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Sample records for cells expression design

  1. Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

    Science.gov (United States)

    Zhong, Guocai; Wang, Haimin; Bailey, Charles C; Gao, Guangping; Farzan, Michael

    2016-01-01

    Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes’s activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules. DOI: http://dx.doi.org/10.7554/eLife.18858.001 PMID:27805569

  2. Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

    Directory of Open Access Journals (Sweden)

    Deutsch Eric W

    2008-05-01

    Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

  3. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  4. Designing a HER2/neu promoter to drive alpha1,3galactosyltransferase expression for targeted anti-alphaGal antibody-mediated tumor cell killing.

    OpenAIRE

    Lanteri, Marion; Ollier, Laurence; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2005-01-01

    INTRODUCTION: Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-alphaGal antibodies by using a murine alpha1,3galactosyltransferase (malphaGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the alphaGalT activity that promotes Galalpha1,3Galbeta1,4GlcNAc-R (alphaGal) epitope expression has been mutationally disrupted during the course of evoluti...

  5. Combining eicosapentaenoic acid, decosahexaenoic acid and arachidonic acid, using a fully crossed design, affect gene expression and eicosanoid secretion in salmon head kidney cells in vitro.

    Science.gov (United States)

    Holen, Elisabeth; He, Juyun; Espe, Marit; Chen, Liqiou; Araujo, Pedro

    2015-08-01

    Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses.

  6. Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

    Science.gov (United States)

    Lanteri, Marion; Ollier, Laurence; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2005-01-01

    Introduction Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man. Method Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4. Results We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression. Conclusion Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression

  7. New Strategies for Designing Inexpensive but Selective Bioadsorbants for Environmental Pollutants: Selection of specific Ligands and Their Cell Surface Expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent L. Iverson; George Georgiou; Mohammad M. Ataai; Richard R. Koepsel

    2001-02-22

    The Broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in our laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules we will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal.

  8. iVisDesigner: Expressive Interactive Design of Information Visualizations.

    Science.gov (United States)

    Ren, Donghao; Höllerer, Tobias; Yuan, Xiaoru

    2014-12-01

    We present the design, implementation and evaluation of iVisDesigner, a web-based system that enables users to design information visualizations for complex datasets interactively, without the need for textual programming. Our system achieves high interactive expressiveness through conceptual modularity, covering a broad information visualization design space. iVisDesigner supports the interactive design of interactive visualizations, such as provisioning for responsive graph layouts and different types of brushing and linking interactions. We present the system design and implementation, exemplify it through a variety of illustrative visualization designs and discuss its limitations. A performance analysis and an informal user study are presented to evaluate the system.

  9. Expression regulation of design process gene in product design

    DEFF Research Database (Denmark)

    Fang, Lusheng; Li, Bo; Tong, Shurong

    2011-01-01

    is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed......To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications....

  10. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  11. Global gene expression analysis for evaluation and design of biomaterials

    Directory of Open Access Journals (Sweden)

    Nobutaka Hanagata, Taro Takemura and Takashi Minowa

    2010-01-01

    Full Text Available Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data.

  12. Designing genes for successful protein expression.

    Science.gov (United States)

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2011-01-01

    DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.

  13. Investigation by microarray analysis of effects of cigarette design characteristics on gene expression in human lung mucoepidermoid cancer cells NCI-H292 exposed to cigarette smoke.

    Science.gov (United States)

    Sekine, Takashi; Sakaguchi, Chikako; Fukano, Yasuo

    2015-02-01

    The effects of tobacco leaf types and the presence or absence of charcoal in the cigarette filters on gene expression were investigated using cigarette prototypes made of either flue-cured (FC) leaf or burley (BLY) leaf and Kentucky Reference 2R4F as a representative blend cigarette with cellulose acetate filters or charcoal filters. NCI-H292, human lung mucoepidermoid carcinoma cell line, was exposed to the total particulate matter (TPM) and gas/vapor phase (GVP) from each prototype for 8h and then the changes in gene expression from microarray data were analyzed. A number of genes associated with oxidative stress, inflammation, DNA damage and xenobiotic response were modified by the two fractions, TPM and GVP, from the three prototypes with cellulose acetate filters. Both TPM and GVP fractions strongly enhanced the gene expression of HMOX1, which is encoding the limiting enzyme in heme degradation and a key regulator of oxidative stress and inflammatory process. Comparing the effects of TPM and GVP fraction, TPM strongly activated Nrf2 pathway-mediated anti-oxidative stress reaction, whereas GVP caused notable DNA damage response. In comparison of FC and BLY, TPM from FC more strongly induced the expression of histone family proteins than that from BLY. GVP from FC markedly induced gene expression associated with HSP70-mediated inflammation relative to that from BLY. Charcoal included in the filter strongly reduced the effects of GVP from each cigarette on gene expression. However, charcoal did not modified the effects of TPM. As a whole, charcoal is a useful material for reducing the biological effects of GVP.

  14. Calreticulin: Roles in Cell-Surface Protein Expression

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    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  15. Rational design, fabrication, characterization and in vitro testing of biodegradable microparticles that generate targeted and sustained transgene expression in HepG2 liver cells

    Science.gov (United States)

    Intra, Janjira; Salem, Aliasger K.

    2017-01-01

    Poly (lactide-co-glycolide) (PLGA) microparticles have significant potential for sustained delivery of plasmid DNA. However, unmodified PLGA microparticles have poor transfection efficiencies. In this study, we use several approaches to enhance the transfection efficiencies of PLGA microparticles in a HepG2 liver cell line. Polyethylenimine is used to condense the plasmid DNA prior to loading into the PLGA microparticles. This provides enhanced loading efficiencies and greater protection to the plasmid DNA (pDNA) during the entrapment process. In addition, the pDNA used (ApoE) incorporates a hybrid liver-specific Murine Albumin enhancer/α-1 antitrypsin promoter (AlbE/hAAT) to enhance transgene expression in human liver (HepG2) cells. The percentage of surfactant used in the preparation of the microparticles, the polymer composition of the PLGA, the ratio of the (polyethylenimine) PEI to pDNA (N/P), the structure of the PEI and the potential utility of a galactose targeting ligand were then investigated to further optimize the efficacy of the cationic microparticle non-viral delivery system in transfecting HepG2 cells. For each PLGA PEI pDNA microparticle formulation prepared, we evaluated particle size, zeta-potential, loading of pDNA, cytotoxicity and transgene expression in HepG2 cells and control human embryonic kidney (HEK293) and Monkey African green kidney fibroblast-like (COS7) cells. Loading PLGA particles with PEI-ApoE pDNA complexes resulted in a significant reduction in particle size when compared to PLGA microparticles loaded with ApoE pDNA alone. Scanning electron microscopy images showed that all the particle formulations were smooth and spherical in appearance. Incorporation of the cationic PEI in the PLGA particles changed the zeta potential from negative to positive. Complexing PEI with ApoE pDNA increased the loading efficiency of the ApoE pDNA into the PLGA microparticles. The cytotoxicity of PLGA particles loaded with PEI-ApoE pDNA complexes

  16. CHO gene expression profiling in biopharmaceutical process analysis and design.

    Science.gov (United States)

    Schaub, Jochen; Clemens, Christoph; Schorn, Peter; Hildebrandt, Tobias; Rust, Werner; Mennerich, Detlev; Kaufmann, Hitto; Schulz, Torsten W

    2010-02-01

    Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization.

  17. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  18. Rho GTPase expression in human myeloid cells.

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    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  19. Gene expression profile of renal cell carcinoma clear cell type

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    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  20. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  1. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  2. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  3. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

    2013-01-01

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized...

  4. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito;

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...

  5. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    Science.gov (United States)

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  6. CNPase Expression in Olfactory Ensheathing Cells

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    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  7. Adipose tissue-derived stromal cells express neuronal phenotypes

    Institute of Scientific and Technical Information of China (English)

    杨立业; 刘相名; 孙兵; 惠国桢; 费俭; 郭礼和

    2004-01-01

    Background Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons.Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE).Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies.Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

  8. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  9. Expression in aneuploid Drosophila S2 cells.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2010-02-01

    Full Text Available Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A. Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.

  10. New strategies for designing inexpensive but selective bioadsorbants for environmental pollutants: Selection of specific ligands and their cell surface expression. 1998 annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Iverson, B.L.; Georgiou, G. [Univ. of Texas, Austin, TX (US); Ataai, M.M.; Koepsel, R.R. [Univ. of Pittsburgh, PA (US)

    1998-06-01

    'The broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in the laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules the authors will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal. The authors are currently in year two of a three year program. Work has been focused on preparing the molecular biology constructs needed to carry out the optimization of a metal complex binding antibody, and on the isolation of a metal binding peptide.'

  11. Design, synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells.

    Science.gov (United States)

    Shiokawa, Zenyu; Hashimoto, Kentaro; Saito, Bunnai; Oguro, Yuya; Sumi, Hiroyuki; Yabuki, Masato; Yoshimatsu, Mie; Kosugi, Yohei; Debori, Yasuyuki; Morishita, Nao; Dougan, Douglas R; Snell, Gyorgy P; Yoshida, Sei; Ishikawa, Tomoyasu

    2013-12-15

    We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC50 23 nM and cellular IAP [cIAP]: IC50 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI50 2.8 nM) with high permeability and low potential of MDR1 substrate.

  12. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...... studies of vitamin D's effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients....

  13. Detection of gene expression in an individual cell type within a cell mixture using microarray analysis.

    Directory of Open Access Journals (Sweden)

    Penelope A Bryant

    Full Text Available BACKGROUND: A central issue in the design of microarray-based analysis of global gene expression is the choice between using cells of single type and a mixture of cells. This study quantified the proportion of lipopolysaccharide (LPS induced differentially expressed monocyte genes that could be measured in peripheral blood mononuclear cells (PBMC, and determined the extent to which gene expression in the non-monocyte cell fraction diluted or obscured fold changes that could be detected in the cell mixture. METHODOLOGY/PRINCIPAL FINDINGS: Human PBMC were stimulated with LPS, and monocytes were then isolated by positive (Mono+ or negative (Mono- selection. The non-monocyte cell fraction (MonoD remaining after positive selection of monocytes was used to determine the effect of non-monocyte cells on overall expression. RNA from LPS-stimulated PBMC, Mono+, Mono- and MonoD samples was co-hybridised with unstimulated RNA for each cell type on oligonucleotide microarrays. There was a positive correlation in gene expression between PBMC and both Mono+ (0.77 and Mono- (0.61-0.67 samples. Analysis of individual genes that were differentially expressed in Mono+ and Mono- samples showed that the ability to detect expression of some genes was similar when analysing PBMC, but for others, differential expression was either not detected or changed in the opposite direction. As a result of the dilutional or obscuring effect of gene expression in non-monocyte cells, overall about half of the statistically significant LPS-induced changes in gene expression in monocytes were not detected in PBMC. However, 97% of genes with a four fold or greater change in expression in monocytes after LPS stimulation, and almost all (96-100% of the top 100 most differentially expressed monocyte genes were detected in PBMC. CONCLUSIONS/SIGNIFICANCE: The effect of non-responding cells in a mixture dilutes or obscures the detection of subtle changes in gene expression in an individual

  14. Melanopsin expressing human retinal ganglion cells

    DEFF Research Database (Denmark)

    Hannibal, Jens; Christensen, Anders Tolstrup; Heegaard, Steffen

    2017-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex and sleep/wake cycles. The different functions seem...

  15. Dental enamel cells express functional SOCE channels.

    Science.gov (United States)

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  16. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  17. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    Science.gov (United States)

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment.

  18. Toward stable gene expression in CHO cells

    Science.gov (United States)

    Mariati; Koh, Esther YC; Yeo, Jessna HM; Ho, Steven CL; Yang, Yuansheng

    2014-01-01

    Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific. PMID:25482237

  19. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  20. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    Science.gov (United States)

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  1. Direct Cell Lysis for Single-Cell Gene Expression Profiling

    OpenAIRE

    David eSvec; Daniel eAndersson; Milos ePekny; Robert eSjöback; Mikael eKubista; Anders eStåhlberg

    2013-01-01

    The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously express...

  2. Induction of T Cell Development In Vitro by Delta-Like (Dll)-Expressing Stromal Cells.

    Science.gov (United States)

    Mohtashami, Mahmood; Zarin, Payam; Zúñiga-Pflücker, Juan Carlos

    2016-01-01

    Recreating the thymic microenvironment in vitro poses a great challenge to immunologists. Until recently, the only approach was to utilize the thymic tissue in its three-dimensional form and to transfer the hematopoietic progenitors into this tissue to generate de novo T cells. With the advent of OP9-DL cells (bone marrow-derived cells that are transduced to express Notch ligand, Delta-like), hematopoietic stem cells (HSC) could be induced to differentiate into T cells in culture for the first time outside of the thymic tissue on a monolayer. We, as well as others, asked whether the ability to support T cell development in vitro in a monolayer is unique to BM-derived OP9 cells, and showed that provision of Delta-like expression to thymic epithelial cells and fibroblasts also allowed for T cell development. This provides the opportunity to design an autologous coculture system where the supportive stromal and the hematopoietic components are both derived from the same individual, which has obvious clinical implications. In this chapter, we describe methods for establishing a primary murine dermal fibroblast cell population that is transduced to express Delta-like 4, and describe the conditions for its coculture with HSCs to support T cell lineage initiation and expansion, while comparing it to the now classic OP9-DL coculture.

  3. Study of Contemporary Jewelry Design Emotional Expression Skills

    Directory of Open Access Journals (Sweden)

    Bai-hui Gong

    2017-02-01

    Full Text Available With the development of the society, people gradually realize the importance of design, design also has played a more and more important role in the life.More and more people are willing to choose the design work with unique emotions.Designers gives the new concept of life into the jewelry through the material, color, craft technique, etc. Jewelry as emotional carrier, can better deliver humanistic ideas behind the design in silence.This paper studies the emotional expression skills of contemporary jewelry design.

  4. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian;

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon......-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear...

  5. Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

    Science.gov (United States)

    Yoon, Jeongheon; Wang, Hongsheng; Kim, Yong Chan; Yoshimoto, Momoko; Abbasi, Sadia; Morse Iii, Herbert C

    2016-09-01

    Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

  6. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  7. Probe design for expression arrays using OligoWiz

    DEFF Research Database (Denmark)

    Wernersson, Rasmus

    2014-01-01

    Since all measurements from a DNA microarray is dependant on the probes used, a good choice of probes is of vital importa nce when designing custom micro-arrays. This chapter describes how to de sign expression arrays using the “ OligoWiz ” software suite. The general desired features of good...... probes and the issues which probe design must address are introduced and a conceptual (rather than mathematical) description of how OligoWiz scores the quality of th e potential probes is presented. This is followed by a detailed step-by-step guide to designing expression arrays with OligoWiz....

  8. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  9. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  10. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    David eSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  11. Graphics Innovation Design Based on Cell Intuition Simulation Method

    Institute of Scientific and Technical Information of China (English)

    XIA Zhi-qiu; REN Na; ZHAO Ting-ting; CUI Lun-feng

    2014-01-01

    This paper proposes a new cell intuition simulation method which is a combination of intuitive simulation calculation method and the operation of binary image, and applied it in the innovation of the graphic design process. First of all, we study how to express a variety of graphics, and establish the definition of cell intuitive model, work out the cell intuitive operation process and many new cellular operators such as a variety of matrix blocks crossover operator, a variety of matrix blocks mutation operator, matrix blocks replace operator, matrix blocks compression operator, matrix blocks extension operator. By choosing two or more cells and selecting the artificial selection or fitness selection, we can set up and visualize the design and pick the best design results .Finally, validation is made on this algorithm by an example, and a innovation graphic is also represented.

  12. Gene expression profiles in irradiated cancer cells

    Science.gov (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  13. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  14. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Directory of Open Access Journals (Sweden)

    Kimitoshi Kohno

    2011-10-01

    Full Text Available We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143 regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1, aurora kinase B (AURKB and some minichromosome maintenance complex components (MCM. However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  15. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Energy Technology Data Exchange (ETDEWEB)

    Izumi, Hiroto, E-mail: h-izumi@med.uoeh-u.ac.jp; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan)

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  16. Expression of basal cell keratins in human prostate cancer metastases and cell lines.

    NARCIS (Netherlands)

    Leenders, G.J.L.H. van; Aalders, M.W.; Hulsbergen-van de Kaa, C.A.; Ruiter, D.J.; Schalken, J.A.

    2001-01-01

    Within normal human prostate epithelium, basal and luminal cells can be discriminated by their expression of keratins (K). While basal cells express K5/14, luminal cells show expression of K8/18 and an intermediate cell population can be identified by co-expression of K5/18. Prostate cancer is predo

  17. Rational design of efficient modular cells.

    Science.gov (United States)

    Trinh, Cong T; Liu, Yan; Conner, David J

    2015-11-01

    The modular cell design principle is formulated to devise modular (chassis) cells. These cells can be assembled with exchangeable production modules in a plug-and-play fashion to build microbial cell factories for efficient combinatorial biosynthesis of novel molecules, requiring minimal iterative strain optimization steps. A modular cell is designed to be auxotrophic, containing core metabolic pathways that are necessary but insufficient to support cell growth and maintenance. To be functional, it must tightly couple with an exchangeable production module containing auxiliary metabolic pathways that not only complement cell growth but also enhance production of targeted molecules. We developed a MODCELL (modular cell) framework based on metabolic pathway analysis to implement the modular cell design principle. MODCELL identifies genetic modifications and requirements to construct modular cell candidates and their associated exchangeable production modules. By defining the degree of similarity and coupling metrics, MODCELL can evaluate which exchangeable production module(s) can be tightly coupled with a modular cell candidate. We first demonstrated how MODCELL works in a step-by-step manner for example metabolic networks, and then applied it to design modular Escherichia coli cells for efficient combinatorial biosynthesis of five alcohols (ethanol, propanol, isopropanol, butanol and isobutanol) and five butyrate esters (ethyl butyrate, propyl butyrate, isopropyl butyrate, butyl butyrate and isobutyl butyrate) from pentose sugars (arabinose and xylose) and hexose sugars (glucose, mannose, and galactose) under anaerobic conditions. We identified three modular cells, MODCELL1, MODCELL2 and MODCELL3, that can couple well with Group 1 of modules (ethanol, isobutanol, butanol, ethyl butyrate, isobutyl butyrate, butyl butyrate), Group 2 (isopropanol, isopropyl butyrate), and Group 3 (propanol, isopropanol), respectively. We validated the design of MODCELL1 for anaerobic

  18. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Robert W. [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Ghert, Michelle [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Singh, Gurmit, E-mail: gurmit.singh@jcc.hhsc.ca [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  19. Quality cell therapy manufacturing by design.

