[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.

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.

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.

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.

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 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.

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

PubMed

Kamachi, Yasuharu; Omasa, Takeshi

2018-04-01

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

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

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.

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.

Transport and Metabolism of Radiolabeled Choline in Hepatocellular Carcinoma

PubMed Central

Kuang, Yu; Salem, Nicolas; Corn, David J.; Erowku, Bernadette; Tian, Haibin; Wang, Fangjing; Lee, Zhenghong

2010-01-01

Objectives Altered choline (Cho) metabolism in cancerous cells can be used as a basis for molecular imaging with PET using radiolabeled Cho. In this study, the metabolism of tracer Cho was investigated in a woodchuck hepatocellular carcinoma (HCC) cell line (WCH17) and in freshly-derived rat hepatocytes. The transporter responsible for [11C]-Cho uptake in HCC was also characterized in WCH17 cells. The study helped to define the specific mechanisms responsible for radio-Cho uptake seen on the PET images of primary liver cancer such as HCC. Methods Cells were pulsed with [14C]-Cho for 5 min and chased for varying durations in cold media to simulate the rapid circulation and clearance of [11C]-Cho. Radioactive metabolites were extracted and analyzed by radio-HPLC and radio-TLC. The Cho transporter (ChoT) was characterized in WCH17 cells. Results WCH17 cells showed higher 14C uptake than rat primary hepatocytes. [14C]-Phosphocholine (PC) was the major metabolite in WCH17. In contrast, the intracellular Cho in primary hepatocytes was found to be oxidized to betaine (partially released into media) and to a less degree, phosphorylated to PC. [14C]-Cho uptake by WCH17 cells was found to have both facilitative transport and non-facilitative diffusion components. The facilitative transport was characterized by Na+ dependence and low affinity (Km = 28.59 ± 6.75 μM) with partial energy dependence. In contrast, ChoT in primary hepatocytes is Na+ independent and low affinity. Conclusions Our data suggest that transport and phosphorylation of Cho are responsible for the tracer accumulation during [11C]-Cho PET imaging of HCC. WCH17 cells incorporate [14C]-Cho preferentially into PC. Conversion of [14C]-PC into phosphatidylcholine occurred slowly in vitro. Basal oxidation and phosphorylation activities in surrounding hepatic tissue contribute to the background seen in [11C]-Cho PET images. PMID:20698576

DNA methylation in CHO cells.

PubMed

Wippermann, Anna; Noll, Thomas

2017-09-20

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

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.

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.

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.

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.

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.

[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.

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

PubMed

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

2015-10-01

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

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.

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

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

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

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.

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.

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.

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).

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.

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 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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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

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

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.

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

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.

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.

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.

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.

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.

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.

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

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

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.

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 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.

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.

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.

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.

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.

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.

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.

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

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.

[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.

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.

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.

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

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.

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.

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.

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

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

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.

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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

PubMed

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

2016-09-01

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

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.

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.

Phosphatidylcholine catabolism in the MCF-7 cell cycle.

PubMed

Lin, Weiyang; Arthur, Gilbert

2006-10-01

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

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.

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

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

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

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.

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.

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

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.

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

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.

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.

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.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

PubMed Central

Sanchez-Lopez, E; Zimmerman, T; Gomez del Pulgar, T; Moyer, M P; Lacal Sanjuan, J C; Cebrian, A

2013-01-01

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction. PMID:24287694

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

PubMed

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

2001-11-01

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

Spatiotemporal relationship of embryonic cholinesterases with cell proliferation in chicken brain and eye.

PubMed Central

Layer, P G; Sporns, O

1987-01-01

Close relationships between acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, true cholinesterase, EC, 3.1.1.7) and butyrylcholinesterase (BtChoEase, acylcholine acylhydrolase, pseudocholinesterase, EC, 3.1.1.8) with cell proliferation were observed in the early chicken brain. These include the following: BtChoEase is transiently accumulating in patchy fashion on the ventricular side of the neuroepithelium shortly before AcChoEase appears in cell bodies along the opposing mantle layer. The amount of BtChoEase in retina and brain is greatest in the early phase (E3-E5, or incubation periods of 3-5 days); in retina it decreases about 2 days later than in brain. However, AcChoEase expression increases with time, in inverse order to that of BtChoEase. In both tissues decrease of cell proliferation is closely followed by decrease in BtChoEase. A double-labeling technique of cholinesterase staining together with [3H]thymidine autoradiography reveals proliferation zones that are diffusely stained by BtChoEase but not by AcChoEase. Patches intensely stained for BtChoEase accompany clusters of cells in final stages of mitosis on their way to the differentiation zone, where they begin expressing AcChoEase. By applying different thymidine pulses, we identify an 11-hr lag from the last thymidine-uptake to full AcChoEase expression. (iv) These findings are confirmed by studying lens development, where areas of proliferation and differentiation are well separated. The spatiotemporal pattern of the transition of neuroblasts from a proliferating into a differentiating state correlates with the expression of BtChoEase just before and during mitosis and that of AcChoEase about 11 hr after mitosis. Thus cholinesterases could be involved in the regulation of this transition. Images PMID:3467355

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

PubMed

Han, Seora; Rhee, Won Jong

2018-05-01

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

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.

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.

Cerebral visual impairment and intellectual disability caused by PGAP1 variants.

PubMed

Bosch, Daniëlle G M; Boonstra, F Nienke; Kinoshita, Taroh; Jhangiani, Shalini; de Ligt, Joep; Cremers, Frans P M; Lupski, James R; Murakami, Yoshiko; de Vries, Bert B A

2015-12-01

Homozygous variants in PGAP1 (post-GPI attachment to proteins 1) have recently been identified in two families with developmental delay, seizures and/or spasticity. PGAP1 is a member of the glycosylphosphatidylinositol anchor biosynthesis and remodeling pathway and defects in this pathway are a subclass of congenital disorders of glycosylation. Here we performed whole-exome sequencing in an individual with cerebral visual impairment (CVI), intellectual disability (ID), and factor XII deficiency and revealed compound heterozygous variants in PGAP1, c.274_276del (p.(Pro92del)) and c.921_925del (p.(Lys308Asnfs*25)). Subsequently, PGAP1-deficient Chinese hamster ovary (CHO)-cell lines were transfected with either mutant or wild-type constructs and their sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment was measured. The mutant constructs could not rescue the PGAP1-deficient CHO cell lines resistance to PI-PLC treatment. In addition, lymphoblastoid cell lines (LCLs) of the affected individual showed no sensitivity to PI-PLC treatment, whereas the LCLs of the heterozygous carrier parents were partially resistant. In conclusion, we report novel PGAP1 variants in a boy with CVI and ID and a proven functional loss of PGAP1 and show, to our knowledge, for the first time this genetic association with CVI.

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

PubMed

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

2018-04-01

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

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region

PubMed Central

1991-01-01

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system. PMID:1757462

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.

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.

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

PubMed

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

2012-01-01

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

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.

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

PubMed Central

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

2016-01-01

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

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

PubMed

Yang, Harry; Wei, Ziping; Schenerman, Mark

2015-01-01

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

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.

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.

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

PubMed

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

2017-08-01

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

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.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed

Hunt, L A

1980-08-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed Central

Hunt, L A

1980-01-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein. Images PMID:6255177

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.

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

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.

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.

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

Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas.

PubMed

Jaeschke, Holger; Mueller, Sandra; Eszlinger, Markus; Paschke, Ralf

2010-12-01

Constitutively activating mutations (CAMs) of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas. Mutations L677V and T620I identified in hot thyroid carcinomas were previously characterized in CHO and in 3T3-Vill cell lines, respectively, stably expressing the mutant without determination of TSHR expression. F666L identified in a patient with hot thyroid nodules, I691F in a family with nonautoimmune hyperthyroidism and F631I identified in a hot thyroid carcinoma were not characterized for their in vitro function. Therefore, we decided to (re)evaluate the in vitro function of these five TSHR variants by determination of cell surface expression, and intracellular cAMP and inositol phosphate levels and performed additionally linear regression analyses to determine basal activity independently from the mutant's cell surface expression in COS-7 and HEK(GT) cells. Only one (F631I) of the five investigated TSHR variants displayed constitutive activity for G(α) s signalling and showed correlation with the clinical phenotype. The previous false classification of T620I and L677V as CAMs is most likely related to the fact that both mutations were characterized in cell lines stably expressing the mutated receptor construct without assessing the respective receptor number per cell. Other molecular aetiologies for the nonautoimmune hyperthyroidism and/or hot thyroid carcinomas in these three patients and one family should be elucidated. © 2010 Blackwell Publishing Ltd.

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

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.

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.

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.

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

PubMed

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

2004-11-20

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

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.

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.

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

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

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.

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

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

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

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.

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

Recombinant follicle-stimulating hormone: new biotechnology for infertility.

PubMed

Prevost, R R

1998-01-01

The frequency of infertility in developed countries is approximately 8-10%. New drugs are available for assisted reproduction techniques. Two recombinant follicle-stimulating hormone (FSH) products, follitropin-beta (Follistim in the United States, Puregon in Europe) and follitropin-alpha (Gonal-F), join compounds derived through transfecting nonhuman cell lines with genetic material capable of replicating identical amino acid sequences to human compounds. The cell line used for recombinant (r)-FSH production is the Chinese hamster ovary (CHO). Previously, the only agents that showed benefit in controlled ovulatory stimulation were derived from the urine of menopausal women. Those compounds contain additional substances, such as urinary proteins and various amounts of luteininzing hormone. The amino acid sequence of r-FSH is identical to that of human FSH, but the two recombinant products exist in many different isoforms and differ from each other and from human FSH due to varied carbohydrate side chains. Due to variation in the carbohydrate side chains, follitropin-beta in solution has a higher pH than urine-derived FSH, which enhances receptor affinity and therefore is a greater inducer of folliculogenesis. Follitropin-beta does not cause endogenous production of anti-CHO or anti-FSH antibodies, and is well tolerated.

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.

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.

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.

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

Assay development of inducible human renal phosphate transporter Npt2A (SLC34A1) in Flp-In-Trex-HEK293 cells.

PubMed

Wu, Hongzhong; Mao, Chonghong; Duenstl, Georg; Su, Wan; Qian, Su

2013-12-05

Hyperphosphatemia is associated with severe decline of renal function in chronic kidney disease and elevates cardiovascular mortality. Type II sodium dependent phosphate transporter 2A (Npt2A) plays a major role in renal phosphate reabsorption and could be explored as a target for anti-hyperphosphatemia therapy. Human Npt2A transporter activity was examined upon transfection into CHO, MDCK, HEK293, Flp-In-CHO and Flp-In-HEK293 cells. Only kidney-derived cells expressed functional Npt2A. HEK293 and Flp-In-HEK293 cell lines stably transfected with hNpt2A could be selected, but these cells were inactive in phosphate transport. This suggests that high-level, constitutive Npt2A expression has deleterious effects on the cell. By using the conditional promoter in the Flp-In-Trex vector, functional expression of Npt2A was achieved by doxycycline induction in HEK293 cells. The EGFP tagged and non-tagged, inducible stable hNpt2A-HEK293 cell lines afforded development of a robust phosphate uptake assay mediated by hNpt2A, which can be used to screen hNpt2A inhibitors and inducers of hNpt2A expression. Using this assay, the small molecule LC-1 was identified as a potent inhibitor of hNpt2A, suggesting that it is feasible to develop potent specific hNpt2A inhibitors to control phosphate overloading for hyperphosphatemia therapy. © 2013 Elsevier B.V. All rights reserved.

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.

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.

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

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.

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.

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.

Expression of human FcgammaRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding.

PubMed

Armour, Kathryn L; Smith, Cheryl S; Clark, Michael R

2010-03-31

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3- to 4-fold stronger that the interaction with FcgammaRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG. 2010 Elsevier B.V. All rights reserved.

[A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

PubMed

Kim, Yong Kyu; Kim, Tae Ue; Joung, In Sil; Im, Kyung Il

1988-06-01

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta-N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80% of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.

Identification of various cell culture models for the study of Zika virus

PubMed Central

Himmelsbach, Kiyoshi; Hildt, Eberhard

2018-01-01

AIM To identify cell culture models supportive for Zika virus (ZIKV) replication. METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis. RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines. CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research. PMID:29468137

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

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

PubMed

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

2015-06-01

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

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

Model-based strategy for cell culture seed train layout verified at lab scale.

PubMed

Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

2016-08-01

Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

PubMed Central

2014-01-01

Background In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes. PMID:25077436

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis.

PubMed

Lohr, Verena; Hädicke, Oliver; Genzel, Yvonne; Jordan, Ingo; Büntemeyer, Heino; Klamt, Steffen; Reichl, Udo

2014-07-30

In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes.

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

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

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

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.

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.

