears necessary to maintain accuracy in carbohydrate estimations.

The Effect of Carbohydrate Ingestion on Performance during a Simulated Soccer Match

PubMed Central

Goedecke, Julia H.; White, Nicholas J.; Chicktay, Waheed; Mahomed, Hafsa; Durandt, Justin; Lambert, Michael I.

2013-01-01

Aim: This study investigated how performance was affected after soccer players, in a postprandial state, ingested a 7% carbohydrate (CHO) solution compared to a placebo (0% CHO) during a simulated soccer match. Methods: Using a double-blind placebo-controlled design, 22 trained male league soccer players (age: 24 ± 7 years, wt: 73.4 ± 12.0 kg, VO2max: 51.8 ± 4.3 mL O2/kg/min) completed two trials, separated by 7 days, during which they ingested, in random order, 700 mL of either a 7% CHO or placebo drink during a simulated soccer match. Ratings of perceived exertion (RPE), agility, timed and run to fatigue were measured during the trials. Results: Change in agility times was not altered by CHO vs. placebo ingestion (0.57 ± 1.48 vs. 0.66 ± 1.00, p = 0.81). Timed runs to fatigue were 381 ± 267 s vs. 294 ± 159 s for the CHO and placebo drinks, respectively (p = 0.11). Body mass modified the relationship between time to fatigue and drink ingestion (p = 0.02 for drink × body mass), such that lower body mass was associated with increased time to fatigue when the players ingested CHO, but not placebo. RPE values for the final stage of the simulated soccer match were 8.5 ± 1.7 and 8.6 ± 1.5 for the CHO and placebo drinks respectively (p = 0.87). Conclusions: The group data showed that the 7% CHO solution (49 g CHO) did not significantly improve performance during a simulated soccer match in league soccer players who had normal pre-match nutrition. However, when adjusting for body mass, increasing CHO intake was associated with improved time to fatigue during the simulated soccer match. PMID:24352094

Quantitative mapping of total choline in healthy human breast using proton echo planar spectroscopic imaging (PEPSI) at 3 Tesla.

PubMed

Zhao, Chenguang; Bolan, Patrick J; Royce, Melanie; Lakkadi, Navneeth; Eberhardt, Steven; Sillerud, Laurel; Lee, Sang-Joon; Posse, Stefan

2012-11-01

To quantitatively measure tCho levels in healthy breasts using Proton-Echo-Planar-Spectroscopic-Imaging (PEPSI). The two-dimensional mapping of tCho at 3 Tesla across an entire breast slice using PEPSI and a hybrid spectral quantification method based on LCModel fitting and integration of tCho using the fitted spectrum were developed. This method was validated in 19 healthy females and compared with single voxel spectroscopy (SVS) and with PRESS prelocalized conventional Magnetic Resonance Spectroscopic Imaging (MRSI) using identical voxel size (8 cc) and similar scan times (∼7 min). A tCho peak with a signal to noise ratio larger than 2 was detected in 10 subjects using both PEPSI and SVS. The average tCho concentration in these subjects was 0.45 ± 0.2 mmol/kg using PEPSI and 0.48 ± 0.3 mmol/kg using SVS. Comparable results were obtained in two subjects using conventional MRSI. High lipid content in the spectra of nine tCho negative subjects was associated with spectral line broadening of more than 26 Hz, which made tCho detection impossible. Conventional MRSI with PRESS prelocalization in glandular tissue in two of these subjects yielded tCho concentrations comparable to PEPSI. The detection sensitivity of PEPSI is comparable to SVS and conventional PRESS-MRSI. PEPSI can be potentially used in the evaluation of tCho in breast cancer. A tCho threshold concentration value of ∼0.7 mmol/kg might be used to differentiate between cancerous and healthy (or benign) breast tissues based on this work and previous studies. Copyright © 2012 Wiley Periodicals, Inc.

Quantitative Mapping of Total Choline in Healthy Human Breast Using Proton Echo Planar Spectroscopic Imaging (PEPSI) at 3 Tesla

PubMed Central

Zhao, Chenguang; Bolan, Patrick J.; Royce, Melanie; Lakkadi, Navneeth; Eberhardt, Steven; Sillerud, Laurel; Lee, Sang-Joon; Posse, Stefan