    Science.gov (United States)

    Lipsitz, Yonatan Y; Timmins, Nicholas E; Zandstra, Peter W

    2016-04-01

    Transplantation of live cells as therapeutic agents is poised to offer new treatment options for a wide range of acute and chronic diseases. However, the biological complexity of cells has hampered the translation of laboratory-scale experiments into industrial processes for reliable, cost-effective manufacturing of cell-based therapies. We argue here that a solution to this challenge is to design cell manufacturing processes according to quality-by-design (QbD) principles. QbD integrates scientific knowledge and risk analysis into manufacturing process development and is already being adopted by the biopharmaceutical industry. Many opportunities to incorporate QbD into cell therapy manufacturing exist, although further technology development is required for full implementation. Linking measurable molecular and cellular characteristics of a cell population to final product quality through QbD is a crucial step in realizing the potential for cell therapies to transform healthcare.

  20. Fuel cells principles, design, and analysis

    CERN Document Server

    Revankar, Shripad T

    2014-01-01

    ""This book covers all essential themes of fuel cells ranging from fundamentals to applications. It includes key advanced topics important for understanding correctly the underlying multi-science phenomena of fuel cell processes. The book does not only cope with traditional fuel cells but also discusses the future concepts of fuel cells. The book is rich on examples and solutions important for applying the theory into practical use.""-Peter Lund, Aalto University, Helsinki""A good introduction to the range of disciplines needed to design, build and test fuel cells.""-Nigel Brandon, Imperial Co

  1. Interleukin-2 expression and glioma cell proliferation following Vaceinia vector gene transfection in vivo

    Institute of Scientific and Technical Information of China (English)

    Xiaogang Wang; Xuezhong Wei; Jiangqiu Liu

    2008-01-01

    BACKGROUND: The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression.OBJECTIVE: This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 (rVV-IL-2) gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN: Experimental observation. SETTING: Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS: The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. MAIN OUTCOME MEASURES: IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS: IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups (P > 0.05).CONCLUSION: Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.

  2. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  3. EXPRESSION OF IMMUNE-RELATED MOLECULES IN GLIOBLASTOMA MULTIFORM CELLS

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective: To investigate the expression of immune- related molecules in glioblastoma multiform(GBM) cells. Methods: The expression of major histocompatibility complex (MHC), β2-microglobulin, Fas, CD80 and CD86 molecules on the surface of GBM cells were evaluated by flow cytometry. The expression of TAP-1, TAP-2 and Tapasin in the GBM cells were evaluated by RT-PCR method. Results: MHC class Ⅰ, β2 microglobulin, TAP-1, TAP-2 and tapasin were expressed in most GBM cell lines. Except U87, there was no MHC class Ⅱ molecule expression on any of the other GBM cell lines. Fas was expressed on all the GBM cell lines examined. Conclusion: The mechanism by which GBM escapes immune surveillance may involve down regulation of expression of MHC class Ⅰ molecules and MHC class Ⅱ molecules. MHC class Ⅰpositive GBM may be the suitable target of immunotherapy.

  4. Flow cell design for effective biosensing

    OpenAIRE

    Pike, DJ; Kapur, N; Millner, PA; Stewart, DI

    2012-01-01

    The efficiency of three different biosensor flow cells is reported. All three flow cells featured a central channel that expands in the vicinity of the sensing element to provide the same diameter active region, but the rate of channel expansion and contraction varied between the designs. For each cell the rate at which the analyte concentration in the sensor chamber responds to a change in the influent analyte concentration was determined numerically using a finite element model and experime...

  5. Concave cell design for FTIR measurements

    Institute of Scientific and Technical Information of China (English)

    Xiang Qin Lin; Zhi Xiang Zhang; Wan Qun Hu

    2011-01-01

    Wedged and V-shaped cells have advantages on depressing strong absorption bands and keeping the photo windows wide open without absorption block. Alternatively, this work presents a concave cell design, which is constructed by a plano-convex CaF2 lens in combination with a flat plate. Mathematical equations have been provided for data treatment with Matlab programs. The result indicates that the concave cell has advantages similar to the V-shaped cell. Reflection-absorption measurements of ethanol have been conducted on the cell in mid-IR region for demonstration. The spectrum obtained from the concave cell can easily be interpreted to regenerate its normal spectrum, which is fitting well with the spectrum taken in a conventional thin-layer cell. The concave cell is easier to construct and more convenient to operate, willing to have popular usages.

  6. High expression of markers of apoptosis in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I;

    2003-01-01

    53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...... number of pathological Langerhans cells in apoptosis as estimated by TUNEL was low. CONCLUSIONS: The low number of TUNEL-reactive cells can be explained by the rapid turnover of apoptotic cells in the tissue, not leaving the apoptotic cells long enough in the tissue to be detected. The co...

  7. Relationship between Meaning and Expression in Architecture Design

    Institute of Scientific and Technical Information of China (English)

    LiKun

    2002-01-01

    During the evolvement of architecture,the research on architectural form and structure as well as different ways to express and realize architectural thoughts is constantly the focus of many architects,This paper illustrates the mutual influence of the ways of expression and design of architecture to people's understanding of the meaning of architecture throughout the history of their efforts and exploration.the paper also discusses the extensive usage of virtual-realith approach through computer technology,It points out that the traditional architectural standpoint will undergo breakthrough by the emergence of new expression and design method.In this way,people's understanding of architecture will be more profound and lasting.

  8. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    Energy Technology Data Exchange (ETDEWEB)

    H.C. Maru; M. Farooque

    2003-03-01

    The program efforts are focused on technology and system optimization for cost reduction, commercial design development, and prototype system field trials. The program is designed to advance the carbonate fuel cell technology from full-size field test to the commercial design. FuelCell Energy, Inc. (FCE) is in the later stage of the multiyear program for development and verification of carbonate fuel cell based power plants supported by DOE/NETL with additional funding from DOD/DARPA and the FuelCell Energy team. FCE has scaled up the technology to full-size and developed DFC{reg_sign} stack and balance-of-plant (BOP) equipment technology to meet product requirements, and acquired high rate manufacturing capabilities to reduce cost. FCE has designed submegawatt (DFC300A) and megawatt (DFC1500 and DFC3000) class fuel cell products for commercialization of its DFC{reg_sign} technology. A significant progress was made during the reporting period. The reforming unit design was optimized using a three-dimensional stack simulation model. Thermal and flow uniformities of the oxidant-In flow in the stack module were improved using computational fluid dynamics based flow simulation model. The manufacturing capacity was increased. The submegawatt stack module overall cost was reduced by {approx}30% on a per kW basis. An integrated deoxidizer-prereformer design was tested successfully at submegawatt scale using fuels simulating digester gas, coal bed methane gas and peak shave (natural) gas.

  9. From the Cover: Design of artificial cell-cell communication using gene and metabolic networks

    Science.gov (United States)

    Bulter, Thomas; Lee, Sun-Gu; Waichun Wong, Wilson; Fung, Eileen; Connor, Michael R.; Liao, James C.

    2004-02-01

    Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

  10. Clinical Implications of Phosphorylated STAT3 Expression in de novo Diffuse Large B-cell Lymphoma

    DEFF Research Database (Denmark)

    Ok, Chi Y; Chen, Jiayu; Xu-Monette, Ziju

    2014-01-01

    of phosphorylated STAT3 (pSTAT3) on prognosis are limited. EXPERIMENTAL DESIGN: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma...

  11. Mammalian designer cells: Engineering principles and biomedical applications.

    Science.gov (United States)

    Xie, Mingqi; Fussenegger, Martin

    2015-07-01

    Biotechnology is a widely interdisciplinary field focusing on the use of living cells or organisms to solve established problems in medicine, food production and agriculture. Synthetic biology, the science of engineering complex biological systems that do not exist in nature, continues to provide the biotechnology industry with tools, technologies and intellectual property leading to improved cellular performance. One key aspect of synthetic biology is the engineering of deliberately reprogrammed designer cells whose behavior can be controlled over time and space. This review discusses the most commonly used techniques to engineer mammalian designer cells; while control elements acting on the transcriptional and translational levels of target gene expression determine the kinetic and dynamic profiles, coupling them to a variety of extracellular stimuli permits their remote control with user-defined trigger signals. Designer mammalian cells with novel or improved biological functions not only directly improve the production efficiency during biopharmaceutical manufacturing but also open the door for cell-based treatment strategies in molecular and translational medicine. In the future, the rational combination of multiple sets of designer cells could permit the construction and regulation of higher-order systems with increased complexity, thereby enabling the molecular reprogramming of tissues, organisms or even populations with highest precision.

  12. Insights Gained from Testing Alternate Cell Designs

    Energy Technology Data Exchange (ETDEWEB)

    J. E. O' Brien; C. M. Stoots; J. S. Herring; G. K. Housley; M. S. Sohal; D. G. Milobar; Thomas Cable

    2009-09-01

    The Idaho National Laboratory (INL) has been researching the application of solid-oxide electrolysis cell for large-scale hydrogen production from steam over a temperature range of 800 to 900ºC. The INL has been testing various solid oxide cell designs to characterize their electrolytic performance operating in the electrolysis mode for hydrogen production. Some results presented in this report were obtained from cells, initially developed by the Forschungszentrum Jülich and now manufactured by the French ceramics firm St. Gobain. These cells have an active area of 16 cm2 per cell. They were initially developed as fuel cells, but are being tested as electrolytic cells in the INL test stands. The electrolysis cells are electrode-supported, with ~10 µm thick yttria-stabilized zirconia (YSZ) electrolytes, ~1400 µm thick nickel-YSZ steam-hydrogen electrodes, and manganite (LSM) air-oxygen electrodes. The experiments were performed over a range of steam inlet mole fractions (0.1 to 0.6), gas flow rates, and current densities (0 to 0.6 A/cm2). Steam consumption rates associated with electrolysis were measured directly using inlet and outlet dewpoint instrumentation. On a molar basis, the steam consumption rate is equal to the hydrogen production rate. Cell performance was evaluated by performing DC potential sweeps at 800, 850, and 900°C. The voltage-current characteristics are presented, along with values of area-specific resistance as a function of current density. Long-term cell performance is also assessed to evaluate cell degradation. Details of the custom single-cell test apparatus developed for these experiments are also presented. NASA, in conjunction with the University of Toledo, has developed another fuel cell concept with the goals of reduced weight and high power density. The NASA cell is structurally symmetrical, with both electrodes supporting the thin electrolyte and containing micro-channels for gas diffusion. This configuration is called a bi

  13. Direct fuel cell product design improvement

    Energy Technology Data Exchange (ETDEWEB)

    Maru, H.C.; Farooque, M. [Energy Research Corp., Danbury, CT (United States)

    1996-12-31

    Significant milestones have been attained towards the technology development field testing and commercialization of direct fuel cell power plant since the 1994 Fuel Cell Seminar. Under a 5-year cooperative agreement with the Department of Energy signed in December 1994, Energy Research Corporation (ERC) has been developing the design for a MW-scale direct fuel cell power plant with input from previous technology efforts and the Santa Clara Demonstration Project. The effort encompasses product definition in consultation with the Fuel Cell Commercialization Group, potential customers, as well as extensive system design and packaging. Manufacturing process improvements, test facility construction, cell component scale up, performance and endurance improvements, stack engineering, and critical balance-of-plant development are also addressed. Major emphasis of this product design improvement project is on increased efficiency, compactness and cost reduction to establish a competitive place in the market. A 2.85 MW power plant with an efficiency of 58% and a footprint of 420 m{sup 2} has been designed. Component and subsystem testing is being conducted at various levels. Planning and preparation for verification of a full size prototype unit are in progress. This paper presents the results obtained since the last fuel cell seminar.

  14. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  15. Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

    Science.gov (United States)

    Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

    2015-01-01

    Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple

  16. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    Science.gov (United States)

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  17. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I;

    2006-01-01

    directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... the umbilical cords. Methods Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Main outcome measures Fold changes in gene expression levels between treated and control cultures were calculated from the microarray...

  18. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)

    LIJUNMIN; JINGCHULUO; 等

    1999-01-01

    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  19. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  20. Targeted repression of AXIN2 and MYC gene expression using designer TALEs

    Energy Technology Data Exchange (ETDEWEB)

    Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    2014-04-18

    Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacent to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.

  1. Expression of HOX C homeobox genes in lymphoid cells.

    Science.gov (United States)

    Lawrence, H J; Stage, K M; Mathews, C H; Detmer, K; Scibienski, R; MacKenzie, M; Migliaccio, E; Boncinelli, E; Largman, C

    1993-08-01

    The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

  2. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  3. Advantages and Applications of CAR-Expressing Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Wolfgang eGlienke

    2015-02-01

    Full Text Available In contrast to donor T cells, natural killer (NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD. In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/ on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

  4. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  5. Expression of Pol(t) in tissues and cell lines of transitional cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To explore the expression of DNA polymerase iota in transitional cell carcinoma cells and tissues; Methods: RT-PCR was applie to detect the expression of polymerase iota in BIU87 and T24 cells, then the expression of polymerase iota was also detected in the same way in transitional cell carcinoma which was derived from clinical bladder carcinoma and renal pelvic carcinoma. Results: The expression of Polt was low in bladder normal membrana mucosa but significantly elevated in transitional cell carcinoma cells. Compared with the expression of polymerase iota in bladder normal mucous membranes, the expression of polymerase iota was significantly increased in transitional cell carcinoma tissue (P<0.01)and associated with the grade of transitional cell carcinoma. Conclusion: The significantly increased expression of polymerase iota may be associated with the generation and development of transitional cell carcinoma, even with its high heterogenicity.

  6. Prostaglandin E₂ receptor EP2 mediates Snail expression in hepatocellular carcinoma cells.

    Science.gov (United States)

    Cheng, Shan-Yu; Zhang, Hai; Zhang, Min; Xia, Shu-Kai; Bai, Xiao-Ming; Zhang, Li; Ma, Juan; Rong, Rong; Wang, Yi-Pin; Du, Ming-Zhan; Wang, Jie; Chen, Meng; Shi, Feng; Yang, Qin-Yi; Leng, Jing

    2014-05-01

    Prostaglandin E2 (PGE2) has been shown to influence cell invasion and metastasis in several types of cancer, including hepatocellular carcinoma (HCC). however, the molecular mechanisms underlying it remain to be further elucidated. Snail, as one of key inducers of epithelial-mesenchymal transition (EMT), plays pivotal roles in HCC invasion and metastasis. The present study was designed to evaluate the possible signaling pathways through which PGE2 regulates Snail protein expression in HCC cell lines. PGE2 markedly enhanced Huh-7 cell invasion and migration ability by upregulating the expression level of Snail protein, and EP2 receptor played an important role in this process. Src, EGFR, Akt and mTOR were all activated and involved in the regulation of snail protein expression. Our findings suggest that PGE2 could upregulate the expression level of Snail protein through the EP2/Src/EGFR/Akt/mTOR pathway in Huh-7 cells, which promotes HCC cell invasion and migration.

  7. Probing the effect of promoters on noise in gene expression using thousands of designed sequences.

    Science.gov (United States)

    Sharon, Eilon; van Dijk, David; Kalma, Yael; Keren, Leeat; Manor, Ohad; Yakhini, Zohar; Segal, Eran

    2014-10-01

    Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

  8. MOLTEN CARBONATE FUEL CELL PRODUCT DESIGN IMPROVEMENT

    Energy Technology Data Exchange (ETDEWEB)

    H.C. Maru; M. Farooque

    2005-03-01

    The program was designed to advance the carbonate fuel cell technology from full-size proof-of-concept field test to the commercial design. DOE has been funding Direct FuelCell{reg_sign} (DFC{reg_sign}) development at FuelCell Energy, Inc. (FCE, formerly Energy Research Corporation) from an early state of development for stationary power plant applications. The current program efforts were focused on technology and system development, and cost reduction, leading to commercial design development and prototype system field trials. FCE, in Danbury, CT, is a world-recognized leader for the development and commercialization of high efficiency fuel cells that can generate clean electricity at power stations, or at distributed locations near the customers such as hospitals, schools, universities, hotels and other commercial and industrial applications. FCE has designed three different fuel cell power plant models (DFC300A, DFC1500 and DFC3000). FCE's power plants are based on its patented DFC{reg_sign} technology, where a hydrocarbon fuel is directly fed to the fuel cell and hydrogen is generated internally. These power plants offer significant advantages compared to the existing power generation technologies--higher fuel efficiency, significantly lower emissions, quieter operation, flexible siting and permitting requirements, scalability and potentially lower operating costs. Also, the exhaust heat by-product can be used for cogeneration applications such as high-pressure steam, district heating and air conditioning. Several sub-MW power plants based on the DFC design are currently operating in Europe, Japan and the US. Several one-megawatt power plant design was verified by operation on natural gas at FCE. This plant is currently installed at a customer site in King County, WA under another US government program and is currently in operation. Because hydrogen is generated directly within the fuel cell module from readily available fuels such as natural gas and

  9. Chemokine receptor expression by inflammatory T cells in EAE.

    Science.gov (United States)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

  10. Expressing functional siRNAs in mammalian cells using convergent transcription

    Directory of Open Access Journals (Sweden)

    Dawes Ian W

    2003-11-01

    Full Text Available Abstract Background The use of small interfering RNAs (siRNAs as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi-mediated gene silencing. Results With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. Conclusion The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences

  11. RNA interference by expression of short hairpin RNAs suppresses bcl-xL gene expression in nasopharyngeal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Fang LIU; Cheng-wei HE; Yue-fei ZHANG; Ke-yuan ZHOU

    2005-01-01

    Aim: To evaluate a new plasmid mediated RNA interference (RNAi) system and investigate whether knock-down of bcl-xL by short hairpin RNA (shRNA) can induce apoptosis of human nasopharyngeal carcinoma (NPC) cell line CNE-2Z in vitro. Methods: The plasmid containing mU6 promoter was subcloned to yield the pmU6 plasmid, recombinant plasmid expressing shRNA targeting bcl-xL gene was designed and constructed, and were co-transfected cells with green fluorescence protein expressing plasmid. Flow cytometry was used to evaluate transfection efficiency, and RT-PCR and Western blot were applied to analyze bcl-xL mRNA and protein levels, respectively. Results: The shRNA expressed by the recombi nant plasmid efficiently suppressed bcl-xL gene expression and induced apoptosis .of NPC cells in vitro. Conclusion: The recombinant plasmid can sufficiently mediate RNAi in CNE-2Z cells, and knock-down of the bcl-xL expression by shRNA significantly induced apoptosis in CNE-2Z cells. The results suggest this new system, mediated RNAi can be used as a tool for the study of gene function and gene therapy.