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

PubMed

Tissot, Stéphanie; Oberbek, Agata; Reclari, Martino; Dreyer, Matthieu; Hacker, David L; Baldi, Lucia; Farhat, Mohamed; Wurm, Florian M

2011-07-01

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Anticancer and apoptosis-inducing effects of quercetin in vitro and in vivo

PubMed Central

Hashemzaei, Mahmoud; Far, Amin Delarami; Yari, Arezoo; Heravi, Reza Entezari; Tabrizian, Kaveh; Taghdisi, Seyed Mohammad; Sadegh, Sarvenaz Ekhtiari; Tsarouhas, Konstantinos; Kouretas, Dimitrios; Tzanakakis, George; Nikitovic, Dragana; Anisimov, Nikita Yurevich; Spandidos, Demetrios A.; Tsatsakis, Aristides M.; Rezaee, Ramin

2017-01-01

The present study focused on the elucidation of the putative anticancer potential of quercetin. The anticancer activity of quercetin at 10, 20, 40, 80 and 120 µM was assessed in vitro by MMT assay in 9 tumor cell lines (colon carcinoma CT-26 cells, prostate adenocarcinoma LNCaP cells, human prostate PC3 cells, pheocromocytoma PC12 cells, estrogen receptor-positive breast cancer MCF-7 cells, acute lymphoblastic leukemia MOLT-4 T-cells, human myeloma U266B1 cells, human lymphoid Raji cells and ovarian cancer CHO cells). Quercetin was found to induce the apoptosis of all the tested cancer cell lines at the utilized concentrations. Moreover, quercetin significantly induced the apoptosis of the CT-26, LNCaP, MOLT-4 and Raji cell lines, as compared to control group (P<0.001), as demonstrated by Annexin V/PI staining. In in vivo experiments, mice bearing MCF-7 and CT-26 tumors exhibited a significant reduction in tumor volume in the quercetin-treated group as compared to the control group (P<0.001). Taken together, quercetin, a naturally occurring compound, exhibits anticancer properties both in vivo and in vitro. PMID:28677813

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

PubMed

Mollet, Mike; Godoy-Silva, Ruben; Berdugo, Claudia; Chalmers, Jeffrey J

2008-06-01

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

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

PubMed

Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

2012-03-01

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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

PubMed

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

2015-01-01

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

PubMed

Zhou, Yizhou; Shaw, David; Lam, Cynthia; Tsukuda, Joni; Yim, Mandy; Tang, Danming; Louie, Salina; Laird, Michael W; Snedecor, Brad; Misaghi, Shahram

2017-09-23

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

SN38 conjugated hyaluronic acid gold nanoparticles as a novel system against metastatic colon cancer cells.

PubMed

Hosseinzadeh, Hosniyeh; Atyabi, Fatemeh; Varnamkhasti, Behrang Shiri; Hosseinzadeh, Reza; Ostad, Seyed Nasser; Ghahremani, Mohammad Hossein; Dinarvand, Rassoul

2017-06-30

Combination of chemotherapy and photothermal therapy has been proposed for better treatment of metastatic colon cancer. In this study SN38, a highly potent cytotoxic agent, was conjugated to negatively charged hyaluronic acid (HA), which was deposited on the surface of the positively charged gold nanoparticles via electrostatic interaction. The drug conjugation and its interaction with gold nanoparticles were verified by 1 H NMR and UV-vis spectroscopies, respectively. The prepared SN38-HA gold NPs are negatively charged spherical nanoparticles with an average size of 75±10nm. In vitro release study revealed that drug release in acidic conditions (pH 5.2) was faster than that in physiological pH. Red light emitting diode (LED, 630nm, 30mW) was used as a light source for photothermal experiments. The drug release in acidic conditions was increased up to 30% using red LED illumination (6min) in comparison with experiment carried out indark. The cytotoxicity study on MUC1 positive HT29, SW480 colon cancer cells and MUC1 negative CHO cells, showed higher toxicity of the nanoparticles on HT29 and SW480 cell lines compared to CHO cells. Confocal microscopy images along with flow cytometry analysis confirm the cytotoxicity results. The incubation time for reaching IC50 decreases from 48h to 24h by LED illumination after nanoparticle treatment. Migratory potential of the HT29 and SW480 cell lines was reduced by co-application of SN38-HA gold NPs and LED radiation. Also anti-proliferative study indicates that LED radiation has increased the cytotoxicity of the nanoparticles and this effect is remained up to 8days. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

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.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

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.

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.

A Different Perspective: How Much Innovation Is Really Needed for Monoclonal Antibody Production Using Mammalian Cell Technology?

PubMed

Kelley, Brian; Kiss, Robert; Laird, Michael

2018-05-03

As biopharmaceutical companies have optimized cell line and production culture process development, titers of recombinant antibodies have risen steadily to 3-8 g/L for fed-batch mammalian cultures at production scales of 10 kL or larger. Most new antibody products are produced from Chinese Hamster Ovary (CHO) cell lines, and there are relatively few alternative production hosts under active evaluation. Many companies have adopted a strategy of using the same production cell line for early clinical phases as well as commercial production, which reduces the risk of product comparability issues during the development lifecycle. Product quality and consistency expectations rest on the platform knowledge of the CHO host cell line and processes used for the production of many licensed antibodies. The lack of impact of low-level product variants common to this platform on product safety and efficacy also builds on the established commercial history of recombinant antibodies, which dates back to 1997.Efforts to increase titers further will likely yield diminishing returns. Very few products would benefit significantly from a titer greater than 8 g/L; in many cases, a downstream processing bottleneck would preclude full recovery from production-scale bioreactors for high titer processes. The benefits of a process platform based on standard fed-batch production culture include predictable scale-up, process transfer, and production within a company's manufacturing network or at a contract manufacturing organization. Furthermore, the confidence in an established platform provides key support towards regulatory flexibility (e.g., design space) for license applications following a quality-by-design strategy.These factors suggest that novel technologies for antibody production may not provide a substantial return on investment. What, then, should be the focus of future process development efforts for companies that choose to launch antibody products using their current platform? This review proposes key focus areas in an effort to continually improve process consistency, assure acceptable product quality, and establish appropriate process parameter limits to enable flexible manufacturing options.

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.

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.

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.

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin.

PubMed

Sánchez, O; Montesino, R; Toledo, J R; Rodríguez, E; Díaz, D; Royle, L; Rudd, P M; Dwek, R A; Gerwig, G J; Kamerling, J P; Harvey, D J; Cremata, J A

2007-08-15

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

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.

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.

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.

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

PubMed

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

2002-11-01

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

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

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 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.

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.

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

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

Polycation nanostructured lipid carrier, a novel nonviral vector constructed with triolein for efficient gene delivery.

PubMed

Zhang, Zhiwen; Sha, Xianyi; Shen, Anle; Wang, Yongzhong; Sun, Zhaogui; Gu, Zheng; Fang, Xiaoling

2008-06-06

A novel nonviral gene transfer vector was developed by modifying nanostructured lipid carrier (NLC) with cetylated polyethylenimine (PEI). Polycation nanostructured lipid carrier (PNLC) was prepared using the emulsion-solvent evaporation method. Its in vitro gene transfer properties were evaluated in the human lung adenocarcinoma cell line SPC-A1 and Chinese Hamster Ovary (CHO) cells. Enhanced transfection efficiency of PNLC was observed after the addition of triolein to the PNLC formulation and the transfection efficiency of the optimized PNLC was comparable to that of Lipofectamine 2000. In the presence of 10% serum the transfection efficiency of the optimal PNLC was not significantly changed in either cell line, whereas that of Lipofectamine 2000 was greatly decreased in both. Thus, PNLC is an effective nonviral gene transfer vector and the gene delivery activity of PNLC was enhanced after triolein was included into the PNLC formulation.

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.

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.

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.

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

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

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.

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

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology.

PubMed

Hu, Jianwen; Han, Jizhong; Li, Haoran; Zhang, Xian; Liu, Lan Lan; Chen, Fei; Zeng, Bin

2018-01-01

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications. © 2018 S. Karger AG, Basel.

Guanidinylated polyethyleneimine-polyoxypropylene-polyoxyethylene conjugates as gene transfection agents.

PubMed

Bromberg, Lev; Raduyk, Svetlana; Hatton, T Alan; Concheiro, Angel; Rodriguez-Valencia, Cosme; Silva, Maite; Alvarez-Lorenzo, Carmen

2009-05-20

Conjugates of linear and branched polyethyleneimine (PEI) and monoamine polyether Jeffamine M-2070 (PO/EO mol ratio 10/31, 2000 Da) were synthesized through polyether activation by cyanuric chloride followed by attachment to PEI and guanidinylation by 1H-pyrazole-carboxamidine hydrochloride. The resulting guanidinylated PEI-polyether conjugates (termed gPEI-Jeffamine) efficiently complexed plasmid DNA, and their polyplexes possessed enhanced colloidal stability in the presence of serum proteins. In vitro studies with mammalian CHO-1, 3T3, and Cos-7 cell lines demonstrated improved transfection efficiency of the pCMVbeta-gal plasmid/gPEI-Jeffamine polyplexes. The guanidinylation of the amino groups of PEI and the conjugation of PEI with the Jeffamine polyether enhanced the conjugates' interaction with genetic material and reduced the cytotoxicity of the polyplexes in experiments with the L929 cell line.

Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells.

PubMed

Ackerstaff, E; Pflug, B R; Nelson, J B; Bhujwalla, Z M

2001-05-01

In this study, a panel of normal human prostate cells (HPCs) and tumor cells derived from metastases were studied by (1)H NMR spectroscopy to determine whether the malignant transformation of HPCs results in the elevation of choline compounds. Although an elevated choline signal has been observed previously in clinical studies, the contribution of the different Cho compounds to this elevation, as well as their quantification, has not been established until now. Here we have shown that HPCs derived from metastases exhibit significantly higher phosphocholine as well as glycerophosphocholine levels compared with normal prostate epithelial and stromal cells. Thus the elevation of the choline peak observed clinically in prostate cancer is attributable to an alteration of phospholipid metabolism and not simply to increased cell density, doubling time, or other nonspecific effects. Androgen deprivation of the androgen receptor-positive cell lines resulted in a significant increase of choline compounds after chronic androgen deprivation of the LNCaP cell line and in a decrease of choline compounds after a more acute androgen deprivation of the LAPC-4 cell line. These data strongly support the use of proton magnetic resonance spectroscopic imaging to detect the presence of prostate cancer for diagnosis, to detect response subsequent to androgen ablation therapy, and to detect recurrence.

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.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process. © 2018 Wiley Periodicals, Inc.

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.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

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 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

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

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.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

PubMed

Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

2011-08-15

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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.

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.

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.

Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

PubMed

De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-06-01

Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') 2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

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

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.

NADE (p75NTR-associated cell death executor) suppresses cellular growth in vivo.

PubMed

Tong, Xiangjun; Xie, Dong; Roth, Wilfried; Reed, John; Koeffler, H Phillip

2003-06-01

NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74. It was reported to mediate nerve growth factor-induced apoptosis. We independently isolated human NADE (pHGR74) from breast cancer cell lines. Expression of NADE in various human cancer cell lines, and human and murine tissues was examined. NADE was highly expressed in human endocrine-related organs and embryotic murine tissues. Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo. We used the yeast two-hybrid system to search for NADE binding protein. Dynactin was identified as a candidate. The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE. Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF. Thus, human and murine NADE appear to have different functions.

Synthesis and Anticancer Activity of 3-(Substituted Aroyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole Derivatives.

PubMed

Zhan, Xiao-Ping; Lan, Lan; Wang, Shuai; Zhao, Kai; Xin, Yu-Xuan; Qi, Qi; Wang, Yao-Lin; Mao, Zhen-Min

2017-02-01

A series of 3-(substituted aroyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole derivatives were synthesized and determined for their anticancer activity against eleven cancer cell lines and two normal tissue cell lines using MTT assay. Among the synthesized compounds, compound 3f was the most potent compound against A375, CT-26, HeLa, MGC80-3, NCI-H460 and SGC-7901 cells (IC 50  = 8.2 - 31.7 μm); 3g, 3n and 3a were the most potent compounds against CHO (IC 50  = 8.2 μm), HCT-15 (IC 50  = 21 μm) and MCF-7 cells (IC 50  = 18.7 μm), respectively. Importantly, all the target compounds showed no cytotoxicity towards the normal tissue cell (IC 50  > 100 μm). Thus, these compounds with the potent anticancer activity and low toxicity have potential for the development of new anticancer chemotherapy agents. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

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

Proton magnetic resonance spectroscopy (MRS) in on-line game addiction

PubMed Central

Han, Doug Hyun; Lee, Young Sik; Shi, Xianfeng; Renshaw, Perry F.

2015-01-01

Recent brain imaging studies suggested that both the frontal and temporal cortices are important candidate areas for mediating the symptoms of internet addiction. We hypothesized that deficits of prefrontal and temporal cortical function in patients with on-line game addiction (PGA) would be reflected in decreased levels of N-acetyl aspartate (NAA) and cytosolic, choline containing compound (Cho). Seventy three young PGA and 38 age and sex matched healthy control subjects were recruited in the study. Structural MR and 1H MRS data were acquired using a 3.0 T MRI scanner. Voxels were sequentially placed in right frontal cortex and right medial temporal cortices. In the right frontal cortex, the levels of NAA in PGA were lower than those in healthy controls. In the medial temporal cortex, the levels of Cho in PGA participants were lower than those observed in healthy controls. The Young Internet Addiction Scale (YIAS) scores and perseverative responses in PGA were negatively correlated with the level of NAA in right frontal cortex. The Beck Depressive Inventory (BDI) scores in the PGA cohort were negatively correlated with Cho levels in the right temporal lobe. To the best of our knowledge, this is the first MRS study of individuals with on-line game addiction. Although, the subjects with on-line game addiction in the current study were free from psychiatric co-morbidity, patients with on-line game addiction appear to share characteristics with ADHD and MDD in terms of neurochemical changes in frontal and temporal cortices. PMID:25088284

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.