2012-01-01

Purpose To quantitatively measure tCho levels in healthy breasts using Proton-Echo-Planar-Spectroscopic-Imaging (PEPSI). Material and Methods The 2-dimensional mapping of tCho at 3 Tesla across an entire breast slice using PEPSI and a hybrid spectral quantification method based on LCModel fitting and integration of tCho using the fitted spectrum were developed. This method was validated in 19 healthy females and compared with single voxel spectroscopy (SVS) and with PRESS prelocalized conventional Magnetic Resonance Spectroscopic Imaging (MRSI) using identical voxel size (8 cc) and similar scan times (~7 min). Results A tCho peak with a signal to noise ratio larger than 2 was detected in 10 subjects using both PEPSI and SVS. The average tCho concentration in these subjects was 0.45 ± 0.2 mmol/kg using PEPSI and 0.48±0.3 mmol/kg using SVS. Comparable results were obtained in 2 subjects using conventional MRSI. High lipid content in the spectra of 9 tCho negative subjects was associated with spectral line broadening of more than 26 Hz, which made tCho detection impossible. Conventional MRSI with PRESS prelocalization in glandular tissue in two of these subjects yielded tCho concentrations comparable to PEPSI. Conclusion The detection sensitivity of PEPSI is comparable to SVS and conventional PRESS-MRSI. PEPSI can be potentially used in the evaluation of tCho in breast cancer. A tCho threshold concentration value of ~0.7mmol/kg might be used to differentiate between cancerous and healthy (or benign) breast tissues based on this work and previous studies. PMID:22782667

The Effect of a Moderately Low and High Carbohydrate Intake on Crossfit Performance

PubMed Central

ESCOBAR, KURT A.; MORALES, JACOBO; VANDUSSELDORP, TRISHA A.

2016-01-01

CrossFit is a metabolically demanding strength and conditioning method which performance may benefit from a carbohydrate (CHO)-rich diet. This study investigated the effect of three consecutive days of high CHO intake on CrossFit performance and corresponding metabolically -related variables in strength trained individuals. Eighteen subjects with a CHO intake of <6 g/kg/day were randomly assigned into a CHO (n = 9) or control (C) group (n =9) and underwent a 9-day training protocol. During days 1, 5, and 9, performance was measured as repetitions completed during a 12 minute CrossFit workout. Oxygen consumption (VO2), respiratory exchange ratio (RER), and blood lactate (BL) were also measured. Days 6–8, the CHO group increased CHO intake from <6 g/kg/day to 6–8 g/kg/day; the C group maintained their current intake of <6 g/kg/day. On days 6 and 7 both groups performed CrossFit workouts followed by a day of rest prior to day 9. There was a significant increase in repetitions completed in both groups in day 9 (vs. means score of day 1 + 5) (p = 0.002), but no differences between C and CHO groups (p = 0.111). However, the CHO group displayed a 15.2 repetition increase (+10.9%) in day 9, compared to 5.7 (+4.2%) by the C group. VO2, RER, and BL were not influenced by the experimental intervention. Our results suggest that the CrossFit-embraced practice of moderately-low CHO diets may be adequate in CHO during short periods of training, however, given the noted trend, extended training periods may be effected. PMID:27766133

The Effect of a Moderately Low and High Carbohydrate Intake on Crossfit Performance.

PubMed

Escobar, Kurt A; Morales, Jacobo; Vandusseldorp, Trisha A

2016-01-01

CrossFit is a metabolically demanding strength and conditioning method which performance may benefit from a carbohydrate (CHO)-rich diet. This study investigated the effect of three consecutive days of high CHO intake on CrossFit performance and corresponding metabolically -related variables in strength trained individuals. Eighteen subjects with a CHO intake of <6 g/kg/day were randomly assigned into a CHO (n = 9) or control (C) group (n =9) and underwent a 9-day training protocol. During days 1, 5, and 9, performance was measured as repetitions completed during a 12 minute CrossFit workout. Oxygen consumption (VO 2 ), respiratory exchange ratio (RER), and blood lactate (BL) were also measured. Days 6-8, the CHO group increased CHO intake from <6 g/kg/day to 6-8 g/kg/day; the C group maintained their current intake of <6 g/kg/day. On days 6 and 7 both groups performed CrossFit workouts followed by a day of rest prior to day 9. There was a significant increase in repetitions completed in both groups in day 9 (vs. means score of day 1 + 5) (p = 0.002), but no differences between C and CHO groups (p = 0.111). However, the CHO group displayed a 15.2 repetition increase (+10.9%) in day 9, compared to 5.7 (+4.2%) by the C group. VO 2 , RER, and BL were not influenced by the experimental intervention. Our results suggest that the CrossFit-embraced practice of moderately-low CHO diets may be adequate in CHO during short periods of training, however, given the noted trend, extended training periods may be effected.

Ingestion of carbohydrate or carbohydrate plus protein does not enhance performance during endurance exercise: a randomized cross-over placebo-controlled clinical trial.

PubMed

Finger, Débora; Lanferdini, Fábio Juner; Farinha, Juliano Boufleur; Brusco, Clarissa Müller; Helal, Lucas; Boeno, Francesco Pinto; Cadore, Eduardo Lusa; Pinto, Ronei Silveira