  12. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    Science.gov (United States)

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2015-07-01

    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment.

  13. Molten carbonate fuel cell product design improvement

    Energy Technology Data Exchange (ETDEWEB)

    P. Voyentzie; T. Leo; A. Kush; L. Christner; G. Carlson; C. Yuh

    1998-12-20

    Drawing on the manufacture, field test, and post-test experience of the sixteen Santa Clara Demonstration Project (SCDP) stacks, ERC is finalizing the next generation commercial entry product design. The second generation cells are 50% larger in area, 40% lighter on equal geometric area basis, and 30% thinner than the earlier design. These improvements have resulted in doubling of the full-height stack power. A low-cost and high-strength matrix has also been developed for improving product ruggedness. The low-cost advanced cell design incorporating these improvements has been refined through six short stack tests. Power production per cell of two times the SCDP maximum power operation, over ten thermal cycles, and overall operating flexibility with respect to load and thermal changes have been demonstrated in these short stack tests. An internally insulated stack enclosure has been designed and fabricated to eliminate the need for an inert gas environment during operation. ERC has acquired the capability for testing 400kW full-height direct fuel ceil (DFC) stack and balance-of-plant equipment. With the readiness of the power plant test facility, the cell package design, and the stack module, full-height stack testing has begun. The first full- height stack incorporating the post-SCDP second generation design was completed. The stack reached a power level of 253 kW, setting a world record for the highest power production from the advanced fuel cell system. Excellent performance uniformity at this power level affirmed manufacturing reproducibility of the components at the factory. This unoptimized small size test has achieved pipeline natural gas to DC electricity conversion efficiency of 47% (based on lower heating value - LHV) including the parasitic power consumed by the BOP equipment; that should translate to more than 50% efficiency in commercial operation, before employing cogeneration. The power plant system also operated smoothly. With the success of this

  14. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

  15. Expression of a secreted protein in mammalian cells using baculovirus particles.

    Science.gov (United States)

    Jardin, Barbara Ann; Elias, Cynthia B; Prakash, Satya

    2012-01-01

    There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

  16. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

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

  17. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    Science.gov (United States)

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

    2009-01-01

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

  18. Expressed protein ligation for metalloprotein design and engineering.

    Science.gov (United States)

    Clark, Kevin M; van der Donk, Wilfred A; Lu, Yi

    2009-01-01

    Metalloproteins contain highly specialized metal-binding sites that are designed to accept specific metal ions to maintain correct function. Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands. Attaining a more thorough analysis of individual amino acid function within metalloproteins has been realized using expressed protein ligation (EPL). Here we describe our recent efforts using EPL to incorporate nonproteinogenic cysteine and methionine analogues into the type 1 copper site found in Pseudomonas aeruginosa azurin.

  19. Blackmagic DesignMedia Express2.0

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Blackmagic Design宣布为Mac OS X、Windows、Linux平台推出Media ExPress2.0。Media ExPress是一款视频采集和回放软件,兼容所有Blackmagic DesignDeckLink、Multibridge和Intensity产品。新版本是一次重要更新,添加了DPX、AVI和QuickTime文件的直接采集和回放支持,以及利用列表进行批采集和回放的支持。

  20. TRPM5-expressing microvillous cells in the main olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Liman Emily R

    2008-11-01

    Full Text Available Abstract Background The main olfactory epithelium (MOE in the nasal cavity detects a variety of air borne molecules that provide information regarding the presence of food, predators and other relevant social and environmental factors. Within the epithelium are ciliated sensory neurons, supporting cells, basal cells and microvillous cells, each of which is distinct in morphology and function. Arguably, the least understood, are the microvillous cells, a population of cells that are small in number and whose function is not known. We previously found that in a mouse strain in which the TRPM5 promoter drives expression of the green fluorescent protein (GFP, a population of ciliated olfactory sensory neurons (OSNs, as well as a population of cells displaying microvilli-like structures is labeled. Here we examined the morphology and immunocytochemical properties of these microvillous-like cells using immunocytochemical methods. Results We show that the GFP-positive microvillous cells were morphologically diversified and scattered throughout the entire MOE. These cells immunoreacted to an antibody against TRPM5, confirming the expression of this ion channel in these cells. In addition, they showed a Ca2+-activated non-selective cation current in electrophysiological recordings. They did not immunoreact to antibodies that label cell markers and elements of the transduction pathways from olfactory sensory neurons and solitary chemosensory cells of the nasal cavity. Further, the TRPM5-expressing cells did not display axon-like processes and were not labeled with a neuronal marker nor did trigeminal peptidergic nerve fibers innervate these cells. Conclusion We provide morphological and immunocytochemical characterization of the TRPM5-expressing microvillous cells in the main olfactory epithelium. Our data demonstrate that these cells are non-neuronal and in terms of chemosensory transduction do not resemble the TRPM5-expressing olfactory sensory neurons

  1. Lab-specific gene expression signatures in pluripotent stem cells.

    Science.gov (United States)

    Newman, Aaron M; Cooper, James B

    2010-08-06

    Pluripotent stem cells derived from both embryonic and reprogrammed somatic cells have significant potential for human regenerative medicine. Despite similarities in developmental potential, however, several groups have found fundamental differences between embryonic stem cell (ESC) and induced-pluripotent stem cell (iPSC) lines that may have important implications for iPSC-based medical therapies. Using an unsupervised clustering algorithm, we further studied the genetic homogeneity of iPSC and ESC lines by reanalyzing microarray gene expression data from seven different laboratories. Unexpectedly, this analysis revealed a strong correlation between gene expression signatures and specific laboratories in both ESC and iPSC lines. Nearly one-third of the genes with lab-specific expression signatures are also differentially expressed between ESCs and iPSCs. These data are consistent with the hypothesis that in vitro microenvironmental context differentially impacts the gene expression signatures of both iPSCs and ESCs.

  2. Oct-4 expression in pluripotent cells of the rhesus monkey.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Hennebold, Jon D; Wolf, Don P

    2003-12-01

    The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests

  3. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  4. Expression and function of FERMT genes in colon carcinoma cells.

    Science.gov (United States)

    Kiriyama, Kenji; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Kubo, Terufumi; Tamura, Yasuaki; Kanaseki, Takayuki; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Ragnarsson, Charlotte; Nakatsugawa, Munehide; Inoda, Satoko; Asanuma, Hiroko; Takasu, Hideo; Hasegawa, Tadashi; Yasoshima, Takahiro; Hirata, Koichi; Sato, Noriyuki

    2013-01-01

    Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

  5. Rationally designed BCL6 inhibitors target activated B cell diffuse large B cell lymphoma.

    Science.gov (United States)

    Cardenas, Mariano G; Yu, Wenbo; Beguelin, Wendy; Teater, Matthew R; Geng, Huimin; Goldstein, Rebecca L; Oswald, Erin; Hatzi, Katerina; Yang, Shao-Ning; Cohen, Joanna; Shaknovich, Rita; Vanommeslaeghe, Kenno; Cheng, Huimin; Liang, Dongdong; Cho, Hyo Je; Abbott, Joshua; Tam, Wayne; Du, Wei; Leonard, John P; Elemento, Olivier; Cerchietti, Leandro; Cierpicki, Tomasz; Xue, Fengtian; MacKerell, Alexander D; Melnick, Ari M

    2016-09-01

    Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

  6. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  7. HCMV Infection Depress NGF Expression in Human Glioma Cells

    Institute of Scientific and Technical Information of China (English)

    Hai-tao WANG; Bin WANG; Zhi-jun LIU; Zhi-qiang BAI; Ling LI; Dong-meng QIAN; Zhi-yong YAN; Xu-xia SONG

    2009-01-01

    Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

  8. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  9. Low power and reliable SRAM memory cell and array design

    CERN Document Server

    Ishibashi, Koichiro

    2011-01-01

    Success in the development of recent advanced semiconductor device technologies is due to the success of SRAM memory cells. This book addresses various issues for designing SRAM memory cells for advanced CMOS technology. To study LSI design, SRAM cell design is the best materials subject because issues about variability, leakage and reliability have to be taken into account for the design.

  10. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  11. Gamma-irradiation enhances transgene expression in leukemic cells.

    Science.gov (United States)

    Vereecque, R; Saudemont, A; Wickham, T J; Gonzalez, R; Hetuin, D; Fenaux, P; Quesnel, B

    2003-02-01

    The majority of immunotherapy-based gene therapy protocols consist of ex vivo gene transfer in tumor cells. To prevent further in vivo growth, modified cells must be irradiated before reinjection into patients. The present study examines the effects of gamma-irradiation on transgene expression in transduced leukemic cells. Human and murine leukemic cells were transfected with retroviral vectors or plasmids carrying beta-galactosidase, GM-CSF or CD80 genes. Fresh leukemic cells from patients with acute myeloid leukemia (AML) were transfected with AdZ.F(pK7) adenoviral vector. gamma-irradiation at various lethal doses enhanced transgene expression in leukemic cell lines and fresh AML cells when the gene of interest was under CMV promoter but not when SV40 promoter was used. Oxidative stress also enhanced transgene expression and both irradiation and oxidative stress effects were inhibited by addition of N-acetyl-L-cysteine, a thiol anti-oxidant, indicating the involvement of reactive oxygen species. Transgene expression was also enhanced in vivo 48 and 120 h after subcutaneous injection of irradiated leukemic cells in syngeneic mice. These results show that a cell vaccine protocol using ex vivo gene transfer of transduced cells might be feasible in acute leukemia even if leukemic cells must be irradiated at lethal doses prior to reinjection to patients.

  12. MicroRNA expression profiles in avian haemopoietic cells

    Directory of Open Access Journals (Sweden)

    Yongxiu eYao

    2013-08-01

    Full Text Available MicroRNAs (miRNAs are small, abundant, non-coding RNAs that modulate gene expression by interfering with translation or stability of mRNA transcripts in a sequence-specific manner. A total of 734 precursor and 996 mature miRNAs have so far been identified in the chicken genome. A number of these miRNAs are expressed in a cell type-specific manner, and understanding their function requires detailed examination of their expression in different cell types. We carried out deep sequencing of small RNA populations isolated from stimulated or transformed avian haemopoietic cell lines to determine the changes in the expression profiles of these important regulatory molecules during these biological events. There were significant changes in the expression of a number of miRNAs, including miR-155, in chicken B cells stimulated with CD40 ligand. Similarly, avian leukosis virus (ALV-transformed DT40 cells also showed changes in miRNA expression in relation to the naïve cells. Embryonic stem cell line BP25 demonstrated a distinct cluster of upregulated miRNAs, many of which were shown previously to be involved in embryonic stem cell development. Finally, chicken macrophage cell line HD11 showed changes in miRNA profiles, some of which are thought to be related to the transformation by v-myc transduced by the virus. This work represents the first publication of a catalog of microRNA expression in a range of important avian cells and provides insights into the potential roles of miRNAs in the hematopoietic lineages of cells in a model non-mammalian species.

  13. Modulation of Vascular Cell Function by Bim Expression

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    Margaret E. Morrison

    2013-01-01

    Full Text Available Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim−/− mice. Bim−/− endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim−/− endothelial cells with Bim−/− pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

  14. Modulation of vascular cell function by bim expression.

    Science.gov (United States)

    Morrison, Margaret E; Palenski, Tammy L; Jamali, Nasim; Sheibani, Nader; Sorenson, Christine M

    2013-01-01

    Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim(-/-)) mice. Bim(-/-) endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim(-/-) endothelial cells with Bim(-/-) pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

  15. Impact of Annexin A3 expression in gastric cancer cells.

    Science.gov (United States)

    Yu, S Y; Li, Y; Fan, L Q; Zhao, Q; Tan, B B; Liu, Y

    2014-01-01

    Annexin A3 participates in various biological processes, including tumorigenesis, drug resistance, and metastasis. The aim of this study was to investigate the expression of Annexin A3 in gastric cancer and its relationship with cell differentiation, migration, and invasion of gastric cancer cells. Annexin A3 expression in gastric cancer tissues was detected by quantitative real-time PCR and Western blotting. The proliferation of gastric cancer cells was measured by the MTT assay. Cell migration and invasion were determined via wound healing and transwell assays, respectively. Knock down of endogenous Annexin A3 in gastric cancer BGC823 cells was performed using siRNA technology. The expression of Annexin A3 was significantly upregulated in gastric cancer tissues, and negatively correlated with the differentiation degree. Silencing of endogenous Annexin A3 suppressed the proliferation, migration, and invasion of BGC823 cells. Additionally, the expression of p21, p27, TIMP-1, and TIMP-2 was upregulated, and the expression of PCNA, cyclin D1, MMP-1, and MMP-2 decreased in cells treated with Annexin A3-siRNA. Annexin A3 was upregulated in gastric cancer cells. Deletion of endogenous Annexin A3 significantly inhibited gastric cancer cell proliferation, migration, and invasion.

  16. Expression and clinical significance of sulfiredoxin expression in cervical squamous cell carcinoma tissue

    Directory of Open Access Journals (Sweden)

    Xiao-yan CHEN

    2015-10-01

    Full Text Available Objective To inquire into the expression and its clinical significance of sulfiredoxin (Srx in cervical squamous cell carcinoma tissue. Methods SABC immunohistochemical method was used to detect the expression levels of Srx in specimens of 104 cervical squamous cell carcinoma and the corresponding adjacent tissues, 15 cervical intraepithelial neoplasm (CIN Ⅲ, and 20 normal cervical squamous cell epithelium tissue. The relationship between the expression of Srx protein and clinical pathological parameters of the cancer was also analyzed. Results The positive expression rates of Srx in CIN Ⅲ and cervical squamous cell carcinoma [73.3%(11/15 and 82.7%(86/104, respectively] were significantly higher than that in normal cervical tissue [35.0%(7/20, χ2=17.778, P=0.000]. Meanwhile, Srx expression in cervical cancer specimens was significantly higher than that in normal adjacent tissues (χ2=56.224, P=0.000. The positive expression of Srx in cervical squamous cell carcinoma was significantly correlated with lymph node metastasis, the depth of cancer invasion, and the infiltration of blood vessels (P0.05. Conclusion The higher expression of Srx protein might be a valuable marker for the early diagnosis and evaluation of prognosis in patients with cervical squamous cell carcinoma. DOI: 10.11855/j.issn.0577-7402.2015.08.11

  17. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma*

    Science.gov (United States)

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado Filho, Carlos D'Apparecida Santos

    2016-01-01

    Background Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment. PMID:27828631

  18. Changes of number of cells expressing proliferation and progenitor cell markers with age in rabbit intervertebral discs

    Institute of Scientific and Technical Information of China (English)

    Miersalijiang Yasen; Qinming Fei; William C Hutton; Jian Zhang; Jian Dong; Xiaoxing Jiang; Feng Zhang

    2013-01-01

    Basic knowledge about the normal regeneration process within the intervertebral disc (IVD) is important to the understanding of the underlying biology.The presence of progenitor and stem cells in IVD has been verified.However,changes of number of progenitor and stem cells with age are still unknown.In this study,changes of cell proliferation and progenitor cell markers with age in IVD cells from rabbits of two different ages were investigated using flow cytometry,immunohistochemistry,real-time polymerase chain reaction,and western blot analysis.Proliferating cell nuclear antigen (PCNA) was chosen as a marker for proliferation,and Notch1,Jagged1,C-KIT,CD166 were chosen as stem/progenitor cell markers.Cell cycle analysis showed that cell number in the G2/M phase of the young rabbits was significantly higher than that of mature rabbits.Immunohistochemical staining demonstrated the expression of PCNA,C-KIT,CD166,Notch1,and Jagged1 in both young and mature annulus fibrosus (AF).Protein expressions of these cell markers in the young rabbits were all significantly higher than those in the mature rabbits.The expression levels of PCNA,CD166,C-KIT,Jagged1 were significantly higher in the AF,and PCNA,C-KIT in the nucleus pulposus from young rabbits than those from the mature rabbits.These findings demonstrated that both proliferation and progenitor cells exist in rabbit IVDs and the number of cells expressing proliferation and progenitor cell markers decreases with age in the rabbit IVD cells.Methods that are designed to maintain the endogenous progenitor cells and stimulate their proliferation could be successful in preventing or inhibiting degenerative disc disease.

  19. Salmonella induces PD-L1 expression in B cells.

    Science.gov (United States)

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2015-10-01

    Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.

  20. Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongxia SHUAI; Ji ZHANG; Yikai YU; Muxun ZHANG

    2008-01-01

    In order to explore the expression of erythropoietin receptor (EPOR) in pancreatic cell ine NIT-1 and its effect on cell apoptosis after binding with erythropoietin (EPO), NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recom- binant human EPO (rHuEPO) had no effect on cell apoptosis but significantly inhibited apoptosis in- duced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT-1 cells and EPO could protect N1T-1 cells from apoptosis induced by cytokines.

  1. Expression of aquaporin-1 in SMMC-7221 liver carcinoma cells promotes cell migration

    Institute of Scientific and Technical Information of China (English)

    LI Yongming; FENG Xuechao; YANG Hong; MA Tonghui

    2006-01-01

    Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we provide evidence that aquaporin expression is involved in tumor cell migration. RT-PCR, immunofluorescence and Western blot analysis demonstrated the AQP1 protein expression on the plasma membrane of SMMC-7221 human hepatoma cells. SMMC-7221 cell clones with high (SMMC-7221hPf) and low (SMMC-7221/Pf) water permeability were identified by functional assays with corresponding high and low AQP1 expression. Cell migration rate was remarkably higher in SMMC-7221hPf cells than SMMC-7221/Pf cells, assessed by Boyden chamber and wound healing assays, whereas cell growth and adhesion were not different. Adenovirus-mediated AQP1 expression in SMMC-7221/Pf cells increased their water permeability and migration rate. These results provide the first evidence that aquaporin-mediated membrane water permeability enhances tumor cell migration and may be associated with tumor invasion and metastasis.