"Antimicrobial and antiproliferative activity of essential oil, aqueous and ethanolic extracts of Ocimum micranthum Willd leaves".

PubMed

Caamal-Herrera, Isabel O; Carrillo-Cocom, Leydi M; Escalante-Réndiz, Diana Y; Aráiz-Hernández, Diana; Azamar-Barrios, José A

2018-02-08

Ocimum micranthum Willd is a plant used in traditional medicine practiced in the region of the Yucatan peninsula. In particular, it is used for the treatment of cutaneous infections and wound healing, however there are currently no existing scientific studies that support these applications. The aim of the present study was to evaluate the antimicrobial and the in vitro proliferative activity (on healthy mammalian cell lines) of the essential oil and extracts (aqueous and ethanolic) of this plant. The minimal inhibitory concentration (MIC) of essential oil and aqueous and ethanolic extracts of Ocimum micranthum leaves against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans was determined using the microdilution technique. The in vitro proliferative activity of human fibroblast (hFB) and Chinese hamster ovary (CHO-K1) cells treated with these extracts was evaluated using the MTT test. The hFB cell line was also evaluated using Trypan Blue assay. Candida albicans was more susceptible to the ethanolic extract and the aqueous extract (MIC value of 5 μL/mL and 80 μL/mL respectively). In the case of Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa, the MIC of the aqueous and ethanolic extract was 125 μL/mL. The aqueous extract showed a significant (p < 0.05) antiproliferative effect on hFB cells at a concentration of 4%, with cell proliferation percentage values of 73.56% and 20.59% by MTT method and Trypan Blue assay, respectively; the same effect was observed for the ethanolic extract at concentration from 0.06% to 0.25% using MTT method and at a concentration from 0.125% to 0.25% using Trypan Blue assay. In CHO-K1 cells an antiproliferative effect was observed at a concentration of 8% of aqueous extract and from 0.06% to 0.25% of ethanolic extract using the MTT method. These assays showed that low concentrations of essential oil and extracts of Ocimum micranthum leaves are sufficient to cause an antiproliferative effect on the hFB cell line but do not produce an antimicrobial effect against the microorganisms evaluated. More studies are necessary to improve understanding of the mechanism of action of the compounds implicated in the bioactivities shown by the crude extracts.

Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation

PubMed Central

Wang, Wenchao; Li, Zhiyuan; Liu, Juanjuan; Yang, Xingxing; Ji, Xinmiao; Luo, Yan; Hu, Chen; Hou, Yubin; He, Qianqian; Fang, Jun; Wang, Junfeng; Liu, Qingsong; Li, Guohui; Lu, Qingyou; Zhang, Xin

2016-01-01

Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs. PMID:27223425

Transient transfection of mammalian cells using a violet diode laser

NASA Astrophysics Data System (ADS)

Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

2010-07-01

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

Antitumor activity of ginseng sapogenins, 25-OH-PPD and 25-OCH3-PPD, on gastric cancer cells.

PubMed

Zhao, Chen; Su, Guangyue; Wang, Xude; Zhang, Xiaoshu; Guo, Shuang; Zhao, Yuqing

2016-01-01

25-Hydroxyprotopanaxadiol (25-OH-PPD) and 25-methoxylprotopanaxadiol (25-OCH3-PPD), two ginseng sapogenins, have potent antitumor activity and their effects on gastric cancer (BGC-823, SGC-7901, MKN-28) cells and a gastric mucosa (GES-1) cell line are reported. Both compounds significantly inhibited the growth of gastric cancer cells, while having lesser inhibitory effects on GES-1 cells by MTT assay. A mechanistic study revealed that the two ginseng sapogenins could induce apoptosis in BGC-823 cells by morphological observation, DNA fragmentation, flow cytometry and western blot analysis. Besides, the apoptosis was inhibited by Ac-DEVD-CHO, a caspase 3 inhibitor, which was confirmed by cell viability analysis. These results indicate that 25-OH-PPD and 25-OCH3-PPD have potential to be promising agents for the treatment of gastric cancer.

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

PubMed Central

Crites, Travis J.; Maddox, Michael; Padhan, Kartika; Muller, James; Eigsti, Calvin; Varma, Rajat

2015-01-01

Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology. PMID:26331983

Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

PubMed

Scarcelli, John J; Shang, Tanya Q; Iskra, Tim; Allen, Martin J; Zhang, Lin

2017-11-01

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017. © 2017 American Institute of Chemical Engineers.

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.

(Q)SAR studies to design new human choline kinase inhibitors as antiproliferative drugs.

PubMed

Campos, J M; Sánchez-Martín, R M; Conejo-García, A; Entrena, A; Gallo, M A; Espinosa, A

2006-01-01

Most of the signal transduction pathways are mediated by protein kinases regulating every aspect of cell function. Mutations which deregulate their expression or their function or both result in cancers. Therefore, protein kinase inhibitors have become the focus of development of new therapies for cancer. A comprehensive review of Choline kinase (ChoK) was published by us in 2003. Since then, molecular information of ChoK inhibitors has been accumulated. In this review, we intend to summarize the new lines of evidence that will include the design of the most active antiproliferative agents so far described against ChoK. Studies have been aimed at the establishment of structure-activity relationships and the structural parameters that define ChoK inhibitory and antiproliferative activities of a set of twenty-five acyclic biscationic pyridophane and forty acyclic biscationic quinolinephane compounds. The corresponding QSAR equation was obtained for the whole set of bisquinolinium compounds for the antiproliferative activity, taking into consideration the electronic parameter sigma(R) of R(4), the molar refractivity (MR) of R(8), and the lipophilic parameters clog P and pi(linker). The most potent antiproliferative agent shows an IC(50) = 0.45 microM, predicted by the QSAR equation, whilst its experimental value is IC(50) = 0.20 microM. Finally, toxicity assays were performed for the most promising compounds because of their interesting antiproliferative activities [IC(50 HT-29) = 0.70, 0.80, 1.50 and 1.90 microM] and low toxicity [LD(50) = 16.7, 12.5, > 25 and > 20 mg/kg of mouse]. These biological activities justify further analysis for antitumoral assays under in vivo conditions.

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).

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

PubMed

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

2016-01-05

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

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.

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.

Antiproliferative Activity of Fucan Nanogel

PubMed Central

Dantas-Santos, Nednaldo; Almeida-Lima, Jailma; Vidal, Arthur Anthunes Jacome; Gomes, Dayanne Lopes; Oliveira, Ruth Medeiros; Santos Pedrosa, Silvia; Pereira, Paula; Gama, Francisco Miguel; Oliveira Rocha, Hugo Alexandre

2012-01-01

Sulfated fucans comprise families of polydisperse natural polysaccharides based on sulfated L-fucose. Our aim was to investigate whether fucan nanogel induces cell-specific responses. To that end, a non toxic fucan extracted from Spatoglossum schröederi was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution with hydrophobic chains was close to 100%, as estimated by elemental analysis. SNFfuc in aqueous media had a mean diameter of 123 nm and zeta potential of −38.3 ± 0.74 mV, as measured by dynamic light scattering. Nanoparticles conserved their size for up to 70 days. SNFuc cytotoxicity was determined using the MTT assay after culturing different cell lines for 24 h. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0%–43.7% at nanogel concentrations of 0.05–0.5 mg/mL and rabbit aorta endothelial cells (RAEC) non-tumor cell line proliferation displayed inhibition of 8.0%–22.0%. On the other hand, nanogel improved Chinese hamster ovary (CHO) and monocyte macrophage cell (RAW) non-tumor cell line proliferation in the same concentration range. The antiproliferative effect against tumor cells was also confirmed using the BrdU test. Flow cytometric analysis revealed that the fucan nanogel inhibited 786 cell proliferation through caspase and caspase-independent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle. PMID:23118717

Evaluation of the genotoxicity of extracts of Houttuynia cordata Thunb.

PubMed

Kang, Chang Keun; Hah, Dae Sik; Kim, Chung Hui; Kim, Euikyung; Kim, Jong Shu

2012-01-01

The present study was conducted to evaluate the activity of methanol extracts from Houttuynia cordata Thunb. (HC) in a reverse mutation assay in Salmonella typhimurium, and a chromosome aberration assay in the Chinese hamster ovary (CHO) cell line and to evaluate its effect on the occurrence of polychromatic erythrocytes in mice. In the reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2urvA(-), methanol extracts of HC (5, 2.5, 1.25, 0.62, or 0.312 mg/plate) did not induce reverse mutations in the presence or absence of an S9 metabolic activation mixture. In the chromosome aberration test using CHO cells, methanol extracts (1.25, 2.5 or 5 μg/ml) caused a few incidences of structural and numerical aberrations, in both of absence or presence of an S9 metabolic activation mixture, but in comparison with the positive control group, these incidences were not significantly increased. In the mouse micronucleus test, no significant increases in the occurrence of micronucleated polychromatic erythrocytes were observed in male ICR mice that were orally administered methanol extracts of HC at doses of 2.0, 1.0, or 0.5 g/kg. From these results, we concluded that the methanol extracts of HC did not induce harmful effects on genes in bacteria, a mammalian cell system or in mouse bone marrow cells. Thus, HC's use for health promotion and/or a sick remedy for humans may be safe.

Proton magnetic resonance spectroscopy (MRS) in on-line game addiction.

PubMed

Han, Doug Hyun; Lee, Young Sik; Shi, Xianfeng; Renshaw, Perry F

2014-11-01

Recent brain imaging studies suggested that both the frontal and temporal cortices are important candidate areas for mediating the symptoms of internet addiction. We hypothesized that deficits of prefrontal and temporal cortical function in patients with on-line game addiction (PGA) would be reflected in decreased levels of N-acetyl aspartate (NAA) and cytosolic, choline containing compound (Cho). Seventy three young PGA and 38 age and sex matched healthy control subjects were recruited in the study. Structural MR and (1)H MRS data were acquired using a 3.0 T MRI scanner. Voxels were sequentially placed in right frontal cortex and right medial temporal cortices. In the right frontal cortex, the levels of NAA in PGA were lower than those in healthy controls. In the medial temporal cortex, the levels of Cho in PGA participants were lower than those observed in healthy controls. The Young Internet Addiction Scale (YIAS) scores and perseverative responses in PGA were negatively correlated with the level of NAA in right frontal cortex. The Beck Depressive Inventory (BDI) scores in the PGA cohort were negatively correlated with Cho levels in the right temporal lobe. To the best of our knowledge, this is the first MRS study of individuals with on-line game addiction. Although, the subjects with on-line game addiction in the current study were free from psychiatric co-morbidity, patients with on-line game addiction appear to share characteristics with ADHD and MDD in terms of neurochemical changes in frontal and temporal cortices. Copyright © 2014 Elsevier Ltd. All rights reserved.

[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.

Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor

PubMed Central

Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching

2013-01-01

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443

Analysis of the in vitro and in vivo effects of photodynamic therapy on prostate cancer by using new photosensitizers, protoporphyrin IX-polyamine derivatives.

PubMed

Fidanzi-Dugas, Chloë; Liagre, Bertrand; Chemin, Guillaume; Perraud, Aurélie; Carrion, Claire; Couquet, Claude-Yves; Granet, Robert; Sol, Vincent; Léger, David Yannick

2017-07-01

Photodynamic therapy, using porphyrins as photosensitizers (PS), has been approved in treatment of several solid tumors. However, commonly used PS induce death but also resistance pathways in cancer cells and an alteration of surrounding normal tissues. Because polyamines (PA) are actively accumulated in cancer cells by the Polyamine Transport System (PTS), they may enable PS to specifically target cancer cells. Here, we investigated whether new protoporphyrin IX-polyamine derivatives were effective PS against prostate cancer and whether PA increased PDT specificity after 630nm irradiation. CHO and CHO-MG cells (differing in their PTS activity) were used to assess efficacy of polyamine vectorization. MTT assays were performed on human prostate non-malignant (RWPE-1) and malignant (PC-3, DU 145 and LNCaP) cell lines to test PS phototoxicity. ROS generation, DNA fragmentation and cell signalling were assessed by ELISA/EIA, western-blots and gel shift assays. Finally, PS effects were studied on tumor growth in nude mice. Our PS were more effective on cancer cells compared to non-malignant cells and more effective than PpIX alone. PpIX-PA generated ROS production involved in induction of apoptotic intrinsic pathways. Different pathways involved in apoptosis resistance were studied: PS inhibited Bcl-2, Akt, and NF-κB but activated p38/COX-2/PGE 2 pathways which were not implicated in apoptosis resistance in our model. In vivo experiments showed PpIX-PA efficacy was greater than results obtained with PpIX. All together, our results showed that PpIX-PA exerted its maximum effects without activating resistance pathways and appears to be a good candidate for prostate cancer PDT treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

In situ Raman spectroscopy for simultaneous monitoring of multiple process parameters in mammalian cell culture bioreactors.