2018-03-15

Protein (PRO) combined with a carbohydrate (CHO) beverage may have an ergogenic effect on endurance performance. However, evidence regarding its efficacy on similar conditions to athletes' race day is still lacking. To compare the effect of three different nutritional supplementation strategies on performance and muscle recovery in a duathlon protocol. , 13 male athletes (29.7 ± 7.7 years) participated in three simulated Olympic-distance duathlons under three different, randomly assigned, supplementation regimens: carbohydrate drink (CHO, 75 g); isocaloric CHO plus protein drink (CHO+PRO, 60.5 g CHO + 14.5 g PRO); and, placebo drink (PLA), offered during the cycling bout. Blood samples were collected before, immediately after and 24 h after each test for creatine kinase (CK) analysis. Isometric peak torque (PT) was measured before and 24 h after each condition. The primary outcome was the time to complete the last 5km running section (t5km) in a self-selected pace. Statistical differences were considered when p<0.05. There was no difference in t5km between CHO (1270.3 ± 130.5 s) vs. CHO+PRO (1267.2 ± 138.9 s) vs. PLA (1275.4 ± 120 s); p = 0.87; ES ≤ 0.1. Pre-post changes for PT and CK values did not show differences in any of three conditions (p = 0.24, ES ≤ 0.4, p = 0.32, 0.3-1.04). For endurance sports lasting up to 2 h, with a pre-meal containing 1.5 g/kg of CHO, CHO or CHO+PRO supplementation does not offer additional benefits when compared to a PLA in performance and muscle recovery.

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.

Preoperative carbohydrate loading in patients undergoing coronary artery bypass or spinal surgery.

PubMed

Tran, Susan; Wolever, Thomas M S; Errett, Lee E; Ahn, Henry; Mazer, C David; Keith, Mary

2013-08-01

Surgical stress creates a state of insulin resistance which may contribute to the development of hyperglycemia and, subsequently, postoperative complications. Consumption of an oral carbohydrate supplement before surgery may improve insulin sensitivity and reduce hyperglycemia. In this trial, we investigated the effects of carbohydrate supplementation on insulin resistance in coronary artery bypass graft and spinal decompression and fusion surgical patients. Twenty-six patients undergoing coronary artery bypass graft and 12 undergoing spine surgery were randomized to receive 800 mL of an oral carbohydrate supplement the evening before and 400 mL 2 hours before surgery (CHO) or to fasting per standard hospital protocol (FAST). Baseline and postoperative measurements of insulin sensitivity were assessed using the short insulin tolerance test and homeostasis model assessment (HOMA). Interleukin-6, C-reactive protein, and free fatty acid levels were determined at baseline, postoperatively, and 24, 48, and 72 hours after surgery. Adiponectin was measured at baseline. Subjective feelings of well-being were measured immediately before surgery, and intra- and postoperative outcomes were documented. Postoperative insulin sensitivity did not differ significantly between the FAST and CHO groups whether measured by the short insulin tolerance test (rate of disappearance of blood glucose: 0.29%/min vs 0.38%/min; 99% confidence interval [CI] for difference, -0.17 to 0.32, P = 0.41) or HOMA (insulin resistance at values >1: 2.3 vs 3.3; 99% CI for difference, -0.8 to 2.8, P = 0.14). Circulating blood glucose levels after surgery in the CHO group, 6.2 mmol/L, tended to be lower than the FAST group, 6.9 mmol/L (99% CI for difference, -1.7 to 0.25, P = 0.05) and postoperative β-cell function, measured by HOMA-β (impaired β-cell function at values <100%), tended to be higher in the CHO group, 87%, vs 47.5% in the FAST group (99% CI for difference, -9.4 to 88.4), but these differences were not significant. Adiponectin levels were not different between groups at baseline, and levels of free fatty acid, interleukin-6 and C-reactive protein were not affected by treatment. Preoperative carbohydrate loading did not improve postoperative insulin sensitivity. However, the observed postoperative blood glucose levels and β-cell function as well as secondary outcomes warrant further study to reevaluate traditional fasting practices in surgical patients.

Effects of Copper and/or Cholesterol Overload on Mitochondrial Function in a Rat Model of Incipient Neurodegeneration

PubMed Central

Castillo, Omar; de Alaniz, María J. T.; Marra, Carlos A.

2013-01-01

Copper (Cu) and cholesterol (Cho) are both associated with neurodegenerative illnesses in humans and animals models. We studied the effect in Wistar rats of oral supplementation with trace amounts of Cu (3 ppm) and/or Cho (2%) in drinking water for 2 months. Increased amounts of nonceruloplasmin-bound Cu were observed in plasma and brain hippocampus together with a higher concentration of ceruloplasmin in plasma, cortex, and hippocampus. Cu, Cho, and the combined treatment Cu + Cho were able to induce a higher Cho/phospholipid ratio in mitochondrial membranes with a simultaneous decrease in glutathione content. The concentration of cardiolipin decreased and that of peroxidation products, conjugated dienes and lipoperoxides, increased. Treatments including Cho produced rigidization in both the outer and inner mitochondrial membranes with a simultaneous increase in permeability. No significant increase in Cyt C leakage to the cytosol was observed except in the case of cortex from rats treated with Cu and Cho nor were there any significant changes in caspase-3 activity and the Bax/Bcl2 ratio. However, the Aβ(1–42)/(1–40) ratio was higher in cortex and hippocampus. These findings suggest an incipient neurodegenerative process induced by Cu or Cho that might be potentiated by the association of the two supplements. PMID:24363953

Role of teichoic acid choline moieties in the virulence of Streptococcus pneumoniae.