  2. PERSPECTIVISM AND DESIGN: EXPRESSIVE OBJECTIVITY PERSPECTIVISMO E DESENHO: OBJETIVIDADE EXPRESSIVA

    Directory of Open Access Journals (Sweden)

    Vânia Dutra de Azeredo

    2011-12-01

    Full Text Available This paper aims at analyzing the design of observation through the notions of interpretation and perspectivism in Nietzsche, trying to show the impossibility of a neutral position referring to the design that could capture its shades in an impartial way. We propose the concept of expressive objectivity as the necessary limit to human production and reading, mainly in the design of observation, making use of the Nietzschean conceptual apparatus.

    O presente artigo visa analisar o desenho de observação a partir das noções de interpretação e de perspectivismo nietzschiano, procurando mostrar a impossibilidade de uma posição neutra, com referência ao desenho, que pudesse captar imparcialmente suas nuanças. Propõe o conceito de objetividade expressiva enquanto limite necessário à produção e à leitura humanas, notadamente, no desenho de observação, desde o conjunto do aparato conceptual de Nietzsche.

  3. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  4. Inducible expression of endomorphins in murine dendritic cells.

    Science.gov (United States)

    Yang, Xiaohuai; Xia, Hui; Chen, Yong; Liu, Xiaofen; Zhou, Cheng; Gao, Qin; Li, Zhenghong

    2012-12-15

    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [(3)H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.

  5. Inducible expression of endomorphins in murine dendritic cells

    Institute of Scientific and Technical Information of China (English)

    Xiaohuai Yang; Hui Xia; Yong Chen; Xiaofen Liu; Cheng Zhou; Qin Gao; Zhenghong Li

    2012-01-01

    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7–8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of μ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of μ-opioid receptors.

  6. Gene expression analysis of in vivo fluorescent cells.

    Directory of Open Access Journals (Sweden)

    Konstantin Khodosevich

    Full Text Available BACKGROUND: The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD. Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters. METHODOLOGY/PRINCIPAL FINDINGS: We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR. CONCLUSIONS/SIGNIFICANCE: Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.

  7. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  8. Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

    Science.gov (United States)

    Chung, Jie-Yu; Mackay, Fabienne; Alderuccio, Frank

    2015-01-01

    Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals.

  9. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  10. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    -derived antigenic peptides, a function which is currently explored in immunotherapeutic approaches against cancer. Additionally, membrane-bound Hsp70 can stimulate antigen presenting cells to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells...... frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...... cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 cell surface expression was confined...

  11. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    R.M. Determann; M. Weisfelt; J. de Gans; A. van der Ende; M.J. Schultz; D. van de Beek

    2006-01-01

    Objective: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. Design: Retrospective study of diagnostic accuracy. Setting and patients: CSF was coll

  12. PICTURESQUE EXPRESSIVENESS OF MINERALS APPLIED FOR THE INTERIOR’ DESIGN

    Directory of Open Access Journals (Sweden)

    Olesya Vladimirovna Pustozerova

    2015-01-01

    Full Text Available The paper considered the interior that represented at all times a symbolic encoding of culture including, in a varying degree, desire for expressing one’s self that stimulated development of that encoding. The interior is unlikely further to fail its guideline to enhance personal expression of an individual. Our time prefers using natural building materials with their beauty exposed to public. Therefore, such a vogue for placing natural stone in the interior will never be outdated, and unusual application of this material will always excite interest to. Be it marble or meteorite, depends only on one’s personal option. Natural stone has a unique pattern revealed while splitting. Each stone is an original product of nature, as the pattern being composed by many factors of nature, namely wind, geographic location, climate, therefore, origins of any stone also play an important part in. Each natural stone is unique by a lot of lines/ layers; - they may be located in the only probable place for them, to create a certain figurative rhythm/ pace. Therefore, the designer should be able to be sensitive to the stone, to some energy it emanates, that means to supplement the interior with an appropriate selection of materials.

  13. Expression of stromelysin 3 in basal cell carcinomas.

    Science.gov (United States)

    Cribier, B; Noacco, G; Peltre, B; Grosshans, E

    2001-01-01

    Stromelysin 3 is a member of the metalloproteinase family, which is expressed in various remodelling processes. The prognosis of breast cancers and squamous cell carcinomas is correlated to the level of expression of this protein. The purpose of the present work was to evaluate the expression of stromelysin 3 in the major types of basal cell carcinomas. We selected cases of primary tumours that were fully excised, without previous biopsy: 40 Pinkus tumors, 40 superficial, 40 nodular, 38 morpheiform basal cell carcinomas and 10 cases showing deep subcutaneous or muscular invasion. Immunohistochemistry was carried out using monoclonal anti-ST3 antibodies (MC Rio, IGBMC Strasbourg), and evaluated on a semi-quantitative scale from 0 to 3. Positively stained cells were restricted to the periphery of the epithelial cells, which, by contrast, never expressed stromelysin 3. The global rate of expression was 27% in Pinkus tumors, 65% in superficial, 72.5% in nodular, 87% in morpheiform and 100% in deeply invasive carcinomas. The rates of tumours showing the highest number of positively stained cells (class 2 or 3) were respectively 7.5%, 20%, 45%, 63% and 100%. This systematic study of stromelysin3 expression in basal cell carcinomas confirms that it is a marker of poor prognosis, because the rate of positive tumours was much higher in aggressive carcinomas. Moreover, the majority of tumours showing an intense expression (i.e. the highest number of positively stained cells in their stroma) were of the morpheiform and deeply invasive types, which are of poor prognosis. Altogether, the studies performed on cutaneous tumours are consistent with the theory of stromelysin 3 playing an active role in tumour progression.

  14. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  15. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  16. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  17. Tff3 is Expressed in Neurons and Microglial Cells

    Directory of Open Access Journals (Sweden)

    Ting Fu

    2014-11-01

    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

  18. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  19. Gene expression markers for Caenorhabditis elegans vulval cells.

    Science.gov (United States)

    Inoue, Takao; Sherwood, David R; Aspöck, Gudrun; Butler, James A; Gupta, Bhagwati P; Kirouac, Martha; Wang, Minqin; Lee, Pei-Yun; Kramer, James M; Hope, Ian; Bürglin, Thomas R; Sternberg, Paul W

    2002-12-01

    The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

  20. CD44 variant expression in cutaneous T-cell lymphoma.

    Science.gov (United States)

    Orteu, C H; Li, W; Allen, M H; Smith, N P; Barker, J N; Whittaker, S J

    1997-07-01

    Expression of the lymphocyte homing receptor CD44 and its splice variants have been linked to tumour dissemination and poor prognosis in non-Hodgkin's lymphoma. Specifically, the in vitro expression of variant exon V6 confers metastatic potential in rat pancreatic carcinoma cell lines. In this study, we investigated the expression of CD44 splice variants in cutaneous T-cell lymphomas, including patients with mycosis fungoides (MF), Sezary syndrome (SS), large-cell anaplastic lymphoma (LCAL) and HTLV1-associated cutaneous lymphoma. In addition, 4 involved lymph nodes from 2 patients with MF and 1 patient with SS were examined. Inflammatory dermatoses, lichen planus and psoriasis, and normal skin were also studied. Immunohistochemistry was performed using a panel of monoclonal antibodies, including those with specificity for CD44H (standard isoform) and variant exons V3, V6 and V8-9. Normal epidermal keratinocytes were consistently CD44H and CD44 V3, V6 and V8-9 positive. In all the different clinicopathological subtypes and stages of cutaneous T-cell lymphomas, including involved lymph nodes, tumour cells consistently expressed CD44H, but were CD44 V3 and V6 negative. CD44 V8-9 was expressed on a majority of tumour cells in 2/5 LCAL and on occasional tumour cells in 2/5 LCAL. Occasional V8-9 positive tumour cells were also identified in 6/13 MF, 1/4 SS and 3/4 HTLV1. In 2/3 lymph node samples from 2 patients with tumour-stage MF, CD44 V8-9 expression was found on a small percentage of atypical mononuclear cells. Scattered V8-9 positive dermal mononuclear cells were present in sections of lichen planus and psoriasis. We have found no evidence to suggest that the metastasis-associated CD44 variant exon (V6) is expressed in cutaneous T-cell lymphoma, or that CD44H expression is associated with an adverse prognostic group. It is not clear whether the strong expression of CD44 V8-9 in 2 patients with CD30 positive LCAL reflects activation status or metastatic potential.

  1. EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    TANG Wei; DENG Wei-wu; Albert CHAN; Stanley CHIK; Adrain WU

    2005-01-01

    Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner(P<0.01). These results were also seen when the cells were stimulated by TNF-α and IL-13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin(P<0.05). Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.

  2. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  3. Nanostructured Semiconductor Device Design in Solar Cells

    Science.gov (United States)

    Dang, Hongmei

    We demonstrate the use of embedded CdS nanowires in improving spectral transmission loss and the low mechanical and electrical robustness of planar CdS window layer and thus enhancing the quantum efficiency and the reliability of the CdS-CdTe solar cells. CdS nanowire window layer enables light transmission gain at 300nm-550nm. A nearly ideal spectral response of quantum efficiency at a wide spectrum range provides an evidence for improving light transmission in the window layer and enhancing absorption and carrier generation in absorber. Nanowire CdS/CdTe solar cells with Cu/graphite/silver paste as back contacts, on SnO2/ITO-soda lime glass substrates, yield the highest efficiency of 12% in nanostructured CdS-CdTe solar cells. Reliability is improved by approximately 3 times over the cells with the traditional planar CdS counterpart. Junction transport mechanisms are delineated for advancing the basic understanding of device physics at the interface. Our results prove the efficacy of this nanowire approach for enhancing the quantum efficiency and the reliability in windowabsorber type solar cells (CdS-CdTe, CdS-CIGS and CdS-CZTSSe etc) and other optoelectronic devices. We further introduce MoO3-x as a transparent, low barrier back contact. We design nanowire CdS-CdTe solar cells on flexible foils of metals in a superstrate device structure, which makes low-cost roll-to-roll manufacturing process feasible and greatly reduces the complexity of fabrication. The MoO3 layer reduces the valence band offset relative to the CdTe, and creates improved cell performance. Annealing as-deposited MoO3 in N 2 reduces series resistance from 9.98 O/cm2 to 7.72 O/cm2, and hence efficiency of the nanowire solar cell is improved from 9.9% to 11%, which efficiency comparable to efficiency of planar counterparts. When the nanowire solar cell is illuminated from MoO 3-x /Au side, it yields an efficiency of 8.7%. This reduction in efficiency is attributed to decrease in Jsc from 25.5m

  4. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    , membrane-bound Hsp70 can stimulate antigen presenting cells (APCs) to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...... hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 surface expression was confined to the apoptotic Annexin V positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis...

  5. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    Directory of Open Access Journals (Sweden)

    Edyta Biskup

    2010-05-01

    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

  6. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo

    Directory of Open Access Journals (Sweden)

    Rasko John EJ

    2010-11-01

    Full Text Available Abstract Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS, we generated clonal cell populations from a human breast cancer (MCF-7 and a mouse melanoma (B16-F10 cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments.

  7. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  8. The regulation of CD5 expression in murine T cells

    Directory of Open Access Journals (Sweden)

    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

  9. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob;

    2003-01-01

    on monolayers of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium. MATERIALS AND METHODS: Colonic biopsies from four UC patients and four controls were examined by cryoimmuno......-electron microscopy using ICAM-1-antibodies. In four other controls, the epithelium was isolated from colonic biopsies, embedded in collagen, and evaluated similarly. Isolated crypts and cultured cancer cells were stimulated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha). RESULTS: ICAM-1......, both colonocytes and HT29 cells were capable of expressing ICAM-1 on their apical membranes in response to supraphysiologic cytokine concentrations. These observations question the justification of extrapolating observations from colon cancer cell lines to in vivo inflammatory conditions....

  10. Designer cell signal processing circuits for biotechnology.

    Science.gov (United States)

    Bradley, Robert W; Wang, Baojun

    2015-12-25

    Microorganisms are able to respond effectively to diverse signals from their environment and internal metabolism owing to their inherent sophisticated information processing capacity. A central aim of synthetic biology is to control and reprogramme the signal processing pathways within living cells so as to realise repurposed, beneficial applications ranging from disease diagnosis and environmental sensing to chemical bioproduction. To date most examples of synthetic biological signal processing have been built based on digital information flow, though analogue computing is being developed to cope with more complex operations and larger sets of variables. Great progress has been made in expanding the categories of characterised biological components that can be used for cellular signal manipulation, thereby allowing synthetic biologists to more rationally programme increasingly complex behaviours into living cells. Here we present a current overview of the components and strategies that exist for designer cell signal processing and decision making, discuss how these have been implemented in prototype systems for therapeutic, environmental, and industrial biotechnological applications, and examine emerging challenges in this promising field.

  11. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    Science.gov (United States)

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  12. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    2009-01-01

    We show that inhibition of HDAC activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC inhibitor-mediated Hsp70 surface expression was confined to the apoptotic Annexin V...... activity selectively induces surface expression of Hsp70 on hematopoietic cancer cells and that this may increase immunorecognition of these cells.......-positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis such as etoposide and camptothecin also led to a robust induction of Hsp70 surface expression. Hsp70 expression was, however, not caused by induction of apoptosis per se, as activated CD4 T cells remained Hsp70...

  13. Expression of cell cycle regulating factor mRNA in small cell lung cancer xenografts

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1998-01-01

    We have investigated the expression of cyclins, cyclin dependent kinases (CDK), and CDK inhibitors (CKI) at the mRNA level in a panel of small-cell lung cancer (SCLC) cell lines in vitro and in vivo as xenografts in nude mice. The results showed that the cell lines expressed varying amounts of most...... cyclin and CDK's but only a few of the cell lines expressed cyclin D1 and/or D2 and some lacked expression of CDK6. Most cell lines expressed mRNA for the CKI's but two cell lines lacked expression of P15INK4B and p16INK4A. The mRNA expression differed for a few of the cell lines regarding cyclin D2...... and CDK6 when in vitro and in vivo data were compared. Two of the cell lines that express the retinoblastoma (Rb) protein had no sign of a deregulated Rb pathway but further studies at the protein level are necessary to demonstrate whether these two cell lines should have a normal Rb pathway or whether...

  14. Regulation of stem cell factor expression in inflammation and asthma

    Directory of Open Access Journals (Sweden)

    Carla A Da Silva

    2005-03-01

    Full Text Available Stem cell factor (SCF is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.

  15. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

    Directory of Open Access Journals (Sweden)

    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  16. Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells.

    Science.gov (United States)

    Koyama, Hiroyuki; Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Hosoda, Hiroshi; Ariyasu, Hiroyuki; Kusakabe, Toru; Son, Choel; Hosoda, Kiminori; Akamizu, Takashi; Kangawa, Kenji; Nakao, Kazuwa

    2016-02-01

    To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-β1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

  17. Expression and Fuactional Role of HERG1, K+ Channels in Leukemic Cells and Leukemic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    LI Huiyu; LIU Liqiong; GUO Tiannan; ZHANG Jiahua; LI Xiaoqing; DU Wen; LIU Wei; CHEN Xiangjun; HUANG Shi'ang

    2007-01-01

    In order to investigate the expression and functional role of HERG1 K+ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K+ channels expression in leukemic cells and LSCs. The functional role of HERG1 K+ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34+/CD38-, CD123+ LSCs but not in circulating CD34+ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K+ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K+ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K+ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy.

  18. Dynamic distribution and stem cell characteristics of Sox1-expressing cells in the cerebellar cortex

    Institute of Scientific and Technical Information of China (English)

    Joelle Alcock; Virginie Sottile

    2009-01-01

    Bergmann glia cells are a discrete radial glia population surrounding Purkinje cells in the cerebellar cortex. Al-though Bergmann glia are essential for the development and correct arborization of Purkinje cells, little is known about the regulation of this cell population after the developmental phase. In an effort to characterize this population at the molecular level, we have analyzed marker expression and established that adult Bergmann glia express Soxl, Sox2 and Sox9, a feature otherwise associated with neural stem cells (NSCs). In the present study, we have further analyzed the developmental pattern of Soxl-expressing cells in the developing cerebellum. We report that before be-coming restricted to the Purkinje cell layer, Soxl-positive cells are present throughout the immature tissue, and that these cells show characteristics of Bergmann glia progenitors. Our study shows that these progenitors express Soxl, Sox2 and Sox9, a signature maintained throughout cerebellar maturation into adulthood. When isolated in culture, the Soxl-expressing cerebellar population exhibited neurosphere-forming ability, NSC-marker characteristics, and demonstrated multipotency at the clonal level. Our results show that the Bergmann glia population expresses Soxl during cerebellar development, and that these cells can be isolated and show stem cell characteristics in vitro, sug-gesting that they could hold a broader potential than previously thought.

  19. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells.

    Science.gov (United States)

    Oyamada, Masahito; Takebe, Kumiko; Endo, Aya; Hara, Sachiko; Oyamada, Yumiko

    2013-01-01

    Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  20. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells

    Directory of Open Access Journals (Sweden)

    Masahito eOyamada

    2013-07-01

    Full Text Available Pluripotent stem cells, i.e., embryonic stem (ES and induced pluripotent stem (iPS cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  1. Clinicopathological Significance of E-cadherin and PCNA Expression in Hunman Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    Jianwei Ma; Kai Fan; Yanli Zhang; Dawei Song; Jianmei Ma

    2008-01-01

    OBJECTIVE This study was designed to assess E-cadherin(E-cad) and proliferating cell nuclear antigen (PCNA) expression as well as their clinicopathological significance in hunman non-small cell lung cancers (NSCLCs). Possible molecular mechanisms of differentiation and metastasis of NSCLCs are discussed.METHODS Immunohistochemical and immunofluorescence double staining were performed to examine the expression of E-cad and PCNA in 68 primary NSCLCs cases.RESULTS The E-cad expression in squamous cell carcinomas and adenocarcinomas showed no significant difference. E-cad expression had a positive correlation with the histological-differentiated grade. A significant difference of Ecad expression was found between metastatic and non-metastatic groups. PCNA expression in squamous cell carcinomas and adenocarcinomas showed no significant difference. The PCNA expression had a reverse correlation with the histological-differentiated grade. A significant difference of PCNA expression was found between metastatic and non- metastatic groups. The E-cad and PCNA expression presented a reverse correlation.CONCLUSION E-cad expression is not associated with the histological type of NSCLC, but is associated with differentiation and metastasis of the cancer. Down-regulation of E-cad expression affects the proliferation of cancer cells. Conjoint analysis of E-cad and PCNA expression is a good way to evaluate tumor biological behavior.