PubMed

Whelan, Jessica; Craven, Stephen; Glennon, Brian

2012-01-01

In this study, the application of Raman spectroscopy to the simultaneous quantitative determination of glucose, glutamine, lactate, ammonia, glutamate, total cell density (TCD), and viable cell density (VCD) in a CHO fed-batch process was demonstrated in situ in 3 L and 15 L bioreactors. Spectral preprocessing and partial least squares (PLS) regression were used to correlate spectral data with off-line reference data. Separate PLS calibration models were developed for each analyte at the 3 L laboratory bioreactor scale before assessing its transferability to the same bioprocess conducted at the 15 L pilot scale. PLS calibration models were successfully developed for all analytes bar VCD and transferred to the 15 L scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

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.

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

PubMed

Barrett, M P; Tetaud, E; Seyfang, A; Bringaud, F; Baltz, T

1995-12-15

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

[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.

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

A multiparallel bioreactor for the cultivation of mammalian cells in a 3D-ceramic matrix.

PubMed

Goralczyk, Vicky; Driemel, Gregor; Bischof, Andreas; Peter, Andrea; Berthold, Almuth; Kroh, Lothar; Blessing, Lucienne; Schubert, Helmut; King, Rudibert

2010-01-01

For adherently growing cells, cultivation is limited by the provided growth surface. Excellent surface-to-volume ratios are found in highly porous matrices, which have to face the challenge of nutrient supply inside the matrices' caverns. Therefore, perfusion strategies are recommended which often have to deal with the need of developing an encompassing bioreactor periphery. We present a modular bioreactor system based on a porous ceramic matrix that enables the supply of cells with oxygen and nutrients by perfusion. The present version of the reactor system focuses on simple testing of various inoculation and operation modes. Moreover, it can be used to efficiently test different foam structures. Protocols are given to set-up the system together with handling procedures for long-time cultivation of a CHO cell line. Experimental results confirm vital growth of cells inside the matrices' caverns.

PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Harada, Hiroyuki; Kagawa, Yumiko; Ichii, Osamu; Konnai, Satoru; Kaneko, Mika K; Kato, Yukinari

2017-10-01

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

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.

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

PubMed Central

Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

2012-01-01

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

Laser scanning cytometry for automation of the micronucleus assay

PubMed Central

Darzynkiewicz, Zbigniew; Smolewski, Piotr; Holden, Elena; Luther, Ed; Henriksen, Mel; François, Maxime; Leifert, Wayne; Fenech, Michael

2011-01-01

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure. PMID:21164197

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)

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

PubMed

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

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality. © 2014 Wiley Periodicals, Inc.

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.

Dual effects of N-acetyl-L-cysteine dependent on NQO1 activity: Suppressive or promotive of 9,10-phenanthrenequinone-induced toxicity

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

Toyooka, Tatsushi; Shinmen, Takuya; Aarts, Jac M.M.J.G.

2012-11-01

A typical antioxidant, N-acetyl-L-cysteine (NAC) generally protects cells from oxidative damage induced by reactive oxygen species (ROS). 9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, produces ROS in redox cycling following two-electron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1), which has been considered as a cause of its cyto- and genotoxicity. In this study, we show that NAC unexpectedly augments the toxicity of 9,10-PQ in cells with low NQO1 activity. In four human skin cell lines, the expression and the activity of NQO1 were lower than in human adenocarcinoma cell lines, A549 and MCF7. In the skin cells, the cytotoxicitymore » of 9,10-PQ was significantly enhanced by addition of NAC. The formation of DNA double strand breaks accompanying phosphorylation of histone H2AX, was also remarkably augmented. On the other hand, the cyto- and genotoxicity were suppressed by addition of NAC in the adenocarcinoma cells. Two contrasting experiments: overexpression of NQO1 in CHO-K1 cells which originally expressed low NQO1 levels, and knock‐down of NQO1 in the adenocarcinoma cell line A549 by transfection of RNAi, also showed that NAC suppressed 9,10-PQ-induced toxicity in cell lines expressing high NQO1 activity and enhanced it in cell lines with low NQO1 activity. The results suggested that dual effects of NAC on the cyto- and genotoxicity of 9,10-PQ were dependent on tissue-specific NQO1 activity. -- Highlights: ► NAC augmented the cytotoxicity of 9,10-PQ in skin cell lines. ► 9,10-PQ-induced DSBs accompanying γ-H2AX were also augmented by NAC. ► NAC suppressed the cyto- and genotoxicity of 9,10-PQ in adenocarcinoma cell lines. ► The dual effects of NAC on toxicity of 9,10-PQ were dependent on NQO1 activity.« less

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2010-10-29

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

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.

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.

Yoda1 analogue (Dooku1) which antagonizes Yoda1‐evoked activation of Piezo1 and aortic relaxation

PubMed Central

Evans, Elizabeth L; Cuthbertson, Kevin; Endesh, Naima; Rode, Baptiste; Blythe, Nicola M; Hyman, Adam J; Hall, Sally J; Gaunt, Hannah J; Ludlow, Melanie J

2018-01-01

Background and Purpose The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small‐molecule agonist, but the pharmacology of these channels is otherwise limited. Experimental Approach Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta. Key Results Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1‐induced Ca2+‐entry with IC50s of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP‐evoked Ca2+ elevation or store‐operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose‐dependent relaxation of aortic rings, which was mediated by an endothelium‐ and NO‐dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1. Conclusion and Implications Chemical antagonism of Yoda1‐evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains. PMID:29498036

Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology.

PubMed

Thomsson, Elisabeth; Persson, Linn; Grahn, Anna; Snäll, Johanna; Ekblad, Maria; Brunhage, Eva; Svensson, Frida; Jern, Christina; Hansson, Gunnar C; Bäckström, Malin; Bergström, Tomas

2011-07-01

A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Nitrophenols isolated from diesel exhaust particles promote the growth of MCF-7 breast adenocarcinoma cells

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

Furuta, Chie; Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509; Suzuki, Akira K.

2008-08-01

Diesel exhaust particles (DEPs) cause many adverse health problems, and reports indicate increased risk of breast cancer in men and women through exposure to gasoline and vehicle exhaust. However, DEPs include vast numbers of compounds, and the specific compound(s) responsible for these actions are not clear. We recently isolated two nitrophenols from DEPs-3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP)-and showed that they had estrogenic and anti-androgenic activities. Here, we tried to clarify the involvement of these two nitrophenols in promoting the growth of the MCF-7 breast cancer cell line. First, comet assay was used to detect the genotoxicity of PNMC andmore » PNMPP in a CHO cell line. At all doses tested, PNMC and PNMPP showed negative genotoxicity, indicating that they had no tumor initiating activity. Next, the estrogen-responsive breast cancer cell line MCF-7 was used to assess cell proliferation. Proliferation of MCF-7 cells was stimulated by PNMC, PNMPP, and estradiol-17{beta} and the anti-estrogens 4-hydroxytamoxifen and ICI 182,780 inhibited the proliferation. To further investigate transcriptional activity through the estrogen receptor, MCF-7 cells were transfected with a receptor gene that allowed expression of luciferase enzyme under the control of the estrogen regulatory element. PNMC and PNMPP induced luciferase activity in a dose-dependent manner at submicromolar concentrations. ICI 182,780 inhibited the luciferase activity induced by PNMC and PNMPP. These results clearly indicate that PNMC and PNMPP do not show genotoxicity but act as tumor promoters in an estrogen receptor {alpha}-predominant breast cancer cell line.« 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

Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-{kappa}B

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

Bin Hafeez, Bilal; Ahmed, Salahuddin; Wang, Naizhen

2006-10-01

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-{kappa}B (NF-{kappa}B) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 {mu}g/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-{kappa}B/p65 and lowering of NF-{kappa}B/p65 and p50 levels in the cytoplasm and nucleus. GTPmore » treatment of cells reduced I{kappa}B-{alpha} phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-{alpha} and IKK-{beta}, the upstream kinases that phosphorylate I{kappa}B-{alpha}. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-{kappa}B.« less

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.

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.

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

PubMed

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

2007-02-01

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

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

PubMed

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

2018-05-31

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

Pre-biotic molecules in shocks: the case of L1157

NASA Astrophysics Data System (ADS)

Mendoza, Edgar; Lefloch, Bertrand; López-Sepulcre, Ana; Ceccarelli, Cecilia; Codella, Claudio; Boechat-Roberty, Heloisa M.; Bachiller, Rafael

2015-08-01

Interstellar molecules with a peptide link -NH-C(=O)-, like formamide (NH2CHO) and isocyanic acid (HNCO) are notably interesting for their potential role in pre-biotic chemistry. For first time, we report the detection of both molecules towards the protostellar shock L1157-B1 and L1157-B2, with the IRAM 30m telescope. Analysis of the line profiles shows that the emission arises from the outflow cavities associated with B1 and B2. From a simple rotational diagram analysis, molecular abundance of ≈ (0.4-1.1)×10-8 and (3.3-8.8)×10-8 are derived for NH2CHO and HNCO, respectively. The shock regions B1 and B2 appear to be among the richest Galactic sources of HNCO and NH2CHO molecules. We have observed a narrow linear correlation between their abundances, suggesting that the two species are chemically related. Comparison with astrochemical models favours molecule formation on ice grain mantles, with NH2CHO generated from hydrogenation of HNCO.

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

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

PubMed

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

2002-01-20

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

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.

Synthesis and Anti-cancer Activity of 3-substituted Benzoyl-4-substituted Phenyl-1H-pyrrole Derivatives.

PubMed

Zhan, Xiaoping; Qin, Weixi; Wang, Shuai; Zhao, Kai; Xin, Yuxuan; Wang, Yaolin; Qi, Qi; Mao, Zhenmin

2017-01-01

Cancer is considered a major public health problem worldwide. The aim of this paper is to design and synthesis of novel anticancer agents with potent anticancer activity and minimum side effects. A series of pyrrole derivatives were synthesized, their anti-cancer activity against nine cancer cell lines and two non-cancer cell lines were evaluated by MTT assay, and their cell cycle progression were determined by flow cytometry analysis. The study of the structure-activity relationships revealed that the introduction of the electron-donation groups at the 4th position of the pyrrole ring increased the anti-cancer activity. Among the synthesized compounds, specially the compounds bearing 3,4-dimethoxy phenyl at the 4th position of the pyrrole ring showed potent anti-cancer activity, cpd 19 was the most potent against MGC 80-3, HCT-116 and CHO cell lines (IC50s = 1.0－1.7 μM), cpd 21 was the most potent against HepG2, DU145 and CT-26 cell lines (IC50s = 0.5－0.9 μM), and cpd 15 was the most potent against A549 (IC50 = 3.6 μM). Moreover, these potent compounds showed weak cytotoxicity against HUVEC and NIH/3T3. Thus, the cpds 15, 19 and 21 show potential anti-cancer for further investigation. Furthermore, the flow cytometry analysis revealed that cpd 21 arrested the CT-26 cells at S phase, and induced the cell apoptosis. Thus, these compounds with the potent anticancer activity and low toxicity have potential for the development of new anticancer chemotherapy agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

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

Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

1993-07-13

A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with themore » enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.« less

Metabolic Imaging of Pancreatic Ductal Adenocarcinoma Detects Altered Choline Metabolism

PubMed Central

Penet, Marie-France; Shah, Tariq; Bharti, Santosh; Krishnamachary, Balaji; Artemov, Dmitri; Mironchik, Yelena; Wildes, Flonné; Maitra, Anirban; Bhujwalla, Zaver M.

2014-01-01

Purpose Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets. Experimental Design We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets. Results We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between HPNE and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter-like protein 1 (CTL1) in the PDAC cell lines and tumors. Conclusions Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging-probe independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. PMID:25370468

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.

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

PubMed

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

2000-07-03

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

Estimation of the initial slope of the cell survival curve after irradiation from micronucleus frequency in cytokinesis-blocked cells

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

Ono, K.; Masunaga, S.; Akaboshi, M.

1994-04-01

We have already reported that the {alpha}/{beta} ratio of the cell survival curve could be estimated from the micronucleus frequency in cytokinesis-blocked cells treated with cytochalasin-B after irradiation. In this paper, we investigate the direct relationship between the {alpha} value and the appearance of micronuclei. Cells of the SCCVII, RIF-1, EMT6, V-79, CHO, HeLa and human esophageal cancer cell lines were used for the study. Low-dose-rate irradiation was used to determine the {alpha} component of the relationship between dose and micronucleus frequency according to the linear-quadratic (LQ) model. A reduction of the dose rate from 3.09 to 0.0142 Gy/min correspondinglymore » decreased the micronucleus frequency; however, the fraction of binucleate cells without micronuclei was not affected in SCCVII and RIF-1 cells. When this fraction was defined as the normal nuclear division fraction, it decreased exponentially as a function of radiation dose. Then dose vs normal nuclear division fraction (NNDF) was fitted as follows: -In NNDF = aD + C, where D is radiation dose in grays and C is constant. The slope of the dose vs normal nuclear division fraction was not affected by dose rate. The correlation was also explored between the slope (a) and the {alpha} value of the cell survival curve determined by the colony formation assay in cells of eight cell lines. These two values showed extremely high agreement: {alpha} = 1.01a + 0.00795 (r = 0.99, P < 0.01). This assay was applied to estimate the {alpha} value of the cell survival curve of human esophageal cancer cell lines established from surgical specimens. 13 refs., 5 figs.« 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.