PubMed

Gehre, Florian; Spisek, Radek; Kharat, Arun S; Matthews, Phillip; Kukreja, Anjli; Anthony, Robert M; Dhodapkar, Madhav V; Vollmer, Waldemar; Tomasz, Alexander

2009-07-01

In recent reports it was shown that genetically modified choline-free strains of Streptococcus pneumoniae (D39Cho(-)licA64 and D39ChiplicB31) expressing the type II capsular polysaccharide were virtually avirulent in the murine sepsis model, in sharp contrast to the isogenic and highly virulent strains D39Cho(-) and D39Chip, which have retained the choline residues at their surface. We now demonstrate that this choline-associated virulence is independent of Toll-like receptor 2 recognition. Also, despite the lack of virulence, choline-free strains of S. pneumoniae were able to activate splenic dendritic cells, induce secretion of proinflammatory cytokines, and produce specific protective immunity against subsequent challenge. However, after this transient engagement of the immune system the choline-free bacteria were rapidly cleared from the blood, while the isogenic virulent strain D39Cho(-) continued to grow, accompanied by prolonged expression of cytokines, eventually killing the experimental animals. The critical contribution of choline residues to the virulence potential of pneumococci appears to be the role that these amino alcohol residues play in a pneumococcal immune evasion strategy, the mechanism of which is unknown at the present time.

Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

PubMed Central

LaPolla, R J; Mayne, K M; Davidson, N

1984-01-01

A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

Effects of electromagnetic radiation produced by 3G mobile phones on rat brains: magnetic resonance spectroscopy, biochemical, and histopathological evaluation.

PubMed

Dogan, M; Turtay, M G; Oguzturk, H; Samdanci, E; Turkoz, Y; Tasdemir, S; Alkan, A; Bakir, S

2012-06-01

The effects of electromagnetic radiation (EMR) produced by a third-generation (3G) mobile phone (MP) on rat brain tissues were investigated in terms of magnetic resonance spectroscopy (MRS), biochemistry, and histopathological evaluations. The rats were randomly assigned to two groups: Group 1 is composed of 3G-EMR-exposed rats (n = 9) and Group 2 is the control group (n = 9). The first group was subjected to EMR for 20 days. The control group was not exposed to EMR. Choline (Cho), creatinin (Cr), and N-acetylaspartate (NAA) levels were evaluated by MRS. Catalase (CAT) and glutathione peroxidase (GSH-Px) enzyme activities were measured by spectrophotometric method. Histopathological analyses were carried out to evaluate apoptosis in the brain tissues of both groups. In MRS, NAA/Cr, Cho/Cr, and NAA/Cho ratios were not significantly different between Groups 1 and 2. Neither the oxidative stress parameters, CAT and GSH-Px, nor the number of apoptotic cells were significantly different between Groups 1 and 2. Usage of short-term 3G MP does not seem to have a harmful effect on rat brain tissue.

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

PubMed

Mailloux, Ryan J; Adjeitey, Cyril Nii-Klu; Harper, Mary-Ellen

2010-10-13

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Anthrax Toxin

DTIC Science & Technology

1984-10-26

focused initially on EF because it seemed possible that this component, like cholera toxin, might cause edema in skin through elevation of cellular cAMP...behavior differed from that seen in cells exposed to cholera toxin, where cellular cAMP levels remain elevated upon toxin removal. Studies in CHO cell...LF, the rat bioassay is not likely to be an appropriate system for studying the cellular and molecular mechanisms of action of LF. Therefore, a survey

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.

Determination of the maximum operating range of hydrodynamic stress in mammalian cell culture.

PubMed

Neunstoecklin, Benjamin; Stettler, Matthieu; Solacroup, Thomas; Broly, Hervé; Morbidelli, Massimo; Soos, Miroslav

2015-01-20

Application of quality by design (QbD) requires identification of the maximum operating range for parameters affecting the cell culture process. These include hydrodynamic stress, mass transfer or gradients in dissolved oxygen and pH. Since most of these are affected by the impeller design and speed, the main goal of this work was to identify a maximum operating range for hydrodynamic stress, where no variation of cell growth, productivity and product quality can be ensured. Two scale-down models were developed operating under laminar and turbulent condition, generating repetitive oscillating hydrodynamic stress with maximum stress values ranging from 0.4 to 420Pa, to compare the effect of the different flow regimes on the cells behavior. Two manufacturing cell lines (CHO and Sp2/0) used for the synthesis of therapeutic proteins were employed in this study. For both cell lines multiple process outputs were used to determine the threshold values of hydrodynamic stress, such as cell growth, morphology, metabolism and productivity. They were found to be different in between the cell lines with values equal to 32.4±4.4Pa and 25.2±2.4Pa for CHO and Sp2/0, respectively. Below the measured thresholds both cell lines do not show any appreciable effect of the hydrodynamic stress on any critical quality attribute, while above, cells responded negatively to the elevated stress. To confirm the applicability of the proposed method, the obtained results were compared with data generated from classical small-scale reactors with a working volume of 3L. Copyright © 2014 Elsevier B.V. All rights reserved.

Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro.

PubMed

Angelieri, Fernanda; Marcondes, Joao Paulo C; de Almeida, Danielle Cristina; Salvadori, Daisy M F; Ribeiro, Daniel A

2011-04-01

The purpose of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro. Genotoxicity was assessed by the single cell gel (comet) assay using Chinese hamster ovary (CHO) cells. The following orthodontic metallic brackets were used: Morelli (Sorocaba, Brazil); Abzil (São José do Rio Preto, Brazil); Dentaurum (Pforzheim, Germany); and 3M Unitek (Puchheim, Germany). Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride at 0.1 M concentration for 1, 3, 7, 14, 21, 35, and 70 days. CHO cells were exposed to eluates for 30 minutes at 37°C. The negative control was treated with the same solution used for corrosion process for 30 minutes at 37°C. Independent positive control was performed with methyl methanesulfonate (MMS) (Sigma Aldrich, St. Louis, Mo) at 1 ug/mL for 1 hour. None of the eluates was found to exhibit genotoxicity, regardless of the different commercial brands of orthodontic appliance used. In summary, our results indicate corrosion eluates obtained from orthodontic brackets do not induce genetic damage as assessed by single cell gel (comet) assay. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Quality and cost assessment of a recombinant antibody fragment produced from mammalian, yeast and prokaryotic host cells: A case study prior to pharmaceutical development.

PubMed

Lebozec, Kristell; Jandrot-Perrus, Martine; Avenard, Gilles; Favre-Bulle, Olivier; Billiald, Philippe

2018-09-25

Monoclonal antibody fragments (Fab) are a promising class of therapeutic agents. Fabs are aglycosylated proteins and so many expression platforms have been developed including prokaryotic, yeast and mammalian cells. However, these platforms are not equivalent in terms of cell line development and culture time, product quality and possibly cost of production that greatly influence the success of a drug candidate's pharmaceutical development. This study is an assessment of the humanized Fab fragment ACT017 produced from two microorganisms (Escherichia coli and Pichia pastoris) and one mammalian cell host (CHO). Following low scale production and Protein L-affinity purification under generic conditions, physico-chemical and functional quality assessments were carried out prior to economic analysis of industrial scale production using a specialized software (Biosolve, Biopharm Services, UK). Results show higher titer production when using E. coli but associated with high heterogeneity of the protein content recovered in the supernatant. We also observed glycoforms of the Fab produced from P. pastoris, while Fab secreted from CHO was the most homogeneous despite a much longer culture time and slightly higher estimated cost of goods. This study may help inform future pharmaceutical development of this class of therapeutic proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

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.

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.

Correlation Between Expression of Recombinant Proteins and Abundance of H3K4Me3 on the Enhancer of Human Cytomegalovirus Major Immediate-Early Promoter.

PubMed

Soo, Benjamin P C; Tay, Julian; Ng, Shirelle; Ho, Steven C L; Yang, Yuansheng; Chao, Sheng-Hao

2017-08-01

Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.

From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors

PubMed Central

Cibiel, Agnes; Nguyen Quang, Nam; Gombert, Karine; Thézé, Benoit; Garofalakis, Anikitos; Ducongé, Frédéric

2014-01-01

Background Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. Methodology/Principal Findings Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. Conclusions/Significance Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application. PMID:24489826

The Role of PD-1 Ligand in Immune Evasion by Breast Cancer

DTIC Science & Technology

2005-05-01

with anti-CD11c-PE and either anti-PD-L1-FITC or anti-PD-L2-FITC and an- alyzed by flow cytometry . 1260 BLOCKADE OF PD-L ON DC ENHANCES T CELL... surface staining. . In order to accomplish these epitope blocking studies, we made 300.19 and CHO cells transfected with PD-L1 or PD-L2. For anti...Key Research Accomplishments 1. PD-L1 but not PD-L2 is highly expressed on the cell surface of breast cancer cells but not on normal breast

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 and are promising approaches for antigen preparation in vaccine development. PMID:25861441

Identification of the receptors for somatostatin (SST) and cortistatin (CST) in chickens and investigation of the roles of cSST28, cSST14, and cCST14 in inhibiting cGHRH1-27NH2-induced growth hormone secretion in cultured chicken pituitary cells.