  2. Regulation of cell-to-cell variability in divergent gene expression

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  3. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  4. Connective Tissue Growth Factor Expression in Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Amrita DOSANJH

    2006-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor β and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of α type I collagen, 5α-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody.The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.

  5. Expression of cyclooxygenase-2 in human esophageal squamous cell carcinomas

    Institute of Scientific and Technical Information of China (English)

    Jian-Gang Jiang; Dao-Wen Wang; Jiang-Bo Tang; Chun-Lian Chen; Bao-Xing Liu; Xiang-Ning Fu; Zhi-Hui Zhu; Wei Qu; Katherine Cianflone; Michael P. Waalkes

    2004-01-01

    AIM: To determine whether cyclooxygenase-2 (COX-2) was expressed in human esophageal squamous cell carcinoma.METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunohistochemistry and immunofluorescence were used to assess the expression level of COX-2 in esophageal tissue.RESULTS: COX-2 mRNA levels were increased by >80-fold in esophageal squamous cell carcinoma when compared to adjacent noncancerous tissue. COX-2 protein was present in 21 of 30 cases of esophageal squamous cell carcinoma tissues, but was undetectable in noncancerous tissue. Immunohistochemistry was performed to directly show expression of COX-2 in tumor tissue.CONCLUSION: These results suggest that COX-2 may be an important factor for esophageal cancer and inhibition of COX-2 may be helpful for prevention and possibly treatment of this cancer.

  6. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  7. Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs.

    Science.gov (United States)

    Gao, Qian; Zhao, Lili; Song, Ziyi; Yang, Gongshe

    2012-05-01

    Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.

  8. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  9. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Science.gov (United States)

    Hutschenreuther, Antje; Bigl, Marina; Hemdan, Nasr Y. A.; Debebe, Tewodros; Gaunitz, Frank; Birkenmeier, Gerd

    2016-01-01

    The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed. PMID:27999356

  10. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Directory of Open Access Journals (Sweden)

    Antje Hutschenreuther

    2016-12-01

    Full Text Available The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO. Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1 that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

  11. Cell Adhesion Regulates Expression of the Androgen Receptor and Coregulators in Different Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sheng Li

    2007-02-01

    Full Text Available Prostate cancer cells adhere to a tumor basement membrane, while secretoryepithelial cells reside in a suprabasal cell compartment. Since tumor cells are derived fromsuprabasal epithelial cells, they experience de-novo substratum adhesion in the context ofoncogenesis. We therefore analyzed whether cell-matrix adhesion could affect the proteinexpression and activity of the AR. In this study, AR protein expression declined uponsuspension of BPH-1-AR cells, but not in PC-3-AR cells shown by Western blot. In a timecourse study, BPH-1 cell lost AR expression within 6 hours, and the synthetic androgen,R1881 reduced the loss of AR expression. We further explored the mechanism of AR loss insuspended BPH-1 cells. BPH-1-AR cells underwent apoptosis (anoikis when suspended for2 - 5 hours. Suspension did not induce significant apoptosis or decreasing of AR expressionin PC-3 cells. Inhibition of apoptosis in suspended BPH-1-AR cells, either by expression ofBcl-2 or Bcl-xl or by treatment with Z-VAD, a caspase inhibitor, prevented loss of ARprotein. In contrast, the calpain protease inhibitor , ALLN, accelerated the loss of AR proteinexpression. Additionally, cell-matrix adhesion changed the expression of coregulators of ARin the mRNA level of prostate cancer cells. Our results demonstrate that AR proteinexpression was reduced through activation of cell death pathways, and thus indirectly through cell suspension in BPH-AR cells. The activity of AR can also be regulated by adhesion in PC-3-AR and LNCaP cells through affecting the coregulators level.

  12. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

    Directory of Open Access Journals (Sweden)

    Dam Phuongan

    2011-06-01

    Full Text Available Abstract Background Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH receptor (LHR expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE carcinoma cells. Methods The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours. Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. Results Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are

  13. Expression of CIDE proteins in clear cell renal cell carcinoma and their prognostic significance.

    Science.gov (United States)

    Yu, Ming; Wang, Hui; Zhao, Jun; Yuan, Yuan; Wang, Chao; Li, Jing; Zhang, Lijun; Zhang, Liying; Li, Qing; Ye, Jing

    2013-06-01

    Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of renal cell carcinoma. It is known to derive its histologic appearance from accumulation of abundant lipids and glycogens. The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the lipid droplet proteins involved in the metabolism of lipid storage droplets. The purpose of this study was to evaluate the expression of CIDE proteins in ccRCC cells and to investigate their prognostic significance. We examined consecutive patients with sporadic ccRCC, who underwent nephrectomy, to measure their mRNA and protein expression of CIDE proteins. We found that Cidec and ADRP expression were significantly up-regulated in ccRCC, compared with normal kidney tissues. Cideb was down-regulated. We also found that Cideb was expressed more in low-grade ccRCC than in high-grade tumors. To further clarify the relationship between Cideb expression and patient prognosis, we evaluated 57 ccRCC patients followed up for 120 months. Reduced ccRCC Cideb expression was associated with a higher Fuhrman nuclear grade. Patients with high Cideb expression had better overall survival rate than those with low expression (p < 0.05). Cideb expression was an independent predictor of survival (p = 0.001). Although the biologic function of Cideb in ccRCC remains unknown, the expression level of Cideb might be a novel predictor of prognosis in ccRCC.

  14. Immunglobulin Expression and Its Biological Significance in Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Duosha Hu; Hui Zheng; Haidan Liu; Ming Li; Wei Ren; Wei Liao; Zhi Duan; Lili Li; Ya Cao

    2008-01-01

    It is generally believed that the expression of a gene iS restricted "within the right place and at the right time".This principle has long been considered applicable as well to the expression of immunoglobulin(Ig)lymphocytes of B cell lineage.However,increasing evidence has shown Ig "paradoxically" expressed in malignant tumors of epitheliaI origin.We reviewed the recent progress in the study of cancer-derived Ig,and also discussed its mechanisms and possible functions,trying to arouse interest and attention to those working in the field of immunology and oncology.

  15. Implication of expression of Nanog in prostate cancer cells and their stem cells.

    Science.gov (United States)

    Gong, Chen; Liao, Hui; Guo, Fengjin; Qin, Liang; Qi, Jun

    2012-04-01

    Recent studies suggested that the prostate cancer may arise from prostate cancer stem cells that share some same characteristics with normal stem cells. The purpose of this study was to detect the differences of Nanog expression between PC3 prostate cancer cell line and its tumor stem cells, and the relationship was preliminarily examined between Nanog and prostate cancer and its tumor stem cells. By using magnetic active cell sorting (MACS), we isolated a population of CD44(+)/CD133(+) prostate cancer cells that display stem cell characteristics from PC3 cell line. Immunohistochemistry revealed positive expressions of CD44, CD133 and α(2)β(1)-integin in the isolated cells. CCK-8 analysis showed that isolated cells had a strong proliferative ability. The formation of the cell spheres in serum-free medium and holoclones in serum-supplied medium showed that the cells were capable of self-renewing, indicating that the isolated cells were a population of cancer stem-like cells derived from PC3 cell line. Western blotting exhibited that the isolated cells had higher experession of Nanog, an embryonic stem marker, as compared with PC3 cells. Our study showed that Nanog might be helpful in sustaining the self-renewal and the undifferentiation of prostate cancer stem cells, and may serve as a marker for prostate cancer stem cells for isolation and identification.

  16. Polyclonal T-Cells Express CD1a in Langerhans Cell Histiocytosis (LCH) Lesions

    Science.gov (United States)

    West, Jennifer A.; Olsen, Sharon L.; Mitchell, Jenée M.; Priddle, Ross E.; Luke, Jennifer M.; Åkefeldt, Selma Olsson; Henter, Jan-Inge; Turville, Christopher; Kannourakis, George

    2014-01-01

    Langerhans cell histiocytosis (LCH) is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a+/CD3+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs). We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH. PMID:25343480

  17. Polyclonal T-cells express CD1a in Langerhans cell histiocytosis (LCH lesions.

    Directory of Open Access Journals (Sweden)

    Jennifer A West

    Full Text Available Langerhans cell histiocytosis (LCH is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a(+/CD3(+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs. We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a(+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH.

  18. Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong Hou; Lisa Rossi; Ying Shan; Jian-Yu Zheng; Richard W Lambrecht; Herbert L Bonkovsky

    2009-01-01

    AIM: To investigate effects of iron on oxidative stress,heme oxygenase-1 (HMOX1) and hepatitis C viral (HCV) expression in human hepatoma cells stably expressing HCV proteins.METHODS: Effects of iron on oxidative stress, HMOX1,and HCV expression were assessed in CON1 cells.Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots.RESULTS: Iron, in the form of ferric nitrilotriacetate,increased oxidative stress and up-regulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOX1. Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2 (Nrf2) by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels. These iron effects were completely blocked by deferoxamine (DFO). Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%,nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells,whereas DFO increased them.CONCLUSION: Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

  19. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

    Directory of Open Access Journals (Sweden)

    Ana Paula Santin Bertoni

    2015-01-01

    Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

  20. Gene expression profiling of chicken primordial germ cell ESTs

    Directory of Open Access Journals (Sweden)

    Lim Dajeong

    2006-08-01

    Full Text Available Abstract Background Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. Results We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. Conclusion Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

  1. Expression of Neural Markers by Undifferentiated Rat Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Dana Foudah

    2012-01-01

    Full Text Available The spontaneous expression of neural markers by mesenchymal stem cells (MSCs has been considered to be a demonstration of MSCs’ predisposition to differentiate towards neural lineages. In view of their application in cell therapy for neurodegenerative diseases, it is very important to deepen the knowledge about this distinctive biological property of MSCs. In this study, we evaluated the expression of neuronal and glial markers in undifferentiated rat MSCs (rMSCs at different culture passages (from early to late. rMSCs spontaneously expressed neural markers depending on culture passage, and they were coexpressed or not with the neural progenitor marker nestin. In contrast, the number of rMSCs expressing mesengenic differentiation markers was very low or even completely absent. Moreover, rMSCs at late culture passages were not senescent cells and maintained the MSC immunophenotype. However, their differentiation capabilities were altered. In conclusion, our results support the concept of MSCs as multidifferentiated cells and suggest the existence of immature and mature neurally fated rMSC subpopulations. A possible correlation between specific MSC subpopulations and specific neural lineages could optimize the use of MSCs in cell transplantation therapy for the treatment of neurological diseases.

  2. ret/PTC-1 expression alters the immunoprofile of thyroid follicular cells

    Directory of Open Access Journals (Sweden)

    Aherne Sinead

    2008-05-01

    Full Text Available Abstract Background Hashimoto Thyroiditis (H.T. is a destructive autoimmune thyroid condition whose precise molecular pathogenesis remains unclear. ret/PTC-1 is a chimeric transcript which has been described in autoimmune thyroid disease (AITD and thyroid neoplasia. The purpose of this study was to observe the immunogenic effect exposure to H.T. and control lymphocyte supernatant would have on normal (Nthy-ori and ret/PTC-1 (TPC-1 expressing thyroid cell line models. Results A 2 × 2 matrix comprising Nthy-ori and TPC-1 cell lines and H.T. and control lymphocyte supernatant was designed and utilised as follows; activated lymphocytic supernatant from a H.T. and normal control were co-cultured with a cell line derived from normal thyroid (Nthy-ori and also a cell line derived from a papillary thyroid carcinoma that endogenously expresses ret/PTC-1 (TPC-1. The co-cultures were harvested at 0, 6 and 18 hour time points. Gene expression analysis was performed on RNA extracted from thyrocytes using TaqMan® Immune profiling Low-Density Arrays (Applied Biosystems, CA, USA comprising gene expression markers for 93 immune related targets plus 3 endogenous controls. Stimulation of the normal thyroid cell line model with activated T cell supernatant from the H.T. donor yielded global up-regulation of immune targets when compared with control supernatant stimulation. In particular, a cohort of targets (granzyme B, CD3, CD25, CD152, CD45 associated with cytotoxic cell death; T cell receptor (TCR and T cell signaling were up-regulated in the normal cell line model. When the ret/PTC-1 expressing thyroid cell line was co-cultured with H.T. lymphocyte supernatant, in comparison to control supernatant stimulation, down-regulation of the same subset of immune targets was seen. Conclusion Co-culturing H.T. lymphocyte supernatant with a normal thyroid cell line model leads to over-expression of a subset of targets which could contribute to the pathogenesis of H

  3. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.

    Directory of Open Access Journals (Sweden)

    Fabrizio Gelain

    Full Text Available Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2. These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with beta-Tubulin(+, GFAP(+ and Nestin(+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.

  4. Iterative experiment design guides the characterization of a light-inducible gene expression circuit.

    Science.gov (United States)

    Ruess, Jakob; Parise, Francesca; Milias-Argeitis, Andreas; Khammash, Mustafa; Lygeros, John

    2015-06-30

    Systems biology rests on the idea that biological complexity can be better unraveled through the interplay of modeling and experimentation. However, the success of this approach depends critically on the informativeness of the chosen experiments, which is usually unknown a priori. Here, we propose a systematic scheme based on iterations of optimal experiment design, flow cytometry experiments, and Bayesian parameter inference to guide the discovery process in the case of stochastic biochemical reaction networks. To illustrate the benefit of our methodology, we apply it to the characterization of an engineered light-inducible gene expression circuit in yeast and compare the performance of the resulting model with models identified from nonoptimal experiments. In particular, we compare the parameter posterior distributions and the precision to which the outcome of future experiments can be predicted. Moreover, we illustrate how the identified stochastic model can be used to determine light induction patterns that make either the average amount of protein or the variability in a population of cells follow a desired profile. Our results show that optimal experiment design allows one to derive models that are accurate enough to precisely predict and regulate the protein expression in heterogeneous cell populations over extended periods of time.

  5. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    Science.gov (United States)

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  6. Differentially expressed genes in giant cell tumor of bone.

    Science.gov (United States)

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  7. Structure-based, rational design of T cell receptors

    Directory of Open Access Journals (Sweden)

    Vincent eZoete

    2013-09-01

    Full Text Available Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce TCR modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction pMHC. Using the well-characterized 2C TCR/SIYR/H-2K(b structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. 54% of the designed sequence replacements exhibited improved pMHC-binding as compared to the native TCR, with up to 150 fold increase in affinity, while preserving specificity. Genetically-engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes. We have complemented the approach with a simplified rigid method to predict the TCR orientation over pMHC. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of

  8. Effects of Trichostatin A on HDAC8 Expression, Proliferation and Cell Cycle of Molt-4 Cells

    Institute of Scientific and Technical Information of China (English)

    HE Jing; LIU Hongli; CHEN Yan

    2006-01-01

    The effects of Trichostatin A (TSA) on histone deacetylase 8 (HDAC8) expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner. The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 μg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 μg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in G2/M phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells (P<0.01). It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.

  9. Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell autonomous mechanisms

    OpenAIRE

    Zolochevska, Olga; Figueiredo, Marxa L

    2009-01-01

    We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell autonomous and/or non-cell autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real time PCR, and in vivo molecular imaging using a novel three-way xenograft animal mod...

  10. Functional bitter taste receptors are expressed in brain cells.

    Science.gov (United States)

    Singh, Nisha; Vrontakis, Maria; Parkinson, Fiona; Chelikani, Prashen

    2011-03-04

    Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain.

  11. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Carlos Medina

    Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

  12. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    Science.gov (United States)

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  13. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    Science.gov (United States)

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  14. PRL-3 expression in nasal sinus squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zi-Hui Chen; Min-Ying Li

    2016-01-01

    Objective:To investigate the relationship between liver regeneration phosphatase-3 (PRL-3) with differentiation extent of nasal sinus squamous cell carcinoma, and molecular biological effects on the pathogenesis of nasal sinus squamous cell carcinoma to comprehend its relevance, so as to make early diagnosis of patients, and to give guidance to the prognosis. Methods:Immunohistochemistry was used to detect PRL-3 in 30 cases of different degrees of sinus nasal squamous cell carcinoma. 20 cases of normal nasal cavity of mucosa tissues were set as control. Results:The PRL-3 in all levels of sinonasal squamous cell carcinoma tissues, there was a significant difference compared with the normal nasal mucosa (P<0.05), squamous cell carcinoma and its expression increased with the grade with enhanced trend. Conclusions:PRL-3 expression increased significantly in sinonasal squamous cell carcinoma than in nasal polyp tissue, showed that it may be associated with squamous cell carcinoma of nasal sinus squamous cell carcinoma, may be the early event.

  15. Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Yan Wusheng

    2012-01-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC, the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB, normal differentiated squamous epithelium (ND, and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets. Results As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA were verified to be dysregulated in the same pattern at both the mRNA and protein levels. Conclusions These data reveal insight into genes and

  16. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad;

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  17. GLUT-1 Expression in Cutaneous Basal and Squamous Cell Carcinomas.

    Science.gov (United States)

    Abdou, Asmaa Gaber; Eldien, Marwa Mohammad Serag; Elsakka, Daliah

    2015-09-01

    Glucose uptake is a key regulating step in glucose metabolism and is mediated by facilitative glucose transporters (GLUTs), and GLUT-1 is the predominant glucose transporter in many types of human cells. Cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) represent the most common skin cancer in Egypt. The present study aimed at evaluation of the pattern and distribution of GLUT-1 in cutaneous BCC (16 cases) and SCC (16 cases) by means of immunohistochemistry. GLUT-1 was expressed in all SCC (100%) and in 62.5% of BCC. Membranous pattern of GLUT-1 was seen in 62.5% of SCC and 31.25% of BCC. Positivity (P = .02) and percentage (P = .000) of GLUT-1 expression were in favor of SCC in comparison to BCC. The high percentage of GLUT-1 expression was associated with high grade in SCC (P = .03). The immunoreactivity for GLUT-1 was more in the periphery of malignant nests of SCC while it was more in the center of BCC nests. GLUT-1 is overexpressed in cutaneous non-melanoma skin cancer. Its expression in SCC is related to differentiation status, and its expression in BCC is intimately associated with squamous metaplastic areas.