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.

Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

PubMed

Jiang, Hong Ning; Li, Yuan; Jiang, Wen Yi; Cui, Zong Jie

2018-01-01

Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm -2 ) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.

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

PubMed

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

2014-02-01

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions. © 2013 Wiley Periodicals, Inc.

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

PubMed Central

Kopera, Huira C.; Moldovan, John B.; Morrish, Tammy A.; Moran, John V.

2011-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase. PMID:21940498

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

PubMed

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

Testing grain-surface chemistry in massive hot-core regions

NASA Astrophysics Data System (ADS)

Bisschop, S. E.; Jørgensen, J. K.; van Dishoeck, E. F.; de Wachter, E. B. M.

2007-04-01

Aims:We study the chemical origin of a set of complex organic molecules thought to be produced by grain surface chemistry in high mass young stellar objects (YSOs). Methods: A partial submillimeter line-survey was performed toward 7 high-mass YSOs aimed at detecting H2CO, CH3OH, CH2CO, CH3CHO, C2H5OH, HCOOH, HNCO and NH2CHO. In addition, lines of CH3CN, C2H5CN, CH3CCH, HCOOCH3, and CH3OCH3 were observed. Rotation temperatures and beam-averaged column densities are determined. To correct for beam dilution and determine abundances for hot gas, the radius and H2 column densities of gas at temperatures >100 K are computed using 850 μm dust continuum data and source luminosity. Results: Based on their rotation diagrams, molecules can be classified as either cold (<100 K) or hot (>100 K). This implies that complex organics are present in at least two distinct regions. Furthermore, the abundances of the hot oxygen-bearing species are correlated, as are those of HNCO and NH2CHO. This is suggestive of chemical relationships within, but not between, those two groups of molecules. Conclusions: .The most likely explanation for the observed correlations of the various hot molecules is that they are "first generation" species that originate from solid-state chemistry. This includes H2CO, CH3OH, C2H5OH, HCOOCH3, CH3OCH3, HNCO, NH2CHO, and possibly CH3CN, and C2H5CN. The correlations between sources implies very similar conditions during their formation or very similar doses of energetic processing. Cold species such as CH2CO, CH3CHO, and HCOOH, some of which are seen as ices along the same lines of sight, are probably formed in the solid state as well, but appear to be destroyed at higher temperatures. A low level of non-thermal desorption by cosmic rays can explain their low rotation temperatures and relatively low abundances in the gas phase compared to the solid state. The CH3CCH abundances can be fully explained by low temperature gas phase chemistry. No cold N-containing molecules are found. Appendices are only available in electronic form at http://www.aanda.org

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

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

PubMed Central

Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Münz, Markus

2015-01-01

Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct. PMID:26510188

Bioactive cinchona alkaloids from Remijia peruviana.

PubMed

Ruiz-Mesia, Lastenia; Ruiz-Mesía, Wilfredo; Reina, Matías; Martínez-Diaz, Rafael; de Inés, Concepción; Guadaño, Ana; González-Coloma, Azucena

2005-03-23

Three known Cinchona alkaloids of the quinine type, quinine (1), cupreine (2), cinchonine (3), and the possible artifact cinchonine-HCl (3-HCl), along with two new ones, acetylcupreine (4) and N-ethylquinine (5), have been isolated from the bark of Remijia peruviana (Rubiaceae). Their stereochemical structures were established by high resolution NMR spectroscopy. Alkaloids 2-4 had antifeedant effects on Leptinotarsa decemlineata with varying potencies. Compound 4 was cytotoxic to both insect Sf9 and mammalian CHO cells after 48 h of incubation, while 3-HCl had stronger and selective cytotoxicity to Sf9. Quinine 1 had a moderate to low effect on Trypanosoma cruzi. Tumoral cells were also affected by these alkaloids, with 4 and 3-HCl being the most cytotoxic to all the cell lines tested. Overall, the 8R, 9S configurations, as in 3 and 3-HCl, as well as the C-6'acetylated alkaloid 4, with an 8S, 9R configuration, showed stronger biological effects.

A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors

PubMed Central

Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G.; Nimmagadda, Sridhar

2016-01-01

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings. PMID:26848870

An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin.

PubMed

Shalel Levanon, Sagit; Aharonovitz, Orit; Maor-Shoshani, Ayelet; Abraham, Gita; Kenett, Dan; Aloni, Yehoshua

2018-06-20

Glycosylation on the Fc region of recombinant Immunoglobulin G (IgG) therapeutic antibodies is a critical protein quality attribute which may affect the efficacy and safety of the molecule. During the development of biosimilar therapeutics, adjustment of the glycosylation profile is required in order to match the reference innovator profile. Deoxymannojirimycin (DMJ), a known inhibitor of mannosidase, was used in this study to modulate the glycosylation pattern of antibodies. The effect of DMJ, at concentrations of 5 μM - 500 μM, on non-fucosylated glycoform levels was tested in the biosynthesis processes of two different IgG1 (IgG1 #A and IgG1 #B) using two Chinese hamster ovary (CHO) cell lines (CHO-DXB-11 and CHOK1SV, respectively) in Erlenmeyer flasks and in lab scale bioreactors. DMJ affected glycan forms in a dose response manner. At the highest concentration tested, DMJ reduced N-linked complex glycoform and core fucose levels by 15 and 14 fold, respectively, and increased high mannose level by 21 fold. 10 μM DMJ decreased IgG1 #A core fucose level in CHO-DXB-11 from 92% to 73% and increased high mannose level from 4% to 22% in Erlenmeyer flasks. Furthermore, in lab scale bioreactors, 15 μM DMJ decreased IgG1 #A core fucose level from 95% to 84% and increased high mannose level from 3% to 13%. Core fucose level of IgG1 #B in CHOK1SV was decreased from 81% to 73% using 10 μM DMJ in lab scale bioreactors while high mannose was increased from 6% to 15%. While affecting core fucose and high mannose levels, DMJ decreased maximum viable cell concentration by 16% and did not significantly affect cell productivity (less than 10%). This study demonstrated that DMJ can enable the control of core fucosylated and high mannose levels of IgG1 antibodies in a defined range. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Application of magnetic carriers to two examples of quantitative cell analysis

NASA Astrophysics Data System (ADS)

Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

2017-04-01

The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

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

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.

Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

PubMed

Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

2014-02-01

The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

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.

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

PubMed Central

Yang, N S; Burkholder, J; Roberts, B; Martinell, B; McCabe, D

1990-01-01

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy. Images PMID:2175906

Metallothionein turnover in mammalian cell lines: implications in drug resistance

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

Monia, B.P.; Butt, T.R.; Ecker, D.J.

1986-05-01

Metallothioneins (MT) are low molecular weight, cysteine-rich proteins believed to participate in metal detoxification. A wide variety of cells in culture have been shown to accumulate MT in response to metal administration. These metal-induced increases in MT levels result from an increased rate of MT gene transcription, MT mRNA accumulation, and MT synthesis. Turnover of Cd-, Zn- and Au-induced MT was studied in a Chinese Hamster Ovary (CHO) cell line which was resistant to Cd and the Au-containing drug Auranofin (AF). Cd, Zn and Au were potent inducers of MT mRNA and accumulated approximately equal amounts of mRNA under themore » conditions employed in this study. Pulse-chase studies utilizing (/sup 35/S)cysteine revealed that the half-life of Au-, Zn- and Cd-induced MT was 0.75, 10 and 24 hrs. respectively. The reported differences in the tertiary structure of Au-MT from that of Cd-MT lead us to propose that the differences in half-lives observed reflect differences in subceptibility to intracellular proteolysis, which in turn, may effect the ability of MT to confer resistance to various metals.« less

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

PubMed

Hinz, J M; Meuth, M

1999-02-01

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

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

Potential Theranostics Application of Bio-Synthesized Silver Nanoparticles (4-in-1 System)

PubMed Central

Mukherjee, Sudip; Chowdhury, Debabrata; Kotcherlakota, Rajesh; Patra, Sujata; B, Vinothkumar; Bhadra, Manika Pal; Sreedhar, Bojja; Patra, Chitta Ranjan

2014-01-01

In this report, we have designed a simple and efficient green chemistry approach for the synthesis of colloidal silver nanoparticles (b-AgNPs) that is formed by the reduction of silver nitrate (AgNO3) solution using Olax scandens leaf extract. The colloidal b-AgNPs, characterized by various physico-chemical techniques exhibit multifunctional biological activities (4-in-1 system). Firstly, bio-synthesized silver nanoparticles (b-AgNPs) shows enhanced antibacterial activity compared to chemically synthesize silver nanoparticles (c-AgNPs). Secondly, b-AgNPs show anti-cancer activities to different cancer cells (A549: human lung cancer cell lines, B16: mouse melanoma cell line & MCF7: human breast cancer cells) (anti-cancer). Thirdly, these nanoparticles are biocompatible to rat cardiomyoblast normal cell line (H9C2), human umbilical vein endothelial cells (HUVEC) and Chinese hamster ovary cells (CHO) which indicates the future application of b-AgNPs as drug delivery vehicle. Finally, the bio-synthesized AgNPs show bright red fluorescence inside the cells that could be utilized to detect the localization of drug molecules inside the cancer cells (a diagnostic approach). All results together demonstrate the multifunctional biological activities of bio-synthesized AgNPs (4-in-1 system) that could be applied as (i) anti-bacterial & (ii) anti-cancer agent, (iii) drug delivery vehicle, and (iv) imaging facilitator. To the best of our knowledge, there is not a single report of biosynthesized AgNPs that demonstrates the versatile applications (4-in-1 system) towards various biomedical applications. Additionally, a plausible mechanistic approach has been explored for the synthesis of b-AgNPs and its anti-bacterial as well as anti-cancer activity. We strongly believe that bio-synthesized AgNPs will open a new direction towards various biomedical applications in near future. PMID:24505239

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

Endocytic function is critical for influenza A virus infection via DC-SIGN and L-SIGN

PubMed Central

Gillespie, Leah; Roosendahl, Paula; Ng, Wy Ching; Brooks, Andrew G.; Reading, Patrick C.; Londrigan, Sarah L.

2016-01-01

The ubiquitous presence of cell-surface sialic acid (SIA) has complicated efforts to identify specific transmembrane glycoproteins that function as bone fide entry receptors for influenza A virus (IAV) infection. The C-type lectin receptors (CLRs) DC-SIGN (CD209) and L-SIGN (CD209L) enhance IAV infection however it is not known if they act as attachment factors, passing virions to other unknown receptors for virus entry, or as authentic entry receptors for CLR-mediated virus uptake and infection. Sialic acid-deficient Lec2 Chinese Hamster Ovary (CHO) cell lines were resistant to IAV infection whereas expression of DC-SIGN/L-SIGN restored susceptibility of Lec2 cells to pH- and dynamin-dependent infection. Moreover, Lec2 cells expressing endocytosis-defective DC-SIGN/L-SIGN retained capacity to bind IAV but showed reduced susceptibility to infection. These studies confirm that DC-SIGN and L-SIGN are authentic endocytic receptors for IAV entry and infection. PMID:26763587

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus

PubMed Central

Ng, Wy Ching; Londrigan, Sarah L.; Nasr, Najla; Cunningham, Anthony L.; Turville, Stuart; Brooks, Andrew G.

2015-01-01

ABSTRACT It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. IMPORTANCE On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. PMID:26468543

A multi-landing pad DNA integration platform for mammalian cell engineering

PubMed Central

Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

2018-01-01

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of membrane dynamics in electrical and infrared neural stimulation

NASA Astrophysics Data System (ADS)

Moen, Erick K.; Beier, Hope T.; Ibey, Bennett L.; Armani, Andrea M.

2016-03-01

We recently developed a nonlinear optical imaging technique based on second harmonic generation (SHG) to identify membrane disruption events in live cells. This technique was used to detect nanoporation in the plasma membrane following nanosecond pulsed electric field (nsPEF) exposure. It has been hypothesized that similar poration events could be induced by the thermal gradients generated by infrared (IR) laser energy. Optical pulses are a highly desirable stimulus for the nervous system, as they are capable of inhibiting and producing action potentials in a highly localized but non-contact fashion. However, the underlying mechanisms involved with infrared neural stimulation (INS) are not well understood. The ability of our method to non-invasively measure membrane structure and transmembrane potential via Two Photon Fluorescence (TPF) make it uniquely suited to neurological research. In this work, we leverage our technique to understand what role membrane structure plays during INS and contrast it with nsPEF stimulation. We begin by examining the effect of IR pulses on CHO-K1 cells before progressing to primary hippocampal neurons. The use of these two cell lines allows us to directly compare poration as a result of IR pulses to nsPEF exposure in both a neuron-derived cell line, and one likely lacking native channels sensitive to thermal stimuli.

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

Synthesis and biological evaluation of a series of non-hemiacetal ester derivatives of artemisinin.