PubMed

Meng, Fengyan; Huang, Guian; Gao, Shunyu; Li, Juan; Yan, Zhenxin; Wang, Yajun

2014-03-25

Somatostatin receptors (SSTRs) are proposed to mediate the actions of somatostatin (SST) and its related peptide, cortistatin (CST), in vertebrates. However, the identity, functionality, and tissue expression of these receptors remain largely unknown in most non-mammalian vertebrates including birds. In this study, five SSTRs (named cSSTR1, cSSTR2, cSSTR3, cSSTR4, cSSTR5) were cloned from chicken brain by RT-PCR. Using a pGL3-CRE-luciferase reporter system, we demonstrated that activation of each cSSTR expressed in CHO cells by cSST28, cSST14 and cCST14 treatment could inhibit forskolin-induced luciferase activity of CHO cells, indicating the functional coupling of all cSSTRs to Gi protein(s). Interestingly, cSSTR1-4 expressed in CHO cells could be activated by cSST28, cSST14 and cCST14 with high potencies, suggesting that they may function as the receptors common for these peptides. In contrast, cSSTR5 could be potently activated by cSST28 only, indicating that it is a cSST28-specific receptor. Using RT-PCR, wide expression of cSSTRs was detected in chicken tissues including pituitary. In accordance with their expression in pituitary, cSST28, cSST14, and cCST14 were demonstrated to inhibit basal and novel cGHRH1-27NH2-induced GH secretion in cultured chicken pituitary cells dose-dependently (0-10nM) by Western blot analysis, suggesting the involvement of cSSTR(s) common for these peptides in mediating their inhibitory actions. Collectively, our study establishes a molecular basis to elucidate the roles of SST/CST in birds and provide insights into the roles of SST/CST in vertebrates, such as their conserved actions on pituitary. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Use of the Real Time xCelligence System for Purposes of Medical Microbiology.

PubMed

Junka, Adam Feliks; Janczura, Adriana; Smutnicka, Danuta; Mączyńska, Beata; Anna, Secewicz; Nowicka, Joanna; Bartoszewicz, Marzenna; Gościniak, Grażyna

2012-09-28

Roche's xCelligence impedance-measuring instrument is one of a few commercially available systems of such type. According to the best knowledge of authors, instrument was tested so far only for eukaryotic cell research. The aim of this work was to estimate xCELLigence suitability for the microbiological tests, including (i) measurement of morphological changes in eukaryotic cells as a result of bacterial toxin activity, (ii) measurement of bacterial biofilm formation and (iii) impact of antiseptics on the biofilm structure. To test the infuence of bacterial LT enterotoxin on eukaryotic cell lines, Chinese Hamster Ovary (CHO) cell line and reference strain Escherichia coli ATTC 35401 were used. To investigate Roche's instrument ability to measure biofilm formation and impact of antiseptics on its development, Staphylococcus aureus ATTC6538 reference strain was used. The data generated during the experiments indicate excellent ability of xCelligence instrument to detect cytopathic effect caused by bacterial LT endotoxin and to detect staphylococcal biofilm formation. However, interpretation of the results obtained during real-time measurement of antiseptic's bactericidal activity against staphylococcal biofilm, caused many difficulties. xCelligence instrument can be used for real-time monitoring of morphological changes in CHO cells treated with bacterial LT enterotoxin and for real-time measurement of staphylococcal biofilm formation in vitro. Further investigation is necessary to confirm suitability of system to analyze antiseptic's antimicrobial activity against biofilm in vitro.

Effects of carbohydrate combined with caffeine on repeated sprint cycling and agility performance in female athletes

PubMed Central

2014-01-01

Background Caffeine (CAF) has been shown to improve performance during early phase of repeated sprint exercise; however some studies show that CAF also increases the magnitude of physical stress represented by augmented blood lactate, glucose, and cortisol concentrations during latter phase of repeated sprint exercise. No studies have investigated the efficacy of combined carbohydrate (CHO) and CAF consumption during repeated sprint exercise (RSE) in female athletes. Thus, the purpose of this study was to investigate the effects of CAF with CHO supplementation on RSE and agility. Methods Eleven female athletes completed four experimental trials performed 7 d apart in a double-blind, randomized, and counter-balanced crossover design. Treatments included CAF + PLA (placebo), CAF + CHO, PLA + CHO, and PLA + PLA. Participants ingested capsules containing 6 mg · kg−1 of CAF or PLA 60-min prior to RSE, and 0.8 g · kg−1 of CHO solution or PLA immediately before the RSE, which consisted of ten sets of 5 × 4-s sprints on the cycle ergometer with 20-s active recovery. The agility T-test (AT-test) was performed before and after the RSE. Blood samples were acquired to assess glucose, lactate, testosterone, and cortisol. Results During Set 6 of RSE, peak power and mean power were significantly higher in PLA + CHO than those in CAF + PLA and PLA + PLA, respectively (p < .05). Total work was significantly increased by 4.8% and 5.9% with PLA + CHO than those of CAF + CHO and CAF + PLA during Set 3. PLA + CHO also increased total work more than CAF + PLA and PLA + PLA did during Set 6 (p < .05). No significant differences in AT-test performance either before or after the RSE were occurred among treatments (p > .05). Blood lactate and glucose concentrations were significantly higher under CAF + CHO, CAF + PLA, and PLA + CHO versus PLA + PLA (p < .05), but no differences in testosterone or cortisol levels were found (p > .05). Conclusions Findings indicate that CAF + PLA or CAF + CHO ingestion did not improve repeated sprint performance with short rest intervals or agility. However, CHO ingested immediately prior to exercise provided a small but significant benefit on RSE performance in female athletes. PMID:24855458

Radical routes to interstellar glycolaldehyde. The possibility of stereoselectivity in gas-phase polymerization reactions involving CH(2)O and ˙CH(2)OH.