  18. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  19. DUAL ROLES OF CANCER CELL-EXPRESSED IMMUNOGLOBULINS IN CANCER IMMUNOLOGY

    Directory of Open Access Journals (Sweden)

    Gregory Lee

    2014-01-01

    Full Text Available While the expression of immunoglobulins and T cell receptors on cancer cells has been well-established for decades, the potential roles and mechanisms of action of these cancerous antigen receptors have not been fully elucidated. A monoclonal antibody designated as RP215, which reacts specifically with the carbohydrate-associated epitope located on the heavy chain region of cancerous immunoglobulins and T cell receptors, was used as a unique probe to study the roles of antigen receptors in the immunology of cancer cells. Through extensive cell-based biological and immunological studies, it was found that both anti-antigen receptors and RP215 demonstrated similar actions on the gene regulations involved in the growth/proliferation of cancer cells, as well as on toll-like receptors involved in innate immunity. In addition, RP215-specific cancerous immunoglobulins are believed to capture or neutralize circulating antigen/antibodies which may be harmful to cancer cells within the human body. In contrast to normal B and T cells and their respective receptors in the conventional immune system, cancer cells co-express both immunoglobulins and T cell receptors and immune protection is exercised by unique mechanisms. For example, these cancer cell-expressed antigen receptors display a lack of class switching, limited hyper-mutation, aberrant glycosylations and a strong influence on the toll-like receptors of cancer cells. Therefore, it is hypothesized that both normal and cancerous immune systems may co-exist and operate simultaneously within the human body. The balance of these two immune factors for respective surveillance and protection may be relevant to the outcome of cancer immunotherapy in humans. A potential therapeutic strategy is being developed by using RP215 as a drug candidate to target cancer cells based on these observations.

  20. Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

    Institute of Scientific and Technical Information of China (English)

    Huiyan WANG; Suhua CHEN

    2008-01-01

    The killing effects of lymphocytes on Hela cells expressing intedeukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24h and 72h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

  1. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    Science.gov (United States)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  2. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  3. Gene expression analysis of dendritic/Langerhans cells and Langerhans cell histiocytosis

    NARCIS (Netherlands)

    Rust, Renata; Kluiver, J.; Visser, Lydia; Harms, G.; Blokzijl, T.; Kamps, W.A.; Poppema, Sibrand; van den Berg, Anke

    2006-01-01

    Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LQ phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbil

  4. Metastasis regulation by PPARD expression in cancer cells

    Science.gov (United States)

    Zuo, Xiangsheng; Xu, Weiguo; Xu, Min; Tian, Rui; Moussalli, Micheline J.; Mao, Fei; Zheng, Xiaofeng; Wang, Jing; Morris, Jeffrey S.; Eng, Cathy; Maru, Dipen M.; Rashid, Asif; Broaddus, Russell; Wei, Daoyan; Hung, Mien-Chie; Sood, Anil K.

    2017-01-01

    Peroxisome proliferator–activated receptor–δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity for metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer. PMID:28097239

  5. Reduced expression of Slit2 in renal cell carcinoma.

    Science.gov (United States)

    Ma, Wei-Jie; Zhou, Yu; Lu, Dan; Dong, Dong; Tian, Xiao-Jun; Wen, Jie-Xi; Zhang, Jun

    2014-01-01

    Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is down-regulated in RCC partially due to promoter methylation.

  6. Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Sherman, Andrew; Chen, Ginny I;

    2009-01-01

    Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how...... information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1-controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2......- and ephrin-B1-expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling...

  7. Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

    Science.gov (United States)

    Mankame, T; Hokanson, R; Fudge, R; Chowdhary, R; Busbee, D

    2006-05-01

    Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

  8. Stem cell antigen 2 expression in adult and developing mice.

    Science.gov (United States)

    Antica, M; Wu, L; Scollay, R

    1997-01-01

    Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

  9. B cell lymphomas express CX3CR1 a non-B cell lineage adhesion molecule

    DEFF Research Database (Denmark)

    Andreasson, U.; Ek, S.; Merz, H.

    2008-01-01

    To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering...... the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX3CR1, in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX3CR1, which...... normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma...

  10. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  11. Batteries and fuel cells: Design, employment, chemistry

    Science.gov (United States)

    Euler, K.-J.

    The history of electrochemical current sources is considered along with primary cells, standard cells, high-energy primary cells, high-energy storage batteries, and fuel cells. Aspects of battery research and development are also discussed, taking into account general considerations related to technological development projects, the introduction of mathematical methods into battery research, resistance measurements, autoradiography and other radiochemical methods, color photography as an aid in research, electron microscopy, X-ray and electron diffraction, spin resonance methods, and electrical measurements involving powders. Attention is given to zinc/manganese dioxide cells, zinc/mercury cells, zinc/silver oxide primary cells, cells utilizing atmospheric oxygen, lead-acid batteries, nickel-iron and nickel-cadmium storage batteries, zinc/silver storage batteries, dry cells with organic depolarizers, dry cells with solid electrolyte, and storage batteries utilizing hydrogen.

  12. Porcine Adipose-Derived Mesenchymal Stem Cells Retain Their Stem Cell Characteristics and Cell Activities While Enhancing the Expression of Liver-Specific Genes after Acute Liver Failure

    Directory of Open Access Journals (Sweden)

    Chenxia Hu

    2016-01-01

    Full Text Available Acute liver failure (ALF is a kind of complicated syndrome. Furthermore, adipose-derived mesenchymal stem cells (ADMSCs can serve as a useful cell resource for autotransplantation due to their abundance and micro-invasive accessability. However, it is unknown how ALF will influence the characteristics of ADMSCs and whether ADMSCs from patients suffering from end-stage liver diseases are potential candidates for autotransplantation. This study was designed to compare various properties of ALF-derived ADMSCs with normal ADMSCs in pig models, with regard to their cellular morphology, cell proliferative ability, cell apoptosis, expression of surface antigens, mitochondrial and lysosomal activities, multilineage potency, and expression of liver-specific genes. Our results showed that ALF does not influence the stem cell characteristics and cell activities of ADMSCs. Intriguingly, the expression levels of several liver-specific genes in ALF-derived ADMSCs are higher than in normal ADMSCs. In conclusion, our findings indicate that the stem cell characteristics and cell activities of ADMSCs were not altered by ALF and these cells can serve as a new source for regenerative medicine.

  13. Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    Sheng-Quan Cheng; Wen-Liang Wang; Wei Yan; Qing-Long Li; Li Wang; Wen-Yong Wang

    2005-01-01

    AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells.METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector,pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sa/I and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidinbiotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi.RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent,apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited.CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

  14. Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen

    2008-01-01

    BACKGROUND:Currently, one of the tough problems for the application of bioartiifcial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxiifcation. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS:hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and ampliifed. Then hGS mRNA was measured by semi-quantitative RT-PCR;hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS:The expression of hGS mRNA in HepG2/pLNChGS cells (8.306±0.336) and HepG2/pLNAFhGS cells (21.358±1.716) was much stronger than in control cells (P CONCLUSION:The constructed hepatocytes (HepG2 cells) with speciifc high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

  15. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  16. Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EAgroup, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to"Zusanli"acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected.The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

  17. Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.

    Science.gov (United States)

    Patterson, Stacey S; Dionisi, Hebe M; Gupta, Rakesh K; Sayler, Gary S

    2005-03-01

    Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

  18. GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives

    Directory of Open Access Journals (Sweden)

    Tim Kao

    2016-09-01

    Full Text Available The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT, in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

  19. Sequential Notch signalling at the boundary of fringe expressing and non-expressing cells.

    Directory of Open Access Journals (Sweden)

    Tobias Troost

    Full Text Available Wing development in Drosophila requires the activation of Wingless (Wg in a small stripe along the boundary of Fringe (Fng expressing and non-expressing cells (FB, which coincides with the dorso-ventral (D/V boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser and Delta (Dl respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.

  20. A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection

    Science.gov (United States)

    Dong, Hui; Sun, Hao

    2016-01-01

    Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine) with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231) are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclin-dependent kinase inhibitor 1A (CDKN1A), and aurora kinase A (AURKA) genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation. PMID:27649175

  1. A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2016-09-01

    Full Text Available Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231 are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, cyclin-dependent kinase inhibitor 1A (CDKN1A, and aurora kinase A (AURKA genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.

  2. Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells

    Science.gov (United States)

    Dumitrache, Alexandru; Klingeman, Dawn M.; Natzke, Jace; Rodriguez Jr, Miguel; Giannone, Richard J.; Hettich, Robert L.; Davison, Brian H.; Brown, Steven D.

    2017-01-01

    Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes. PMID:28240279

  3. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  4. Tumor endothelial cells express high pentraxin 3 levels.

    Science.gov (United States)

    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  5. Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

    1993-01-01

    A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc-gene-family...... expression correlated with proliferative parameters. All tumours expressed at least one myc family member at the mRNA level. Exclusive c-myc mRNA expression was demonstrated in 8 tumours, L-myc in 7 and N-myc in I. Five tumours expressed both c-myc and L-myc, and 2 tumours expressed both c-myc and N...

  6. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer.

    Science.gov (United States)

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R; Tang, Dean G

    2016-02-29

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

  7. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  8. Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

    DEFF Research Database (Denmark)

    Bagdonaite, Ieva; Wandall, Hans H; Litvinov, Ivan V

    2015-01-01

    CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22∆N), which lacks the N-terminal domain as demonstrated by exon...... biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22∆N (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells...

  9. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    Science.gov (United States)

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

  10. HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

    Science.gov (United States)

    Paul, Pascale; Rouas-Freiss, Nathalie; Khalil-Daher, Iman; Moreau, Philippe; Riteau, Beatrice; Le Gal, Frederique Anne; Avril, Marie Francoise; Dausset, Jean; Guillet, Jean Gerard; Carosella, Edgardo D.

    1998-01-01

    Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies. PMID:9539768

  11. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

  12. Differential expression of the klf6 tumor suppressor gene upon cell damaging treatments in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gehrau, Ricardo C.; D' Astolfo, Diego S.; Andreoli, Veronica [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Bocco, Jose L., E-mail: jbocco@fcq.unc.edu.ar [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Koritschoner, Nicolas P. [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-02-10

    The mammalian Krueppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC{sub 50}). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p < 0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC{sub 50} concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable

  13. MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Quinlin, Imelda S; Fogler, William E; Phillips, Catherine A; Townsend, Mary; Robinson, William; Philip, Ramila

    2008-01-01

    Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

  14. Differential gene expression in stromal cells of human giant cell tumor of bone.

    Science.gov (United States)

    Wuelling, M; Delling, G; Kaiser, E

    2004-12-01

    Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.

  15. Characterization of cell subpopulations expressing progenitor cell markers in porcine cardiac valves.

    Directory of Open Access Journals (Sweden)

    Huan Wang

    Full Text Available Valvular interstitial cells (VICs are the main population of cells found in cardiac valves. These resident fibroblastic cells play important roles in maintaining proper valve function, and their dysregulation has been linked to disease progression in humans. Despite the critical functions of VICs, their cellular composition is still not well defined for humans and other mammals. Given the limited availability of healthy human valves and the similarity in valve structure and function between humans and pigs, we characterized porcine VICs (pVICs based on expression of cell surface proteins and sorted a specific subpopulation of pVICs to study its functions. We found that small percentages of pVICs express the progenitor cell markers ABCG2 (~5%, NG2 (~5% or SSEA-4 (~7%, whereas another subpopulation (~5% expresses OB-CDH, a type of cadherin expressed by myofibroblasts or osteo-progenitors. pVICs isolated from either aortic or pulmonary valves express most of these protein markers at similar levels. Interestingly, OB-CDH, NG2 and SSEA-4 all label distinct valvular subpopulations relative to each other; however, NG2 and ABCG2 are co-expressed in the same cells. ABCG2(+ cells were further characterized and found to deposit more calcified matrix than ABCG2(- cells upon osteogenic induction, suggesting that they may be involved in the development of osteogenic VICs during valve pathology. Cell profiling based on flow cytometry and functional studies with sorted primary cells provide not only new and quantitative information about the cellular composition of porcine cardiac valves, but also contribute to our understanding of how a subpopulation of valvular cells (ABCG2(+ cells may participate in tissue repair and disease progression.

  16. Design and fabrication of silver-hydrogen cells

    Science.gov (United States)

    Klein, M. G.

    1975-01-01

    The design and fabrication of silver-hydrogen secondary cells capable of delivering higher energy densities than comparable nickel-cadmium and nickel-hydrogen cells and relatively high cycle life is presented. An experimental task utilizing single electrode pairs for the optimization of the individual electrode components, the preparation of a design for lightweight 20Ahr cells, and the fabrication of four 20Ahr cells in heavy wall test housing containing electrode stacks of the lightweight design are described. The design approach is based on the use of a single cylindrical self-contained cell with a stacked disc sequence of electrodes. The electrode stack design is based on the use of NASA- Astropower Separator Material, PPF fuel cell anodes, an intercell electrolyte reservoir concept and sintered silver electrodes. Results of performance tests are given.

  17. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

    Science.gov (United States)

    Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J; Plante, Jessica A; Frolova, Elena I; Beasley, David W C; Frolov, Ilya

    2014-07-22

    Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

  18. Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.

    Science.gov (United States)

    Schroeder, Insa S; Sulzbacher, Sabine; Nolden, Tobias; Fuchs, Joerg; Czarnota, Judith; Meisterfeld, Ronny; Himmelbauer, Heinz; Wobus, Anna M

    2012-01-01

    Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.

  19. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

    Directory of Open Access Journals (Sweden)

    David M Gravano

    Full Text Available BACKGROUND: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. METHODOLOGY/PRINCIPAL FINDINGS: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. CONCLUSIONS/SIGNIFICANCE: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and

  20. PEM Fuel Cells Redesign Using Biomimetic and TRIZ Design Methodologies

    Science.gov (United States)

    Fung, Keith Kin Kei

    Two formal design methodologies, biomimetic design and the Theory of Inventive Problem Solving, TRIZ, were applied to the redesign of a Proton Exchange Membrane (PEM) fuel cell. Proof of concept prototyping was performed on two of the concepts for water management. The liquid water collection with strategically placed wicks concept demonstrated the potential benefits for a fuel cell. Conversely, the periodic flow direction reversal concepts might cause a potential reduction water removal from a fuel cell. The causes of this water removal reduction remain unclear. In additional, three of the concepts generated with biomimetic design were further studied and demonstrated to stimulate more creative ideas in the thermal and water management of fuel cells. The biomimetic design and the TRIZ methodologies were successfully applied to fuel cells and provided different perspectives to the redesign of fuel cells. The methodologies should continue to be used to improve fuel cells.

  1. Discrimination of meniscal cell phenotypes using gene expression profiles

    Directory of Open Access Journals (Sweden)

    M Son

    2012-03-01

    Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  2. Expression of cubilin in mouse testes and Leydig cells.

    Science.gov (United States)

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

  3. Design-Based Learning for Biology: Genetic Engineering Experience Improves Understanding of Gene Expression

    Science.gov (United States)

    Ellefson, Michelle R.; Brinker, Rebecca A.; Vernacchio, Vincent J.; Schunn, Christian D.

    2008-01-01

    Gene expression is a difficult topic for students to learn and comprehend, at least partially because it involves various biochemical structures and processes occurring at the microscopic level. Designer Bacteria, a design-based learning (DBL) unit for high-school students, applies principles of DBL to the teaching of gene expression. Throughout…

  4. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    Science.gov (United States)

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  5. Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells.

    Science.gov (United States)

    Funakoshi, Tadanao; Spector, Myron

    2010-06-01

    Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones.

  6. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro.

  7. Global regulation of gene expression and cell differentiation in Caulobacter crescentus in response to nutrient availability.

    Science.gov (United States)

    England, Jennifer C; Perchuk, Barrett S; Laub, Michael T; Gober, James W

    2010-02-01

    In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments, Caulobacter crescentus cells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest that Caulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period.

  8. Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

    Institute of Scientific and Technical Information of China (English)

    伍晓菲; 陈智超; 刘仲萍; 周浩; 游泳; 黎纬明; 邹萍

    2004-01-01

    To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry.Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2 O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.

  9. Ultrasound/Microbubble Enhances Foreign Gene Expression in ECV304 Cells and Murine Myocardium

    Institute of Scientific and Technical Information of China (English)

    Dongping GUO; Xiaorong LI; Xiaoyu LI; Ping SUN; Zhiguang WANG; Xiuying CHEN; Qi CHEN; Leming FAN; Bin ZHANG; Lizheng SHAO

    2004-01-01

    Although viral vectors are efficient systems to transfer foreign genes into cells or target tissues,safety issues remain in relation to human gene therapy. Microbubbles currently used as ultrasound contrast agents have been applied in transfection of genes. This study was designed to test the transfection efficiency and the expression of exogenous gene mediated by ultrasound irradiation enhanced air filled albumin microbubbles in ECV304 cell line in vitro and the heart of the mouse in vivo. Air filled microbubbles (2.0-4.0 μm in diameter) were created by sonicating the mixture of human albumin, glucose, mannitol and special additive that was designed for stabilization. Plasmid DNA loading the reporter genes was gently mixed with microbubbles. The mixture of plasmid DNA and microbubbles was administrated to cultured ECV304 cells and BALB/c mice (tail vein injection) under different ultrasound/microbubble conditions, and then the transfection and expression efficiency were examined. The results both in vivo and in vitro demonstrated that microbubble with ultrasound irradiation could significantly elevate the exogenous gene expression as compared with microbubble or ultrasound only. Overall, the present study showed that the ultrasound-target microbubble destruction method enhanced the exogenous gene expression in vivo and in vitro, and provided a gene therapy way not only efficient but also easy to be manipulated and carried out in clinical.