PubMed

Zuma, Nonkululeko H; Smit, Frans J; de Kock, Carmen; Combrinck, Jill; Smith, Peter J; N'Da, David D

2016-10-21

In an attempt to improve the efficacy and stability of current, clinically used artemisinins, a series non-hemiacetal ester derivatives of artemisinin were synthesized and evaluated for their in vitro antiplasmodial and anticancer activities as well as cytotoxicities. These esters were synthesized through the reaction of acid anhydrides, or acid chlorides with artemisinin derived alcohol. In vitro antiplasmodial activity assessments were conducted against intraerythrocytic NF54 and Dd2 Plasmodium falciparum strains. Cytotoxicities were assessed, using normal human fetal lung fibroblast (WI-38) and Chinese hamster ovarian (CHO) mammalian cell lines, while anticancer activities were tested by using panels with three cell lines, consisting of renal (TK10), melanoma (UACC62) and breast (MCF7) cancer cells. Most compounds were found active against the breast cancer cell line. Since antiplasmodial activities for most compounds were found comparable only to that of artesunate, this study did not yield any esters with significantly improved antimalarial efficacies, nor did it deliver any promising antitumor hits. However, from the outcomes of this study, compounds with good safety profiles and increased thermal stabilities, compared to the clinically used artemisinins, were identified. The benzoate derivative 11 was found to have antimalarial activity, comparable to that of dihydroartemisinin and was it subsequently identified as a candidate for further investigation in the urgent search for new, safe and effective antimalarial drugs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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.

Effects of caffeine on radiation-induced phenomena associated with cell- cycle traverse of mammalian cells

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

Walters, R.A.; Gurley, L.R.; Tobby, R.A.

1974-02-01

Caffeine induced a state of G/sub 1/ arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following x-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cellsmore » synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The x-rayinduced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeinetreated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed. (auth)« less

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.

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.

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

Interlab study on nanotoxicology of representative graphene oxide

NASA Astrophysics Data System (ADS)

Durán, Nelson; Martinez, Diego S. T.; Justo, Giselle Z.; de Lima, Renata; Lúcia de Castro, Vera; Umbuzeiro, Gisela A.; Barbieri, Edison; Durán, Marcela; Melo, Patricia S.; Alves, Oswaldo L.; Fávaro, Wagner J.

2015-05-01

The graphene sample GO:Single-layer graphene oxide, purity 99%, thickness 0.7-1.2 nm (AFM); ∼300-800nm X&Y dimensions is the standard size <450 nm & 1-20 μm lateral dimensions. Cheap Tubes Inc., Bratleboro, USA was selected for our study. Exhaustive characterization of GO was afforded. It exhibited thermal stability over 60oC and it was suspended in deionized water after ultrasonication(1 mg/mL) (stable 10 days). All the biological fluids used in the different assays were used as control of the colloidal suspension stability. Then, all the studies were carried out within that stability period. The cytotoxicity assays were carried out by the Alamar Blue (Resazurin) reduction, MTT and flow cytometry assays in mouse embryonic fibroblast cells (3T3), human keratinocytes (HaCaT), colorectal cancer cells (Caco-2/HCT 116), Lewis lung cancer cells (3LL), acute myeloid leukemia cells (KG-1, Jurkat, Kasumi-1) and chronic myeloid leukemia cells (K562, Lucena) and no significant toxicity was found after exposition to 0.1-100 μg/mL for 24 and 48 h. Breast cancer cells, MCF-7, showed a 20% reduction on cell viability at 24 and 48 h. No cytotoxicity were found in lymphocytes, Chinese hamster ovary cells (CHO) and human macrophage cell line (U937) at 0.1-50 μg/mL, but 30-50% survival inhibition was observed at 100 μg/mL. A dose-dependent increase in apoptosis was observed in some cells (Kasumi-1, Jurkatand K562 cells). In the case of CHO and 3T3 cells, greater levels of necrosis with increasing concentrations of GO (>50 μg/mL) were observed. Genotoxic study using the Comet assay showed slight DNA damage in lymphocytes at all concentrations tested, while more significant effects was observed in CHO cells. Econanotoxicity was carried out by lethality assays in the nematode Caenorhabditis elegans, d in the freshwater coelenterate Hydra, Daphania amd in Shrimp with no signs of toxicity at concentrations varying from 0.1-100 μg/mL of GO. However, death and disintegration of Hydra was observed after exposition to 100 μg/mL for 72 h. In in vivo studies, no changes in biochemical parameters of Fischer 344 rats were observed after the i.p. administration of GO. Some black agglomerates were found in the intraperitoneal cavity of rats injected with GO. However, in Fisher 344 rats-bearing prostate tumors, treatment with GO (up to 100 μg/mL) negatively affected the hepatic parameters, whilst in the renal ones, an improvement was observed. Studies are in progress to understand the mechanisms involved in the uptake of GO by RES. GO appears as a potential non-toxic in vitro and in vivo assays at the concentrations used in this interlab experiments.

Study of potential utility of new radiopharmaceuticals based on technetium-99m labeled derivative of glucose

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

Zeltchan, R., E-mail: r.zelchan@yandex.ru; Medvedeva, A.; Sinilkin, I.

Purpose: to study the potential utility of 1-thio-D-glucose labeled with {sup 99m}Tc for cancer imaging in laboratory animals. Materials and method: the study was carried out in cell cultures of normal CHO (Chinese hamster ovary cells CHO) and malignant tissues MCF-7 (human breast adenocarcinoma MCF-7). To evaluate the uptake of {sup 99m}Tc-1-thio-D-glucose in normal and tumor tissue cells, 25 MBq of 1-thio-D-glucose labeled with {sup 99m}Tc was added to the vials with 3 million cells and incubated for 30 min at room temperature. After centrifugation of the vials with cells, the supernatant was removed. The radioactivity in vials with normalmore » and tumor cells was then measured. In addition, the study included 40 mice of C57B1/6j lines with tumor lesion of the right femur. For neoplastic lesions, Lewis lung carcinoma model was used. Following anesthesia, mice were injected intravenously with 25 MBq of {sup 99m}Tc-1-thio-D-glucose. Planar scintigraphy was performed 15 minutes later in a matrix of 512x512 pixels for 5 min. Results: when measuring the radioactivity of normal and malignant cells after incubation with {sup 99m}Tc-1-thio-D-glucose, it was found that the radioactivity of malignant cells was higher than that of normal cells. The mean values of radioactivity levels in normal and malignant cells were 0.3 ± 0.15 MBq and 1.07 ± 0.6 MBq, respectively. All examined animals had increased accumulation of {sup 99m}Tc-1-thio-D-glucose at the tumor site. The accumulation of {sup 99m}Tc-1-thio-D-glucose in the tumor was on average twice as high as compared to the symmetric region. Conclusion: The present study demonstrated that {sup 99m}Tc-1-thio-D-glucose is a prospective radiopharmaceutical for cancer visualization. In addition, high accumulation of {sup 99m}Tc-1-thio-D-glucose in the culture of cancer cells and in tumor tissue of animals demonstrates tumor tropism of the radiopharmaceutical.« less

Experimental study of radiopharmaceuticals based on technetium-99m labeled derivative of glucose for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zeltchan, R.; Medvedeva, A.; Sinilkin, I.; Bragina, O.; Chernov, V.; Stasyuk, E.; Rogov, A.; Il'ina, E.; Larionova, L.; Skuridin, V.; Dergilev, A.

2016-06-01

Purpose: to study the potential utility of 1-thio-D-glucose labeled with 99mTc for cancer imaging in laboratory animals. Materials and method: the study was carried out in cell cultures of normal CHO (Chinese hamster ovary cells CHO) and malignant tissues MCF-7 (human breast adenocarcinoma MCF-7). To evaluate the uptake of 99mTc-1-thio-D-glucose in normal and tumor tissue cells, 25 MBq of 1-thio-D-glucose labeled with 99mTc was added to the vials with 3 million cells and incubated for 30 minutes at room temperature. After centrifugation of the vials with cells, the supernatant was removed. Radioactivity in vials with normal and tumor cells was then measured. In addition, the study included 40 mice of C57B 1/6j lines with tumor lesion of the right femur. For neoplastic lesions, Lewis lung carcinoma model was used. Following anesthesia, mice were injected intravenously with 25MBq of 99mTc-1-thio-D-glucose. Planar scintigraphy was performed 15 minutes later in a matrix of 512x512 pixels for 5 minutes. Results: when measuring the radioactivity of normal and malignant cells after incubation with 99mTc-1-thio-D- glucose, it was found that the radioactivity of malignant cells was higher than that of normal cells. The mean values of radioactivity levels in normal and malignant cells were 0.3±0.15MBq and 1.07±0.6MBq, respectively. All examined animals had increased accumulation of 99mTc-1-thio- D-glucose at the tumor site. The accumulation of 99mTc-1-thio-D-glucose in the tumor was on average twice as high as compared to the symmetric region. Conclusion: The present study demonstrated that 99mTc-1-thio-D-glucose is a prospective radiopharmaceutical for cancer visualization. In addition, high accumulation of 99mTc-1-thio-D-glucose in the culture of cancer cells and in tumor tissue of animals demonstrates tumor tropism of the radiopharmaceutical.

Study of potential utility of new radiopharmaceuticals based on technetium-99m labeled derivative of glucose

NASA Astrophysics Data System (ADS)

Zeltchan, R.; Medvedeva, A.; Sinilkin, I.; Chernov, V.; Stasyuk, E.; Rogov, A.; Il'ina, E.; Larionova, L.; Skuridin, V.

2016-08-01

Purpose: to study the potential utility of 1-thio-D-glucose labeled with 99mTc for cancer imaging in laboratory animals. Materials and method: the study was carried out in cell cultures of normal CHO (Chinese hamster ovary cells CHO) and malignant tissues MCF-7 (human breast adenocarcinoma MCF-7). To evaluate the uptake of 99mTc-1-thio-D-glucose in normal and tumor tissue cells, 25 MBq of 1-thio-D-glucose labeled with 99mTc was added to the vials with 3 million cells and incubated for 30 min at room temperature. After centrifugation of the vials with cells, the supernatant was removed. The radioactivity in vials with normal and tumor cells was then measured. In addition, the study included 40 mice of C57B1/6j lines with tumor lesion of the right femur. For neoplastic lesions, Lewis lung carcinoma model was used. Following anesthesia, mice were injected intravenously with 25 MBq of 99mTc-1-thio-D-glucose. Planar scintigraphy was performed 15 minutes later in a matrix of 512x512 pixels for 5 min. Results: when measuring the radioactivity of normal and malignant cells after incubation with 99mTc-1-thio-D-glucose, it was found that the radioactivity of malignant cells was higher than that of normal cells. The mean values of radioactivity levels in normal and malignant cells were 0.3 ± 0.15 MBq and 1.07 ± 0.6 MBq, respectively. All examined animals had increased accumulation of 99mTc-1-thio-D-glucose at the tumor site. The accumulation of 99mTc-1-thio-D-glucose in the tumor was on average twice as high as compared to the symmetric region. Conclusion: The present study demonstrated that 99mTc-1-thio-D-glucose is a prospective radiopharmaceutical for cancer visualization. In addition, high accumulation of 99mTc-1-thio-D-glucose in the culture of cancer cells and in tumor tissue of animals demonstrates tumor tropism of the radiopharmaceutical.

DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair.

PubMed

Bajinskis, Ainars; Natarajan, Adayapalam T; Erixon, Klaus; Harms-Ringdahl, Mats

2013-08-30

The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by (137)Cs γ-rays or radon progeny α-particles. Irradiation was also performed in the presence of 2M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with γ-rays or α-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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.

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Single-step purification of recombinant Gaussia luciferase from serum-containing culture medium of mammalian cells.

PubMed

Inouye, Satoshi

2018-01-01

A dihydrofolate reductase-deficient Chinese hamster ovary (CHO-K1/dhfr - ) cell line stably expressing Gaussia luciferase with a histidine-tag sequence at the carboxyl terminus (GLase-His) was established. Recombinant GLase-His was purified from serum-containing culture medium by single-step Ni-chelate column chromatography in the presence of 2 M NaCl and 0.01% Tween 20. The protein yield of GLase-His with over 95% purity was 0.5 mg from 0.9 L of the cultured medium. The enzymatic properties of purified GLase-His were characterized. Interestingly, non-ionic detergent Tween 20 stabilized and stimulated GLase-His activity and its luminescence activity was stimulated 2-fold by the synergistic effect of 0.01% Tween 20 and 150 mM NaCl. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells.

PubMed

Ghosh-Choudhury, N; Butcher, M; Ghosh, H P

1990-03-01

A DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.

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

PubMed Central

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

1994-01-01

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

From the Cover: An Investigation of the Genotoxicity and Interference of Gold Nanoparticles in Commonly Used In Vitro Mutagenicity and Genotoxicity Assays.