PubMed

Wang, Tianfang; Bowie, John H

2010-10-21

A previous report that the interstellar molecule glycolaldehyde (HOCH(2)CHO) can be made from hydroxymethylene (HOCH:) and formaldehyde has been revisited at the CCSD(T)/6-311++G(3df,2p)//MP2/6-311++G(3df,2p) level of theory. This reaction competes with the formation of acetic acid and methylformate, molecules which have also been detected in interstellar clouds. Other possible modes of formation of glycolaldehyde by radical/radical reactions have been shown to be viable theoretically as follows: HO˙+˙CH2CHO -->HOCH2CHO [ΔG(Γ)(298K)=-303kJ mol⁻¹] HOCH2˙+˙CHO-->HOCH2CHO (-259kJ mol⁻¹). The species in these two processes are known interstellar molecules. Key radicals ˙CH(2)CHO and ˙CH(2)OH in these sequences have been shown to be stable for the microsecond duration of neutralization/reionization experiments in the dual collision cells of a VG ZAB 2HF mass spectrometer. The polymerization reaction HOCH(2)CH˙OH + nCH(2)O → HOCH(2)[CH(OH)](n)˙CHOH (n = 1 to 3) has been studied theoretically and shown to be energetically feasible, as is the cyclization reaction of HOCH(2)[(CH(2)OH)(4)]˙CHOH (in the presence of one molecule of water at the reacting centre) to form glucose. The probability of such a reaction sequence is small even if polymerization were to occur in interstellar ice containing a significant concentration of CH(2)O. The large number of stereoisomers produced by such a reaction sequence makes the formation of a particular sugar, again for example glucose, an inefficient synthesis. The possibility of stereoselectivity occurring during the polymerization was investigated for two diastereoisomers of HOCH(2)[(CHOH)](2)˙CHOH. No significant difference was found in the transition state energies for addition of CH(2)O to these two diastereoisomers, but a barrier difference of 12 kJ mol(-1) was found for the H transfer reactions ˙OCH(2)[(CHOH)](2)CH(2)OH → HOCH(2)[(CHOH)(2)˙CHOH of the two diastereoisomers.

The Caenorhabditis elegans choline transporter CHO-1 sustains acetylcholine synthesis and motor function in an activity-dependent manner.

PubMed

Matthies, Dawn Signor; Fleming, Paul A; Wilkes, Don M; Blakely, Randy D

2006-06-07

Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.

Oxidative stress and neurodegeneration: The possible contribution of quinone reductase 2.

PubMed

Cassagnes, Laure-Estelle; Chhour, Monivan; Pério, Pierre; Sudor, Jan; Gayon, Régis; Ferry, Gilles; Boutin, Jean A; Nepveu, Françoise; Reybier, Karine

2018-05-20

There is increasing evidence that oxidative stress is involved in the etiology and pathogenesis of neurodegenerative disorders. Overproduction of reactive oxygen species (ROS) is due in part to the reactivity of catecholamines, such as dopamine, adrenaline, and noradrenaline. These molecules are rapidly converted, chemically or enzymatically, into catechol-quinone and then into highly deleterious semiquinone radicals after 1-electron reduction in cells. Notably, the overexpression of dihydronicotinamide riboside:quinone oxidoreductase (QR2) in Chinese hamster ovary (CHO) cells increases the production of ROS, mainly superoxide radicals, when it is exposed to exogenous catechol-quinones (e.g. dopachrome, aminochrome, and adrenochrome). Here we used electron paramagnetic resonance analysis to demonstrate that the phenomenon observed in CHO cells is also seen in human leukemic cells (K562 cells) that naturally express QR2. Moreover, by manipulating the level of QR2 in neuronal cells, including immortalized neuroblast cells and ex vivo neurons isolated from QR2 knockout animals, we showed that there is a direct relationship between QR2-mediated quinone reduction and ROS overproduction. Supporting this result, the withdraw of the QR2 co-factor (BNAH) or the addition of the specific QR2 inhibitor S29434 suppressed oxidative stress. Taken together, these data suggest that the overexpression of QR2 in brain cells in the presence of catechol quinones might lead to ROS-induced cell death via the rapid conversion of superoxide radicals into hydrogen peroxide and then into highly reactive hydroxyl radicals. Thus, QR2 may be implicated in the early stages of neurodegenerative disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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.

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

PubMed Central

Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A.; Fischetti, Vincent A.; McClain, Mark S.; Vartanian, Timothy

2015-01-01

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity. PMID:25993478

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

PubMed

Rumah, Kareem Rashid; Ma, Yinghua; Linden, Jennifer R; Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A; Fischetti, Vincent A; McClain, Mark S; Vartanian, Timothy

2015-05-01

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

Proton Magnetic Resonance Spectroscopy Study on the Metabolism Changes of Cerebellum in Patients with Post-Stroke Depression.