  10. Expression of basement membrane antigens in spindle cell melanoma.

    Science.gov (United States)

    Prieto, V G; Woodruff, J M

    1998-07-01

    Spindle cell melanoma (SCM) is an uncommon form of melanoma that may be confused histologically with other tumors, including malignant peripheral nerve sheath tumors (MPNST). Tumors with neural differentiation and melanocytic nevi may both show basement membrane immunohistochemically and at the ultrastructural level. However, most ultrastructural studies of melanoma have failed to demonstrate well formed basement membrane around tumor cells. The presence of basement membrane has been used by some authors as evidence favoring MPNST, as opposed to SCM. To evaluate this distinction immunohistochemically, 22 primary and metastatic cutaneous melanomas having a spindle cell component (SCM) were studied using monoclonal antibodies against laminin and Type IV collagen. S100 protein and HMB45 antigen expression were also studied. All but one of the SCM were reactive for S100 protein in at least 25% of the cells. Thirteen of 20 tumors (65%) were focally reactive with HMB45. Laminin was expressed in 42% of the tumors (only membranous pattern in 3; cytoplasmic and membranous in 5). Seventeen tumors (77%) expressed type IV collagen (only membranous pattern in 7; cytoplasmic and membranous pattern in 10). Laminin and type IV collagen, known components of basement membrane, are often found in SCM. Therefore, their detection cannot be used to distinguish SCM from MPNST.

  11. Reframed Genome-Scale Metabolic Model to Facilitate Genetic Design and Integration with Expression Data.

    Science.gov (United States)

    Gu, Deqing; Jian, Xingxing; Zhang, Cheng; Hua, Qiang

    2016-06-08

    Genome-scale metabolic network models (GEMs) have played important roles in the design of genetically engineered strains and helped biologists to decipher metabolism. However, due to the complex gene-reaction relationships that exist in model systems, most algorithms have limited capabilities with respect to directly predicting accurate genetic design for metabolic engineering. In particular, methods that predict reaction knockout strategies leading to overproduction are often impractical in terms of gene manipulations. Recently, we proposed a method named LTM (logical transformation of model) to simplify the gene-reaction associations by introducing intermediate pseudo reactions, which makes it possible to generate genetic design. Here, we propose an alternative method to relieve researchers from deciphering complex gene-reactions by adding pseudo gene controlling reactions. In comparison to LTM, this new method introduces fewer pseudo reactions and generates a much smaller model system named as gModel. We showed that gModel allows two seldom reported applications: identification of minimal genomes and design of minimal cell factories within a modified OptKnock framework. In addition, gModel could be used to integrate expression data directly and improve the performance of the E-Fmin method for predicting fluxes. In conclusion, the model transformation procedure will facilitate genetic research based on GEMs, extending their applications.

  12. Binary gene induction and protein expression in individual cells

    Directory of Open Access Journals (Sweden)

    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  13. Mast cells express novel functional IL-15 receptor alpha isoforms.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  14. Characterization of protocadherin-1 expression in primary bronchial epithelial cells : association with epithelial cell differentiation

    NARCIS (Netherlands)

    Koning, Henk; Sayers, Ian; Stewart, Ceri E.; de Jong, Debora; ten Hacken, Nick H. T.; Postma, Dirkje S.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2012-01-01

    Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA an

  15. Design, Modeling, and Development of Microbial Cell Factories

    KAUST Repository

    Kodzius, Rimantas

    2014-03-26

    Using Metagenomic analysis, computational modeling, single cell and genome editing technologies, we will express desired microbial genes and their networks in suitable hosts for mass production of energy, food, and fine chemicals.

  16. Analysis of the regulation of fatty acid binding protein 7 expression in human renal carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Sugiyama Takayuki

    2011-07-01

    Full Text Available Abstract Background Improving the treatment of renal cell carcinoma (RCC will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7, also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system. Results We determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293 were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2 and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines. Conclusions Three putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key

  17. Expression of Odontogenic Genes in Human Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Seyedeh Sara Bagheri

    2013-01-01

    Full Text Available Objective: Tooth loss is a common problem and since current tooth replacement methods cannot counter balance with biological tooth structures, regenerating natural tooth structures has become an ideal goal. A challenging problem in tooth regeneration is to find a proper clinically feasible cell to seed.This study was designed to investigate the odontogenic potential of human bone marrow mesenchymal stem cells (HBMSCs for seeding in tooth regeneration.Materials and Methods: In this experimental study, three pregnant Sprague Dawley (SD rats were used at the eleventh embryonic day and rat fetuses were removed surgically using semilunar flap under general anesthesia. The primary mandible was cut using a stereomicroscope. The epithelial and mesenchymal components were separated and the dissected oral epithelium was cultured for 3 days. We used flow cytometry analysis to confirm presence of mesenchymal stem cells and not hematopoietic cells and to demonstrate the presence of oral epithelium. Bone marrow mesenchymal stem cells (BMSCs and cultured oral epithelium were then co-cultured for 14 days. BMSCs cultured alone were used as controls. Expression of two odontogenic genes Pax9 and DMP1 was assessed using quantitative reverse transcription- polymerase chain reaction (RT-PCR.Results: Expression of two odontogenic genes, Pax9 and DMP1, were detected in BMSCs co-cultured with oral epithelium but not in the control group.Conclusion: Expression of Pax9 and DMP1 by human BMSCs in the proximity of odontogenic epithelium indicates odontogenic potential of these cells.

  18. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors.

    Science.gov (United States)

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan

    2013-11-01

    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

  19. Applications of ``PV Optics`` for solar cell and module design

    Energy Technology Data Exchange (ETDEWEB)

    Sopori, B.L.; Madjdpour, J.; Chen, W. [National Renewable Energy Lab., Golden, CO (United States)

    1998-09-01

    This paper describes some applications of a new optics software package, PV Optics, developed for the optical design of solar cells and modules. PV Optics is suitable for the analysis and design of both thick and thin solar cells. It also includes a feature for calculation of metallic losses related to contacts and back reflectors.

  20. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    Science.gov (United States)

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  1. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞

    2003-01-01

    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  2. Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

    Institute of Scientific and Technical Information of China (English)

    Claudia Lange; Philipp Bassler; Michael V. Lioznov; Helge Bruns; Dietrich Kluth; Axel R. Zander; Henning C. Fiegel

    2005-01-01

    AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore,we assessed the influence of cocultured liver cells on induction of liver-specific gene expression.METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF,EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19,and AFP was performed in the different cell populations.RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18,CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression.CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.

  3. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  4. PAX2 regulates ADAM10 expression and mediates anchorage-independent cell growth of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Sophia Boyoung Lee

    Full Text Available PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression.

  5. Trichostatin A Regulates hGCN5 Expression and Cell Cycle on Daudi Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Hongli; CHEN Yan; CUI Guohui; WU Gang; WANG Tao; HU Jianli

    2006-01-01

    The expression of human general control of amino acid synthesis protein 5 (hGCN5) in human Burkitt's lymphoma Daudi cells in vitro, effects of Trichostatin A (TSA) on cell proliferation and apoptosis and the molecular mechanism of TSA inhibiting proliferation of Daudi cells were investigated. The effects of TSA on the growth of Daudi cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effect of TSA on the cell cycle of Daudi cells was assayed by a propidium iodide method. Immunochemistry and Western blot were used to detect the expression of hGCN5. The proliferation of Daudi cells was decreased in TSA-treated group with a 24 h IC50 value of 415.3979 μg/L. TSA induced apoptosis of Daudi cells in a time- and dose-dependent manner. Treatment with TSA (200 and 400 μg/L) for 24 h, the apoptosis rates of Daudi cells were (14.74±2.04) % and (17.63±1.25) %, respectively. The cell cycle was arrested in G0/G1 phase (50, 100 μtg/L) and in G2/M phase (200 μg/L) by treatment with TSA for 24 h.The expression of hGCN5 protein in Daudi cells was increased in 24 h TSA-treated group by immunochemistry and Western blot (P<0.05). It was suggested that TSA as HDACIs could increase the expression of hGCN5 in Daudi cells, and might play an important role in regulating the proliferation and apoptosis of B-NHL cell line Daudi cells.

  6. Impact of methoxyacetic acid on mouse Leydig cell gene expression

    Directory of Open Access Journals (Sweden)

    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  7. Expression of human adenosine deaminase in murine hematopoietic cells.

    Science.gov (United States)

    Belmont, J W; MacGregor, G R; Wager-Smith, K; Fletcher, F A; Moore, K A; Hawkins, D; Villalon, D; Chang, S M; Caskey, C T

    1988-01-01

    Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells. Images PMID:3072474

  8. Design and testing of the Series III AMTEC cell

    Energy Technology Data Exchange (ETDEWEB)

    Mital, R.; Sievers, R.K.

    1999-07-01

    This paper describes the design and testing of the Series III (S3) Alkali Metal Thermal to Electric Converter (AMTEC) cell which is capable of high efficiency (15--25%) and high power density (100--150 W/kg). Compared to the Series 2 cell which is being developed primarily for space power systems, the Series III cell design provides a significantly higher beta{double_prime}-alumina solid electrolyte (BASE) tube packing density around the heat source thereby increasing cell power and minimizing heat loss. The prototype S3 cell will have 96 BASE tubes and is expected to produce about 150 We. In this cell design the BASE tube assemblies are mounted on a cylindrical tube support plate. The BASE tubes are arranged like spokes on a wheel. The inner cylinder, concentric to the tube support plate, is the hot side of the cell and the outer cylinder is the condenser. Since the prototype S3 cell will be the first of its kind, an engineering cell with same dimensions as the prototype but with 24 BASE tubes was built first. The purpose of this cell was to identify and resolve structural, thermal, manufacturing and sodium management issues before launching into the build of a complete 96 BASE tube cell. The engineering cell has been successfully built and tested. The data of the engineering cells have been used to calibrate the SINDA/FLUINT code to predict the prototype cell performance more accurately. The build of the prototype 96 BASE tube cells is now in progress. This paper presents the design and development of the prototype S3 cell. The fabrication and testing of the first S3 engineering cell is discussed next. Based on the test data of the engineering cell, the anticipated thermal performance of the prototype cells predicted by the calibrated SINDA model are also presented.

  9. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells.

    Science.gov (United States)

    Rieske, Piotr; Augelli, Brian J; Stawski, Robert; Gaughan, John; Azizi, S Ausim; Krynska, Barbara

    2009-02-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

  10. Erythropoietin protects myocardin-expressing cardiac stem cells against cytotoxicity of tumor necrosis factor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Madonna, Rosalinda [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); Institute of Cardiology, and Center of Excellence on Aging, ' G. d' Annunzio' University, Chieti (Italy); Shelat, Harnath; Xue, Qun; Willerson, James T. [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); The Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, Texas (United States); De Caterina, Raffaele [Institute of Cardiology, and Center of Excellence on Aging, ' G. d' Annunzio' University, Chieti (Italy); Geng, Yong-Jian, E-mail: yong-jian.geng@uth.tmc.edu [The Center for Cardiovascular Biology and Atherosclerosis Research, The University of Texas Health Science Center at Houston, Texas (United States); The Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, Texas (United States)

    2009-10-15

    Cardiac stem cells are vulnerable to inflammation caused by infarction or ischemic injury. The growth factor, erythropoietin (Epo), ameliorates the inflammatory response of the myocardium to ischemic injury. This study was designed to assess the role of Epo in regulation of expression and activation of the cell death-associated intracellular signaling components in cardiac myoblasts stimulated with the proinflammatory cytokine tumor necrosis factor (TNF)-{alpha}. Cardiac myoblasts isolated from canine embryonic hearts characterized by expression of myocardin A, a promyogenic transcription factor for cardiovascular muscle development were pretreated with Epo and then exposed to TNF-{alpha}. Compared to untreated cells, the Epo-treated cardiac myoblasts exhibited better morphology and viability. Immunoblotting revealed lower levels of active caspase-3 and reductions in iNOS expression and NO production in Epo-treated cells. Furthermore, Epo pretreatment reduced nuclear translocation of NF-{kappa}B and inhibited phosphorylation of inhibitor of kappa B (I{kappa}B) in TNF-{alpha}-stimulated cardiac myoblasts. Thus, Epo protects cardiac myocyte progenitors or myoblasts against the cytotoxic effects of TNF-{alpha} by inhibiting NF-{kappa}B-mediated iNOS expression and NO production and by preventing caspase-3 activation.

  11. CXCL12 expression by healthy and malignant ovarian epithelial cells

    Directory of Open Access Journals (Sweden)

    Emilie Dominique

    2011-03-01

    Full Text Available Abstract Background CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC, CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. Methods Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study. Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. Results Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47% to absent in 18 cases ( Conclusion Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. Trial Registration ClinicalTrials.gov: NCT00052468

  12. Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    Science.gov (United States)

    Liu, Li; Spurrier, Joshua; Butt, Tauseef R; Strickler, James E

    2008-11-01

    Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.

  13. E-cadherin and c-Met expression in actinic cheilits and lip squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    A Martínez

    2011-12-01

    Full Text Available Objective: The aim of this study was to assess epithelial expression of E-cadherin and c-Met in normal lip, in actinic cheilitis and lip squamous cell carcinoma. Study Design: Biopsies of normal lip vermillion (NL, n=18, actinic cheilitis (AC, n=37, and lip SCC (n=22 were processed for E-cadherin and c-Met immunodetection. Epithelial and tumor cell expression was scored for each sample considering staining intensity and percentage. Results: E-cadherin expression was significantly reduced in AC and lip SCC as compared to normal lip (P<0.05, with a significant reduction in lip SCC as compared to AC (P=0.003. Expression of c-Met was significantly higher in AC and lip SCC as compared to NL (P<0.05, with a significant increase in lip SCC as compared to AC (P<0.0001. Conclusion: The results showed that epithelial E-cadherin expression is reduced and c-Met expression is increased as lip carcinogenesis progresses, suggesting that these proteins may be useful markers of malignant transformation.

  14. Mural granulosa cell gene expression associated with oocyte developmental competence

    Directory of Open Access Journals (Sweden)

    Jiang Jin-Yi

    2010-03-01

    Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

  15. Interfacial polymerization for colorimetric labeling of protein expression in cells.

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    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  16. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  17. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    Science.gov (United States)

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

  18. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

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    Yue Yu

    Full Text Available BACKGROUND: Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. METHODS: Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. RESULTS: Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. CONCLUSIONS: Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  19. Alterations in integrin expression modulates invasion of pancreatic cancer cells.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    2009-01-01

    BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

  20. Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

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    Takahiro Nagatake

    Full Text Available Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4, and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga and other enteroendocrine cell-related genes (Ffar1, Ffar4, Gpr119, and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1. A Cld4+UEA-1- cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip, thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg, pancreatic polypeptide-like peptide with N-terminal tyrosine amide (Pyy, cholecystokinin (Cck, secretin (Sct, and tryptophan hydroxylase 1 (Tph1. In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.

  1. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

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    Anne Mathilde Lund

    Full Text Available A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

  2. Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains

    Science.gov (United States)

    2017-01-01

    A fundamental question in biology is how sharp boundaries of gene expression form precisely in spite of biological variation/noise. Numerous mechanisms position gene expression domains across fields of cells (e.g. morphogens), but how these domains are refined remains unclear. In some cases, domain boundaries sharpen through differential adhesion-mediated cell sorting. However, boundaries can also sharpen through cellular plasticity, with cell fate changes driven by up- or down-regulation of gene expression. In this context, we have argued that noise in gene expression can help cells transition to the correct fate. Here we investigate the efficacy of cell sorting, gene expression plasticity, and their combination in boundary sharpening using multi-scale, stochastic models. We focus on the formation of hindbrain segments (rhombomeres) in the developing zebrafish as an example, but the mechanisms investigated apply broadly to many tissues. Our results indicate that neither sorting nor plasticity is sufficient on its own to sharpen transition regions between different rhombomeres. Rather the two have complementary strengths and weaknesses, which synergize when combined to sharpen gene expression boundaries. PMID:28135279

  3. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    Science.gov (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  4. Expression

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    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  5. Tissue-specific designs of stem cell hierarchies

    NARCIS (Netherlands)

    Visvader, Jane E; Clevers, Hans

    2016-01-01

    Recent work in the field of stem cell biology suggests that there is no single design for an adult tissue stem cell hierarchy, and that different tissues employ distinct strategies to meet their self-renewal and repair requirements. Stem cells may be multipotent or unipotent, and can exist in quiesc

  6. Mutational study of sapovirus expression in insect cells

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    Natori Katsuro

    2005-02-01

    Full Text Available Abstract Human sapovirus (SaV, an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1 in a baculovirus expression system results in the formation of virus-like particles (VLPs. In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct, whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct. While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.

  7. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

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    David T. Ting

    2014-09-01

    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

  8. Analysis of the CD161-expressing cell quantities and CD161 expression levels in peripheral blood natural killer and T cells of systemic lupus erythematosus patients.

    Science.gov (United States)

    Lin, Yi-Lung; Lin, Shih-Chang

    2017-02-01

    Expressed on the cell surface of most of NK cells and some T cells, CD161 has been shown to deliver inhibitory signal in human NK cells. To determine whether the CD161-expressing cell quantities and the cell surface expression levels of CD161 in NK and T cells were altered in systemic lupus erythematosus (SLE) patients, we analyzed the CD3, CD56 and CD161 expression patterns of peripheral blood lymphocytes by flow cytometric analysis to identify different NK and T cell subpopulations. The cell surface expression levels of CD161 were estimated by the mean florescence intensities (MFIs) of CD161. It was found that SLE patients had lower frequencies of CD161+CD56+CD3- and CD161+CD56+CD3+ cells among the lymphocyte population than normal controls, whereas the frequencies of CD161-CD56+CD3- and CD161+CD56-CD3+ cells were not statistically different between two groups. In addition, SLE patients also had decreased absolute counts of all CD161-expressing NK cells and T cells and had reduced frequencies of CD161+ cells in CD56+CD3-, CD56+CD3+ and CD56-CD3+ cell populations. Moreover, SLE patients had reduced MFIs of CD161 in CD161+CD56+CD3+ and CD161+CD56-CD3+, but not CD161+CD56+CD3-, cell populations. Our results indicated that CD161-expressing cell frequency and the CD161 expression levels were reduced in some NK and T cell subpopulations of SLE patients, suggesting possible important role of CD161 and CD161-expressing immune cells in the SLE pathogenesis.