PubMed

George, Jiya M; Magogotya, Millicent; Vetten, Melissa A; Buys, Antoinette V; Gulumian, Mary

2017-03-01

The suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (AuNPs). Specifically, the Ames test was conducted without metabolic activation, where no mutagenic effects were observed. High resolution transmission electron microscopy and Cytoviva dark-field image analysis showed that AuNPs did not enter the bacterial cells, thus confirming the unreliability of the Ames test for nanoparticle mutagenicity studies. In addition, the Chinese hamster ovary (CHO) cell line was used for Comet, Chromosome aberration and Micronucleus assays. CHO cells were treated with AuNPs for 20 h at 37 °C. Cytotoxicity was not detected by cell impedance studies even though AuNP uptake was confirmed using Cytoviva image analysis. The DNA damage was statistically significant in treated cells when assessed by the Comet assay. However, minimal and nonstatistically significant chromosomal DNA damage was observed using the chromosome aberration and micronucleus assays. In this study, we showed that false positive results obtained with Comet assay may have been due to the possibility of direct contact between the residual, intracellular AuNPs and DNA during the assay procedure. Therefore, the chromosome aberration and micronucleus assays are better suited to assess the genotoxic effects of nanoparticles due to low probability of such direct contact occurring. Genotoxic effect of 14 and 20 nm citrate-stabilized, as well as, 14 nm PCOOH AuNPs were also investigated using chromosome aberration and micronucleus assays. Based on our acceptance criteria for a positive genotoxic response, none of the AuNPs were found to be genotoxic in either of these assays. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Abundances of ethylene oxide and acetaldehyde in hot molecular cloud cores

NASA Technical Reports Server (NTRS)

Nummelin, A.; Dickens, J. E.; Bergman, P.; Hjalmarson, A.; Irvine, W. M.; Ikeda, M.; Ohishi, M.

1998-01-01

We have searched for millimetre-wave line emission from ethylene oxide (c-C2H4O) and its structural isomer acetaldehyde (CH3CHO) in 11 molecular clouds using SEST. Ethylene oxide and acetaldehyde were detected through multiple lines in the hot cores NGC 6334F, G327.3-0.6, G31.41+0.31, and G34.3+0.2. Acetaldehyde was also detected towards G10.47+0.03, G322.2+0.6, and Orion 3'N, and one ethylene oxide line was tentatively detected in G10.47+0.03. Column densities and rotational excitation temperatures were derived using a procedure which fits the observed line intensifies by finding the minimum chi 2-value. The resulting rotational excitation temperatures of ethylene oxide and acetaldehyde are in the range 16-38 K, indicating that these species are excited in the outer, cooler parts of the hot cores or that the excitation is significantly subthermal. For an assumed source size of 20", the deduced column densities are (0.6-1)x10(14) cm-2 for ethylene oxide and (2-5)x10(14) cm-2 for acetaldehyde. The fractional abundances with respect to H2 are X[c-C2H4O]=(2-6)xl0(-10), and X[CH3CHO]=(0.8-3)x10(-9). The ratio X[CH3CHO]/X[c-C2H4O] varies between 2.6 (NGC 6334F) and 8.5 (G327.3-0.6). We also detected and analysed multiple transitions of CH3OH, CH3OCH3, C2H5OH, and HCOOH. The chemical, and possibly evolutionary, states of NGC 6334F, G327.3-0.6, G31.41+0.31, and G34.3+0.2 seem to be very similar.

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.

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.

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

Potential antitumor agent from the endophytic fungus Pestalotiopsis photiniae induces apoptosis via the mitochondrial pathway in HeLa cells.

PubMed

Chen, Chuan; Hu, Shu-Yuan; Luo, Du-Qiang; Zhu, Si-Yu; Zhou, Chuan-Qi

2013-10-01

4-(3',3'-Dimethylallyloxy)-5-methyl-6-methoxy-phthalide (DMMP) has previously been isolated from the endophytic fungus Pestalotiopsis photiniae. Although the cytotoxic activities of DMMP have been reported, little is known concerning the molecular mechanism of its cytotoxic effect. In the present study, we investigated the effect of DMMP on the growth of several types of cancer cell lines and investigated the mechanism of its antiproliferative effect. DMMP caused the growth inhibition of human cancer lines HeLa, MCF7 and MDA-MB-231, but had little antiproliferative effect on MRC5 normal lung cells. DMMP also significantly caused cell cycle arrest in the G1 phase and upregulated the cyclin-dependent kinase inhibitor p27KIPI protein in the HeLa cells. Moreover DMMP was able to induce marked nuclear apoptotic morphology in HeLa cells. DMMP induced apoptosis and loss of mitochondrial membrane potential (ΔΨm) in the HeLa cells. Although the activated forms of caspase-9 and -3 in HeLa cells were detected, pretreatment with caspase inhibitors (Ac-DEVD-CHO and Z-VAD-FMK) failed to attenuate DMMP-induced cell death. In addition, protein levels of the p53 family members, p53 and p73, were upregulated, and DMMP significantly increased the mRNA expression of pro-apoptotic Bcl-2 family genes (PUMA, NOXA, Bax, Bad and Bim). HPV E6-E7 mRNA levels were reduced. In conclusion, DMMP demonstrates potential for use in the treatment of cervical 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

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

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.

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.

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

PubMed

Muyan, M; Boime, I

1998-05-01

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGbeta subunit (CGbeta114) with the alpha-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGbeta and CGbeta114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

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

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

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

1982-01-01

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

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.

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus.

PubMed

Ng, Wy Ching; Londrigan, Sarah L; Nasr, Najla; Cunningham, Anthony L; Turville, Stuart; Brooks, Andrew G; Reading, Patrick C

2016-01-01

It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5(+)) but not late (Rab7(+)) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. Copyright © 2015, American Society for Microbiology. 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.

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.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

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

Belakavadi, Madesh; Prabhakar, B.T.; Salimath, Bharathi P.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptoticmore » effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.« less

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

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

NASA Astrophysics Data System (ADS)

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

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

Simulating the Effect of Spectroscopic MRI as a Metric for Radiation Therapy Planning in Patients with Glioblastoma

PubMed Central

Cordova, J. Scott; Kandula, Shravan; Gurbani, Saumya; Zhong, Jim; Tejani, Mital; Kayode, Oluwatosin; Patel, Kirtesh; Prabhu, Roshan; Schreibmann, Eduard; Crocker, Ian; Holder, Chad A.; Shim, Hyunsuk; Shu, Hui-Kuo

2017-01-01

Due to glioblastoma’s infiltrative nature, an optimal radiation therapy (RT) plan requires targeting infiltration not identified by anatomical magnetic resonance imaging (MRI). Here, high-resolution, whole-brain spectroscopic MRI (sMRI) is used to describe tumor infiltration alongside anatomical MRI and simulate the degree to which it modifies RT target planning. In 11 patients with glioblastoma, data from preRT sMRI scans were processed to give high-resolution, whole-brain metabolite maps normalized by contralateral white matter. Maps depicting choline to N-Acetylaspartate (Cho/NAA) ratios were registered to contrast-enhanced T1-weighted RT planning MRI for each patient. Volumes depicting metabolic abnormalities (1.5−, 1.75−, and 2.0-fold increases in Cho/NAA ratios) were compared with conventional target volumes and contrast-enhancing tumor at recurrence. sMRI-modified RT plans were generated to evaluate target volume coverage and organ-at-risk dose constraints. Conventional clinical target volumes and Cho/NAA abnormalities identified significantly different regions of microscopic infiltration with substantial Cho/NAA abnormalities falling outside of the conventional 60 Gy isodose line (41.1, 22.2, and 12.7 cm3, respectively). Clinical target volumes using Cho/NAA thresholds exhibited significantly higher coverage of contrast enhancement at recurrence on average (92.4%, 90.5%, and 88.6%, respectively) than conventional plans (82.5%). sMRI-based plans targeting tumor infiltration met planning objectives in all cases with no significant change in target coverage. In 2 cases, the sMRI-modified plan exhibited better coverage of contrast-enhancing tumor at recurrence than the original plan. Integration of the high-resolution, whole-brain sMRI into RT planning is feasible, resulting in RT target volumes that can effectively target tumor infiltration while adhering to conventional constraints. PMID:28105468

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.

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.

Synchronized mammalian cell culture: part I--a physical strategy for synchronized cultivation under physiological conditions.

PubMed

Barradas, Oscar Platas; Jandt, Uwe; Becker, Max; Bahnemann, Janina; Pörtner, Ralf; Zeng, An-Ping

2015-01-01

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large-scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole-culture methods. Conventional nonsynchronizing methods with subsequent cell-specific, for example, flow cytometric analysis, can only resolve cell-limited effects of the cell cycle. In this work, we demonstrate countercurrent-flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO-K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2/M phase, with enrichment factors (Ysync) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population-resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions. © 2014 American Institute of Chemical Engineers.

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.

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.

Characterization of Relative Biological Effectiveness for Proton Therapy in Human Cancer Cell Lines

NASA Astrophysics Data System (ADS)

Howard, Michelle Erin

Purpose: Relative biological effectiveness (RBE) is utilized to account for the differences in biological effect from different radiation types. The RBE for proton therapy remains uncertain as it has been shown to vary from the clinically used value of 1.1. The purpose of this thesis was to investigate the RBE of protons as compared to X-rays and correlate the biological differences with the underlying physics. Methods: Three cell lines were irradiated (CHO, Chinese hamster ovary; A549, human lung adenocarcinoma; and T98, human glioma) and assessed for cell survival using clonogenic assay. Cells were irradiated with 71 and 160 MeV protons at depths along the Bragg curve and 6 MV X-rays to various doses. To correlate the underlying physics to RBE, both the dose averaged lineal energy (y¯D) and dose averaged LET (LETd) investigated. The microdosimetric quantity y¯D was measured under similar conditions as the cells using a solid state microdosimeter and LETd calculated using Monte Carlo (MC) simulations. Survival data were fit using the linear quadratic model. RBE values were calculated by comparing the physical dose (D6MV/Dp) that results in 50% (RBE0.5), 10% (RBE0.1) cell survival, and survival after 2Gy (RBE2 Gy).. Results: For 10% and 50% survival, the RBE for all three cell lines increased with decreasing proton energy (or increased depth). The RBE at 2Gy also increased with a decrease in proton energy in all cases, within experimental error. Results also showed the experimental end point proved to influence the measured proton RBE as well with larger values corresponding to 50% cell survival. Cell type had the least influence on proton RBE compared to proton energy and end point. Results from this study showed an increase in RBE corresponded to an increase in both LETd and y¯ D. Additionally, the measured y¯D and calculated LET d values did not match for all the points of measurement along the curve for the 71 and 160 MeV proton beams. Conclusion: Proton RBE depends on proton energy, cell type and LET. Cellular response to radiation is varied and can be seen in the data from CHO, A549 and T98 cell lines irradiated with 71 MeV protons. Both A549 and T98 cells generally had higher RBE values, indicating a greater biological response to protons. The RBE values in this study vary from 0.89- 2.40, indicating the clinical value of 1.1 may not be suitable in all cases. Innovation/impact: The rare but devastating complication of brainstem necrosis has occurred recently in pediatric patients treated with proton therapy (PT). Many believe these effects are due to the uncertainty in the RBE of PT, which may be underestimating the biological dose near critical structures. An increased confidence in RBE for PT can lead to a decrease in toxicity to normal tissues and therefore a decrease in secondary or recurrent cancer and better overall patient outcomes. This study is one of the first to consider the intricacies of proton RBE dependence on parameters such as cell type, proton energy and LET. The broader implications of understanding RBE variations in a cell-specific manner will allow for biologically optimized treatment planning and an overall decrease in PT uncertainty which may lead to improved patient outcomes. Further, this was the first study, to our knowledge, to measure y¯D with a solid state detector for the comparison with measured RBE values.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

PubMed Central

Cao, Yuhong; Chen, Haodong; Birey, Fikri; Leal-Ortiz, Sergio A.; Han, Crystal M.; Santiago, Juan G.; Paşca, Sergiu P.; Wu, Joseph C.; Melosh, Nicholas A.

2017-01-01

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation. PMID:28223521

Directional cell migration in an extracellular pH gradient: a model study with an engineered cell line and primary microvascular endothelial cells.

PubMed

Paradise, Ranjani K; Whitfield, Matthew J; Lauffenburger, Douglas A; Van Vliet, Krystyn J

2013-02-15

Extracellular pH (pH(e)) gradients are characteristic of tumor and wound environments. Cell migration in these environments is critical to tumor progression and wound healing. While it has been shown previously that cell migration can be modulated in conditions of spatially invariant acidic pH(e) due to acid-induced activation of cell surface integrin receptors, the effects of pH(e) gradients on cell migration remain unknown. Here, we investigate cell migration in an extracellular pH(e) gradient, using both model α(v)β(3) CHO-B2 cells and primary microvascular endothelial cells. For both cell types, we find that the mean cell position shifts toward the acidic end of the gradient over time, and that cells preferentially polarize toward the acidic end of the gradient during migration. We further demonstrate that cell membrane protrusion stability and actin-integrin adhesion complex formation are increased in acidic pH(e), which could contribute to the preferential polarization toward acidic pH(e) that we observed for cells in pH(e) gradients. These results provide the first demonstration of preferential cell migration toward acid in a pH(e) gradient, with intriguing implications for directed cell migration in the tumor and wound healing environments. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Global brain metabolic quantification with whole-head proton MRS at 3 T.