PubMed

Zhang, Lei; Sui, Ru-Bo

2017-01-01

To study the metabolic changes of cerebellum by proton magnetic resonance Spectroscopy (1H-MRS) and discuss the relationships between the cerebellar changes and depression severity in patients with post-stroke depression. Data of demographic characteristics, individual history and life style of all subjects were collected. 40 patients with stroke and 20 controls were enrolled. All groups received T1WI, T2WI, DWI and 1H-MRS examination. The cerebral infarction volume and the distribution and severity of leukoaraiosis were evaluated. The ratios of NAA/Cr, Cho/Cr and Cho/NAA in the cerebellum were calculated. There were no statistical significant difference in the NAA/Cr, Cho/Cr and Cho/NAA ratios in bilateral cerebellum between CONT group and NORM group. The Cho/Cr and Cho/NAA ratios in the cerebellum contralateral to the stroke region were higher in PSD group than those in NORM and CONT groups, and the Cho/Cr and Cho/NAA ratios in the cerebellum ipsilateral to the stroke region were similar with those in NORM and CONT groups. However, there were no statistical significant difference in the NAA/Cr ratios in bilateral cerebellum among three groups. The result shows preliminarily that the cerebellum involves in the development of post-stroke depression. © 2017 The Author(s). Published by S. Karger AG, Basel.

A Comparative Study on Carbohydrate Estimation: GoCARB vs. Dietitians.

PubMed

Vasiloglou, Maria F; Mougiakakou, Stavroula; Aubry, Emilie; Bokelmann, Anika; Fricker, Rita; Gomes, Filomena; Guntermann, Cathrin; Meyer, Alexa; Studerus, Diana; Stanga, Zeno

2018-06-07

GoCARB is a computer vision-based smartphone system designed for individuals with Type 1 Diabetes to estimate plated meals' carbohydrate (CHO) content. We aimed to compare the accuracy of GoCARB in estimating CHO with the estimations of six experienced dietitians. GoCARB was used to estimate the CHO content of 54 Central European plated meals, with each of them containing three different weighed food items. Ground truth was calculated using the USDA food composition database. Dietitians were asked to visually estimate the CHO content based on meal photographs. GoCARB and dietitians achieved comparable accuracies. The mean absolute error of the dietitians was 14.9 (SD 10.12) g of CHO versus 14.8 (SD 9.73) g of CHO for the GoCARB ( p = 0.93). No differences were found between the estimations of dietitians and GoCARB, regardless the meal size. The larger the size of the meal, the greater were the estimation errors made by both. Moreover, the higher the CHO content of a food category was, the more challenging its accurate estimation. GoCARB had difficulty in estimating rice, pasta, potatoes, and mashed potatoes, while dietitians had problems with pasta, chips, rice, and polenta. GoCARB may offer diabetic patients the option of an easy, accurate, and almost real-time estimation of the CHO content of plated meals, and thus enhance diabetes self-management.

Rat strain differences in brain structure and neurochemistry in response to binge alcohol.

PubMed

Zahr, Natalie M; Mayer, Dirk; Rohlfing, Torsten; Hsu, Oliver; Vinco, Shara; Orduna, Juan; Luong, Richard; Bell, Richard L; Sullivan, Edith V; Pfefferbaum, Adolf

2014-01-01

Ventricular enlargement is a robust phenotype of the chronically dependent alcoholic human brain, yet the mechanism of ventriculomegaly is unestablished. Heterogeneous stock Wistar rats administered binge EtOH (3 g/kg intragastrically every 8 h for 4 days to average blood alcohol levels (BALs) of 250 mg/dL) demonstrate profound but reversible ventricular enlargement and changes in brain metabolites (e.g., N-acetylaspartate (NAA) and choline-containing compounds (Cho)). Here, alcohol-preferring (P) and alcohol-nonpreferring (NP) rats systematically bred from heterogeneous stock Wistar rats for differential alcohol drinking behavior were compared with Wistar rats to determine whether genetic divergence and consequent morphological and neurochemical variation affect the brain's response to binge EtOH treatment. The three rat lines were dosed equivalently and approached similar BALs. Magnetic resonance imaging and spectroscopy evaluated the effects of binge EtOH on brain. As observed in Wistar rats, P and NP rats showed decreases in NAA. Neither P nor NP rats, however, responded to EtOH intoxication with ventricular expansion or increases in Cho levels as previously noted in Wistar rats. Increases in ventricular volume correlated with increases in Cho in Wistar rats. The latter finding suggests that ventricular volume expansion is related to adaptive changes in brain cell membranes in response to binge EtOH. That P and NP rats responded differently to EtOH argues for intrinsic differences in their brain cell membrane composition. Further, differential metabolite responses to EtOH administration by rat strain implicate selective genetic variation as underlying heterogeneous effects of chronic alcoholism in the human condition.