PubMed

Kirov, Ivan I; Wu, William E; Soher, Brian J; Davitz, Matthew S; Huang, Jeffrey H; Babb, James S; Lazar, Mariana; Fatterpekar, Girish; Gonen, Oded

2017-10-01

Total N-acetyl-aspartate + N-acetyl-aspartate-glutamate (NAA), total creatine (Cr) and total choline (Cho) proton MRS ( 1 H-MRS) signals are often used as surrogate markers in diffuse neurological pathologies, but spatial coverage of this methodology is limited to 1%-65% of the brain. Here we wish to demonstrate that non-localized, whole-head (WH) 1 H-MRS captures just the brain's contribution to the Cho and Cr signals, ignoring all other compartments. Towards this end, 27 young healthy adults (18 men, 9 women), 29.9 ± 8.5 years old, were recruited and underwent T 1 -weighted MRI for tissue segmentation, non-localizing, approximately 3 min WH 1 H-MRS (T E /T R /T I  = 5/10/940 ms) and 30 min 1 H-MR spectroscopic imaging (MRSI) (T E /T R  = 35/2100 ms) in a 360 cm 3 volume of interest (VOI) at the brain's center. The VOI absolute NAA, Cr and Cho concentrations, 7.7 ± 0.5, 5.5 ± 0.4 and 1.3 ± 0.2 mM, were all within 10% of the WH: 8.6 ± 1.1, 6.0 ± 1.0 and 1.3 ± 0.2 mM. The mean NAA/Cr and NAA/Cho ratios in the WH were only slightly higher than the "brain-only" VOI: 1.5 versus 1.4 (7%) and 6.6 versus 5.9 (11%); Cho/Cr were not different. The brain/WH volume ratio was 0.31 ± 0.03 (brain ≈ 30% of WH volume). Air-tissue susceptibility-driven local magnetic field changes going from the brain outwards showed sharp gradients of more than 100 Hz/cm (1 ppm/cm), explaining the skull's Cr and Cho signal losses through resonance shifts, line broadening and destructive interference. The similarity of non-localized WH and localized VOI NAA, Cr and Cho concentrations and their ratios suggests that their signals originate predominantly from the brain. Therefore, the fast, comprehensive WH- 1 H-MRS method may facilitate quantification of these metabolites, which are common surrogate markers in neurological disorders. Copyright © 2017 John Wiley & Sons, Ltd.

Biodirected synthesis of Miconazole-conjugated bacterial silver nanoparticles and their application as antifungal agents and drug delivery vehicles.

PubMed

Kumar, C Ganesh; Poornachandra, Y

2015-01-01

The recent strategy to improve the efficacy of drugs is to combine them with metal nanoparticles for the control of microbial infections. Considering this fact, we developed a low cost and eco-friendly method for silver nanoparticles synthesis using the cell free supernatant of Delftia sp. strain KCM-006 and their application as antifungal agents and as a drug carrier. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis revealed the formation of spherical and monodispersed silver nanoparticles with an average size of 9.8 nm. The synthesized nanoparticles were found to be photoluminescent, highly stable and crystalline in nature having a zeta potential of -31 mV. The silver nanoparticles exhibited very good antifungal activity against various pathogenic Candida strains. Furthermore, the efficacy of nanoparticles was increased by conjugating the antifungal drug Miconazole to silver nanoparticles which exhibited significant fungicidal activity, inhibition of ergosterol biosynthesis and biofilm inhibition by increasing ROS levels. In addition, the cell viability and immunocytochemistry analysis against different normal cell lines including Chinese hamster ovary cells (CHO), human lung cell line (MRC5) and human vascular endothelial cells (HUVEC) demonstrated that these nanoparticles were non-toxic up to a concentration of 20 μM. In conclusion, these results suggest that the synthesized nanoparticles find application as both antifungal agents and drug delivery vehicles. This is a first report on the preparation of silver nanoparticles using culture supernatant from Delftia sp. and also on the conjugation of Miconazole, an antifungal drug, to the bacterial silver nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell culture medium improvement by rigorous shuffling of components using media blending.

PubMed

Jordan, Martin; Voisard, Damien; Berthoud, Antoine; Tercier, Laetitia; Kleuser, Beate; Baer, Gianni; Broly, Hervé

2013-01-01

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.

The Surprising Complexity of Diffuse and Translucent Clouds Toward SGR B2: Diatomics and COMs from 4 GHz to 1.2 THz

NASA Astrophysics Data System (ADS)

McGuire, Brett A.; Corby, Joanna F.; Martin-Drumel, Marie-Aline; Schilke, P.; McCarthy, Michael C.; Remijan, Anthony

2017-06-01

Many diffuse and translucent clouds lie along the line of sight between Earth and the Galactic Center that can be probed through molecular absorption at characteristic velocities. We highlight results of a study of diffuse and translucent clouds along the line of sight to Sgr B2, including SOFIA observations of SH near 1.4 THz and GBT PRIMOS observations from 4 to 50 GHz. We find significant variation in the chemical conditions within these clouds, and the abundances do not appear to correlate with the total optical depth. Additionally, from the GBT observations, we report the first detections of multiple complex organic molecules (COMs) in diffuse and translucent clouds, including CH_3CN, HC_3N, CH_3CHO, and NH_2CHO. We compare the GBT results to complementary observations of SH, H_2S, and others at mm, sub-mm, and THz frequencies from the NRAO 12m, Herschel HIFI, and SOFIA facilities, and comment on the insights into interstellar sulfur chemistry which is currently not well constrained.

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.

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

Gold nanoparticles and electroporation impose both separate and synergistic radiosensitizing effects in HT-29 tumor cells: an in vitro study.

PubMed

Rezaee, Zohre; Yadollahpour, Ali; Bayati, Vahid; Negad Dehbashi, Fereshteh

2017-01-01

Radiation therapy (RT) is the gold standard treatment for more than half of known tumors. Despite recent improvements in RT efficiency, the side effects of ionizing radiation (IR) in normal tissues are a dose-limiting factor that restricts higher doses in tumor treatment. One approach to enhance the efficiency of RT is the application of radiosensitizers to selectively increase the dose at the tumor site. Gold nanoparticles (GNPs) and electroporation (EP) have shown good potential as radiosensitizers for RT. This study aims to investigate the sensitizing effects of EP, GNPs, and combined GNPs-EP on the dose enhancement factor (DEF) for 6 MV photon energy. Radiosensitizing effects of EP, GNPs, and combinations of GNPs-EP were comparatively investigated in vitro for intestinal colon cancer (HT-29) and Chinese hamster ovary (CHO) cell lines by MTT assay and colony formation assay at 6 MV photon energy in six groups: IR (control group), GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR. Treatment of both cell lines with EP, GNPs, and combined GNPs-EP significantly enhanced the response of cells to irradiation. However, the HT-29 showed higher DEF values for all groups. In addition, the DEF value for HT-29 cells for GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR was, respectively, 1.17, 1.47, 1.36, 2.61, and 2.89, indicating synergistic radiosensitizing effect for the GNPs (24 h)+EP+IR group. Furthermore, the synergistic effect was observed just for HT-29 tumor cell lines. Combined GNPs-EP protocols induced synergistic radiosensitizing effect in HT-29 cells, and the effect is also tumor specific. This combined therapy can be beneficially used for the treatment of intrinsically less radiosensitive tumors.

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.

Box C/D small nucleolar RNA (snoRNA) U60 regulates intracellular cholesterol trafficking.

PubMed

Brandis, Katrina A; Gale, Sarah; Jinn, Sarah; Langmade, Stephen J; Dudley-Rucker, Nicole; Jiang, Hui; Sidhu, Rohini; Ren, Aileen; Goldberg, Anna; Schaffer, Jean E; Ory, Daniel S

2013-12-13

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.

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

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.

Genotoxic action of an aqueous extract of Heliotropium curassavicum var. argentinum.

PubMed

Carballo, M; Mudry, M D; Larripa, I B; Villamil, E; D'Aquino, M

1992-06-16

Heliotropium curassavicum var. argentinum is widely employed in gout, rheumatism, neuralgias, arteriosclerotic disorders, muscular algias, phlebitis, varix and other illnesses. In order to analyze the genotoxic effect produced in vitro by this medicinal plant, chromosomal aberrations (CA), mitotic index (MI) and anaphase delay (AD) were studied in the CHO cell line, with and without the addition of S9 mix. Prepared according to the Argentine pharmacopeia 0.1, 1, 10 and 100 micrograms/ml plant decoction (aqueous extract) were assayed. One hundred cells per culture were studied for CA and AD, while MI was calculated for 2000 nuclei. The results revealed a significant increase in the percentage of abnormal metaphases (p less than 0.001) and in total aberrations (p less than 0.001). Both the MI and the AD affected the cell cycle. All results were enhanced by the addition of an S9 fraction. The toxic effect could be associated with pyrrolizidine alkaloids and their N-oxides, which through a process of in vitro metabolism become activated by microsomal oxidation and change into pyrrolic derivatives.

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.

Rapid desensitization of vasopressin-stimulated phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis questions the role of these pathways in sustained diacylglycerol formation in A10 vascular-smooth-muscle cells.

PubMed

Plevin, R; Wakelam, M J

1992-08-01

The kinetics of vasopressin-stimulated PtdIns(4,5)P2 and phosphatidylcholine (PtdCho) hydrolysis in relation to sustained diacylglycerol (DAG) formation was investigated in A10 vascular-smooth-muscle cells in culture. Vasopressin stimulated a transient increase in Ins(1,4,5)P3 mass formation, which was mirrored by a decrease in PtdIns(4,5)P2 mass levels. Vasopressin stimulated sustained accumulation of total [3H]inositol phosphates ([3H]IP) in the presence of Li+; however, this was significantly decreased by adding a vasopressin-receptor antagonist at different times after initial stimulation. Vasopressin-stimulated phospholipase D (PLD) activity was found to be a transient phenomenon lasting approx. 2 min. Experiments involving agonist preincubation with subsequent addition of butanol confirmed that vasopressin-stimulated PLD activity was desensitized. Vasopressin stimulated an increase in formation of choline, but not of phosphocholine, suggesting that PLD was the major catalytic route of PtdCho hydrolysis in this cell line. The roles of choline and inositol phospholipid hydrolysis in the prolonged phase of DAG formation was examined by comparing vasopressin-stimulated changes in DAG levels in the presence of butanol, the protein kinase C inhibitor Ro-31-8220 or a V1a-receptor antagonist. Vasopressin-stimulated DAG formation was decreased by 40-50% in the presence of butanol between 1 and 10 min; however, during more prolonged stimulation butanol was without significant effect. In cells pretreated with Ro-31-8220, vasopressin-stimulated DAG formation was decreased by approx. 30% at 2 min, but was significantly potentiated at later times. This coincided with an enhancement of vasopressin-stimulated [3H]IP accumulation. In cells exposed to the V1a-receptor antagonist 5 min after addition of vasopressin, subsequent DAG formation was significantly decreased, indicating that sustained formation of DAG, like [3H]IP accumulation, was dependent on continual agonist receptor activation. The results are discussed in terms of different phospholipid-hydrolytic pathways providing DAG generation.

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

PubMed

Jung, Kwang Bo; Lee, Hana; Son, Ye Seul; Lee, Ji Hye; Cho, Hyun-Soo; Lee, Mi-Ok; Oh, Jung-Hwa; Lee, Jaemin; Kim, Seokho; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

2018-01-01

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5 mCherry ) and intestine-specific homeobox enhanced green fluorescence protein (ISX eGFP ). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. © FASEB.

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

NASA Technical Reports Server (NTRS)

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

1997-01-01

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

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene

NASA Technical Reports Server (NTRS)

Kraemer, S. M.; Vannais, D. B.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

2001-01-01

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.

PubMed

Golsaz Shirazi, Forough; Amiri, Mohammad Mehdi; Mohammadi, Hamed; Bayat, Ali Ahmad; Roohi, Azam; Khoshnoodi, Jalal; Zarnani, Amir Hassan; Jeddi-Tehrani, Mahmood; Kardar, Gholam Ali; Shokri, Fazel

2013-09-01

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Impact of various lipophilic substituents on ruthenium(II), rhodium(III) and iridium(III) salicylaldimine-based complexes: synthesis, in vitro cytotoxicity studies and DNA interactions.

PubMed

Cassells, Irwin; Stringer, Tameryn; Hutton, Alan T; Prince, Sharon; Smith, Gregory S

2018-05-30

A series of bidentate salicylaldimine ligands was prepared and reacted with either [RuCl(µ-Cl)(p-cymene)] 2 , [RhCl(µ-Cl)(Cp*)] 2 or [IrCl(µ-Cl)(Cp*)] 2 . All of the compounds were characterised using an array of spectroscopic and analytical techniques, namely, nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and mass spectrometry. Single crystal X-ray diffraction (XRD) was used to confirm the bidentate coordination mode of the salicylaldimine ligand to the metal centre. The platinum group metal (PGM) complexes were screened against the MCF7 breast cancer cell line. The ruthenium and iridium salicylaldimine complexes showed comparable or greater cytotoxicity than cisplatin against the MCF7 cancer cells, as well as greater cytotoxicity than their rhodium counterparts. Three of the salicylaldimine complexes showed potent activity in the range 18-21 µM. Two of these complexes had a greater affinity for cancerous cells than for CHO non-cancerous cells (SI > 4). Preliminary mechanistic studies suggest that the ruthenium complexes undergo solvation prior to 5'-GMP binding, whereas the iridium complexes were inert to the solvation process.