  9. Expression of T cell factor-4 in non-small-cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    LI Chun-yan; WANG Yan; CUI Ze-shi; WANG En-hua

    2005-01-01

    Background T cell factor- 4 (TCF- 4) plays an important role in development and carcinogenesis. Recently, the role of TCF- 4 has been described in colon cancer and other cancers. However, whether TCF- 4 plays a similar role in lung cancer is unknown. To answer this question, we studied the expression of TCF- 4 protein and mRNA in non-small-cell lung cancer (NSCLC) and the relation of TCF- 4 expression pattern to histological type and cell differentiation. Methods Tissue samples from sixty cases of pathologically diagnosed NSCLC and eight normal tissue samples were obtained between September 2001 and March 2003. Immunohistochemistry was used to investigate the distribution of TCF- 4 protein. The staining patterns of the tumors were divided into 4 categories: nuclear staining alone or nuclear staining greater than cytoplasmic staining; cytoplasmic staining or cytoplasmic staining greater than nuclear staining; equal nuclear and cytoplasmic staining; no nuclear staining or cytoplasmic staining. The integrated optical density (OD) values of all sections were analyzed by UIC MetaMorph image analysis software. The expression of TCF- 4 mRNA was detected by one-step reverse transcription-polymerase chain reaction (RT-PCR). The integrated density values of the PCR products were analyzed semi-quantitatively.Results Immunohistochemistry showed that there was no expression of TCF- 4 in normal tissue. However, TCF- 4 was expressed in 86.7% (52/60) of NSCLC samples, mainly in the nuclei of tumor cells. Furthermore, there was a significant difference in TCF- 4 localization patterns between squamous cell carcinomas and adenocarcinomas (P<0.05). The integrated OD values of TCF- 4 expression was significantly higher in tumors with moderate-poor cell differentiation than in well differentiated tumors (51.63±6.67 vs 46.13±12.31, P<0.01). There was no TCF- 4 mRNA expression in normal tissue. However, 63.9% (23/36) of carcinoma samples expressed TCF- 4 mRNA. TCF- 4 mRNA expression was

  10. Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina.

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    Diana Katsman

    Full Text Available Cell-derived microvesicles (MVs, recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  11. Expression of stem cell markers in the human fetal kidney.

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    Sally Metsuyanim

    Full Text Available In the human fetal kidney (HFK self-renewing stem cells residing in the metanephric mesenchyme (MM/blastema are induced to form all cell types of the nephron till 34(th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2 are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24 in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (50% of HFK cells and predominantly co-express EpCAM(bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM(+EpCAM(- and to a lesser extent in NCAM(+EpCAM(+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM(+EpCAM(+FZD7(+, MM stem cells (NCAM(+EpCAM(-FZD7(+ or both (NCAM(+FZD7(+. These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

  12. A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

    Science.gov (United States)

    Scoville, Steven D; Mundy-Bosse, Bethany L; Zhang, Michael H; Chen, Li; Zhang, Xiaoli; Keller, Karen A; Hughes, Tiffany; Chen, Luxi; Cheng, Stephanie; Bergin, Stephen M; Mao, Hsiaoyin C; McClory, Susan; Yu, Jianhua; Carson, William E; Caligiuri, Michael A; Freud, Aharon G

    2016-05-17

    The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

  13. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

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    Maricla Galetti

    Full Text Available BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

  14. Nucleus- and cell-specific gene expression in monkey thalamus.

    Science.gov (United States)

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  15. Characterization of a new human melanoma cell line with CD133 expression.

    Science.gov (United States)

    Gil-Benso, Rosario; Monteagudo, Carlos; Cerdá-Nicolás, Miguel; Callaghan, Robert C; Pinto, Sandra; Martínez-Romero, Alicia; Pellín-Carcelén, Ana; San-Miguel, Teresa; Cigudosa, Juan C; López-Ginés, Concha

    2012-06-01

    A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

  16. Restoration of p53 Expression in Human Cancer Cell Lines Upregulates the Expression of Notch1: Implications for Cancer Cell Fate Determination after Genotoxic Stress

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    Fatouma Alimirah

    2007-05-01

    Full Text Available Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress.

  17. Restoration of p53 Expression in Human Cancer Cell Lines Upregulates the Expression of Notch1: Implications for Cancer Cell Fate Determination after Genotoxic Stress1

    Science.gov (United States)

    Alimirah, Fatouma; Panchanathan, Ravichandran; Davis, Francesca J; Chen, Jianming; Choubey, Divaker

    2007-01-01

    Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53-mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53-mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress. PMID:17534448

  18. Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels.

    Directory of Open Access Journals (Sweden)

    Naotake Funamizu

    Full Text Available Tetrahydrouridine (THU is a well characterized and potent inhibitor of cytidine deaminase (CDA. Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299 exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.

  19. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    Science.gov (United States)

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  20. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  1. Performance benchmarking of four cell-free protein expression systems.

    Science.gov (United States)

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  2. Design Report for ACP Hot Cell Rear Door

    Energy Technology Data Exchange (ETDEWEB)

    Ku, J. H.; Kwon, K. C.; Choung, W. M.; Cho, I. J.; Kook, D. H.; Lee, W. K.; You, G. S.; Lee, E. P.; Park, S. W

    2005-12-15

    A hot-cell facility was constructed at the IMEF building for the demonstrate ACP process. ACP hot-cell consists of process cell and maintenance cell, and each cell has rear door. Since this facility was constructed at basement floor, all process materials, equipment and radioactive materials are take in and out through the rear door. Also, this door can be an access route of workers for the maintenance works. Therefore ACP hot-cell rear doors must maintain the radiation shielding, sealing, mechanical and structural safety. This report presents design criteria, design contents of each part and driving part. It was confirmed that the rear doors sufficiently maintain the safety through the structural analysis and shielding analysis. Also, it was confirmed that the rear doors were constructed as designed by the gamma scanning test after the installation.

  3. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells.

    Science.gov (United States)

    Wang, Hongtao; Gao, Peng; Zheng, Jie

    2014-09-05

    Arsenic trioxide (As2O3) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As2O3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As2O3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As2O3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As2O3 than HPV 16-positive CaSki and SiHa cells. After As2O3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As2O3 is a potential anticancer drug for cervical cancer.

  4. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C. (Massachusetts Institute of Technology, Cambridge (USA))

    1990-01-01

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.

  5. Expression of lactoperoxidase in differentiated mouse colon epithelial cells.

    Science.gov (United States)

    Kim, Byung-Wook; Esworthy, R Steven; Hahn, Maria A; Pfeifer, Gerd P; Chu, Fong-Fong

    2012-05-01

    Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

  6. Induction in myeloid leukemic cells of genes that are expressed in different normal tissues

    OpenAIRE

    2005-01-01

    Using DNA microarray and cluster analysis of expressed genes in a cloned line (M1-t-p53) of myeloid leukemic cells, we have analyzed the expression of genes that are preferentially expressed in different normal tissues. Clustering of 547 highly expressed genes in these leukemic cells showed 38 genes preferentially expressed in normal hematopoietic tissues and 122 other genes preferentially expressed in different normal non-hematopoietic tissues including neuronal tissues, muscle, liver and te...

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

    Science.gov (United States)

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

    2015-12-01

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

  8. RASSF1A expression inhibits cell growth and enhances cell chemosensitivity to mitomycin in BEL-7402 hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    GUAN Hong-geng; XUE Wan-jiang; QIAN Hai-xin; ZHOU Xiao-jun; QIN Lei; LAN Jing

    2009-01-01

    Background The antitumor role of Ras association domain family 1A (RASSFIA) gene and its potential molecular mechanisms are not well understood. The objective of this study was to observe the antitumor ability of RASSFIA in hepatoceliular carcinoma, and study the mechanisms of cell apoptosis induced by RASSFIA.Methods After stably transfecting a RASSF1A (wild-type or mutant) expression vector into the BEL-7402 hepatocellular carcinoma cell line, RT-PCR and Westem blotting was used to detect the RASSF1A expression levels in recombinant cells. The effects of wild-type RASSF1A on cell growth were observed in vitro by analyzing cell proliferation rate, cell colony formation, and in vivo by analyzing tumorigenesis in nude mice. In addition, the effect of RASSF1A gene expression on the chemosensitivity of human hepatocellular carcinoma cells to antitumor drugs was examined by inhibition of cell proliferation and the percentage of apoptotic cells.Results Wild-type RASSF1A, not the mutant, suppressed cell growth in vitro and in vivo. Re-expression of wild-type RASSF1A could enhance the inhibition of cell proliferation and the percentage of apoptotic cells following cell treatment with mitomycin, but had no significant effect when combined with adriamycin, etoposide, 5-fluorouracil and cisplatJn treatment.Conclusion Wild-type RASSF1A inhibits cell growth and enhances cell chemosensitivity to mitomycin in hepatocellular carcinoma, suggesting that RASSF1A may serve as a new target for gene therapy in hepatocellular carcinoma patients.

  9. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær;

    2014-01-01

    , in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors......., cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple...... construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning...

  10. SREBP inhibits VEGF expression in human smooth muscle cells.

    Science.gov (United States)

    Motoyama, Koka; Fukumoto, Shinya; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

    Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate expression of genes encoding enzymes for lipid biosynthesis. SREBPs are activated by HMG-CoA reductase inhibitors (statins). Statins have been also reported to suppress vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells (VSMCs). Therefore, we hypothesized that SREBPs are involved in statin-mediated regulation of VEGF production in VSMCs. SREBP1 was robustly expressed, and was activated by atorvastatin in VSMCs, as demonstrated by increased levels of the mature nuclear form of SREBP1, and increased promoter activities of a reporter containing sterol regulatory elements by atorvastatin. Moreover, overexpression of SREBP1a dose-dependently suppressed VEGF promoter activity. Site-specific mutation or deletion of the proximal Sp1 sites reduced the inhibitory effects of SREBP1a on VEGF promoter activity. These data demonstrated that SREBP1, activated by atorvastatin, suppressed VEGF expression through the indirect interaction with the proximal tandem Sp1 sites in VSMCs.

  11. MicroRNA Expression in Alzheimer Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Hyman M. Schipper

    2007-01-01

    Full Text Available Various coding genes representing multiple functional categories are downregulated in blood mononuclear cells (BMC of patients with sporadic Alzheimer disease (AD. Noncoding microRNAs (miRNA regulate gene expression by degrading messages or inhibiting translation. Using BMC as a paradigm for the study of systemic alterations in AD, we investigated whether peripheral miRNA expression is altered in this condition. MicroRNA levels were assessed using the microRNA microarray (MMChip containing 462 human miRNA, and the results validated by real time PCR. Sixteen AD patients and sixteen normal elderly controls (NEC were matched for ethnicity, age, gender and education. The expression of several BMC miRNAs was found to increase in AD relative to NEC levels, and may differ between AD subjects bearing one or two APOE4 alleles. As compared to NEC, miRNAs signifi cantly upregulated in AD subjects and confi rmed by qPCR were miR-34a and 181b. Predicted target genes downregulated in Alzheimer BMC that correlated with the upregulated miRNAs were largely represented in the functional categories of Transcription/Translation and Synaptic Activity. Several miRNAs targeting the same genes were within the functional category of Injury response/Redox homeostasis. Taken together, induction of microRNA expression in BMC may contribute to the aberrant systemic decline in mRNA levels in sporadic AD.

  12. Tolerogenic IDO+ dendritic cells are induced by PD-1-expressing mast cells

    Directory of Open Access Journals (Sweden)

    Cecilia Pessoa Rodrigues

    2016-01-01

    Full Text Available Mast cells (MC are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs, on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between immature monocyte-derived DCs (iDCs and MC bends DCs towards tolerance induction. DCs that had direct contact with MC (MC-iDC decreased HLA-DR but increased PD-L1 expression and stimulated regulatory T lymphocytes, which expresses FoxP3+, secrete TGF-β and IL-10, and suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, MC-iDC expressed higher levels of indoleamine-2,3-deoxigenase (IDO, a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or anti-PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs. Contact with MC also increased phosphorylated STAT-3 levels in iDCs. When a STAT-3 inhibitor, JSI-124, was added to the DCs before contact with MC, the MC-iDC recovered their ability to induce allogeneic T cell proliferation and did not increase their IDO expression.

  13. Dpp-expressing and non-expressing cells: two different populations of growing cells in Drosophila.

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    Carolina Arias

    Full Text Available There are different models that explain growth during development. One model is based on insect and amphibian regeneration studies. This model proposes that growth is directed by pattern, and growth takes place by intercalation at a growth discontinuity; therefore, proliferation should surround the discontinuity. Currently, this model, apart from regenerative studies on mostly adult patterning, has not found supporting evidence in Drosophila that shows proliferation surrounding a discontinuity. Despite this lack of evidence, the importance of discontinuities has been shown in different experiments, even under wt conditions, more specifically in the formation of the leg joints because of the occurrence of cell death at their boundaries. Here, we show the existence of a sharp discontinuity in Decapentaplegic (Dpp in the genital discs at the third larvae stage (L3, which determines the upregulation in the Jun-NH2-Terminal-Kinase (JNK pathway, reaper (rpr, head involution defective (hid and active caspases from its boundaries. The proliferation and cell death surrounding the discontinuity suggest that growth can proceed by intercalation and competitive death takes place in this area. Finally, we show that the Rpr, Grim and Hid (RGH products are a few of the factors that define the growth discontinuity because they are negative regulators of growth, a new function that is unique from their known functions in apoptosis.

  14. Thiazolidinediones enhance vascular endothelial growth factor expression and induce cell growth inhibition in non-small-cell lung cancer cells

    Directory of Open Access Journals (Sweden)

    Yoshizaki Yumiko

    2010-03-01

    Full Text Available Abstract Background It is known that thiazolidinediones are involved in regulating the expression of various genes, including the vascular endothelial growth factor (VEGF gene via peroxisome proliferator-activated receptor γ (PPARγ; VEGF is a prognostic biomarker for non-small-cell lung cancer (NSCLC. Methods In this study, we investigated the effects of troglitazone and ciglitazone on the mRNA expression of VEGF and its receptors in human NSCLC cell lines, RERF-LC-AI, SK-MES-1, PC-14, and A549. These mRNA expressions were evaluated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR analysis. We also studied the effect of Je-11, a VEGF inhibitor, on the growth of these cells. Results In NSCLC cells, thiazolidinediones increased the mRNA expression of VEGF and neuropilin-1, but not that of other receptors such as fms-like tyrosine kinase and kinase insert domain receptor-1. Furthermore, the PPARγ antagonist GW9662 completely reversed this thiazolidinedione-induced increase in VEGF expression. Furthermore, the addition of VEGF inhibitors into the culture medium resulted in the reversal of thiazolidinedione-induced growth inhibition. Conclusions Our results indicated that thiazolidinediones enhance VEGF and neuropilin-1 expression and induce the inhibition of cell growth. We propose the existence of a pathway for arresting cell growth that involves the interaction of thiazolidinedione-induced VEGF and neuropilin-1 in NSCLC.

  15. Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas

    DEFF Research Database (Denmark)

    Møller, C J; Christgau, S; Williamson, M R;

    1992-01-01

    (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone......The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin...... in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans...

  16. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Wang

    Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

  17. The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells.

    Science.gov (United States)

    Egusa, Hiroshi; Doi, Masanori; Saeki, Makio; Fukuyasu, Sho; Akashi, Yoshihiro; Yokota, Yoshifumi; Yatani, Hirofumi; Kamisaki, Yoshinori

    2011-08-01

    Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to

  18. Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

    Directory of Open Access Journals (Sweden)

    Valeria Feinshtein

    2013-09-01

    Full Text Available Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD, a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp and Breast Cancer Resistance Protein (BCRP expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp for comparison. Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3 and rhodamine123(rh123. Cyclosporine A (CsA served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3 and rh123 was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

  19. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kati Juuti-Uusitalo

    Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

  20. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    Science.gov (United States)

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis.

  1. LIGHT/TNFSR14 can regulate hepatic lipase expression by hepatocytes independent of T cells and Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Bijoy Chellan

    Full Text Available LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTβR signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTβR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTβR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTβR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTβR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT's costimulatory function.

  2. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    Science.gov (United States)

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  3. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  4. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Shuqiang Li

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  5. Caspases regulate VAMP-8 expression and phagocytosis in dendritic cells.

    Science.gov (United States)

    Ho, Yong Hou Sunny; Cai, Deyu Tarika; Huang, Dachuan; Wang, Cheng Chun; Wong, Siew Heng

    2009-09-18

    During an inflammation and upon encountering pathogens, immature dendritic cells (DC) undergo a maturation process to become highly efficient in presenting antigens. This transition from immature to mature state is accompanied by various physiological, functional and morphological changes including reduction of caspase activity and inhibition of phagocytosis in the mature DC. Caspases are cysteine proteases which play essential roles in apoptosis, necrosis and inflammation. Here, we demonstrate that VAMP-8, (a SNARE protein of the early/late endosomes) which has been shown previously to inhibit phagocytosis in DC, is a substrate of caspases. Furthermore, we identified two putative conserved caspase recognition/cleavage sites on the VAMP-8 protein. Consistent with the up-regulation of VAMP-8 expression upon treatment with caspase inhibitor (CI), immature DC treated with CI exhibits lower phagocytosis activity. Thus, our results highlight the role of caspases in regulating VAMP-8 expression and subsequently phagocytosis during maturation of DC.

  6. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob;

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers ...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium.......Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...

  7. Expression of inducible nitric oxide in human lung epithelial cells.

    Science.gov (United States)

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  8. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  9. NASA Alternative Orion Small Cell Battery Design Support

    Science.gov (United States)

    Haynes, Chuck

    2016-01-01

    The NASA Orion Crew Module Reference Design was produced to address large scale thermal runaway (TR) hazard with specific safety controls for the Orion Spacecraft. The design presented provides the description of a full scale battery design reference for implementation as a drop in replacement to meet all spacecraft energy requirements with compatible 120 Vdc electrical and mechanical interface using small cell technology (18650) packaging. The 32V SuperBrick incorporates unique support features and an electrical bus bar arrangement that allows cells negative can insertion into heat sink that is compressively coupled to the battery enclosure to promote good thermal management. The housing design also provides an internal flame suppression "filter tray" and positive venting path internal to the enclosure to allow hot effluent ejecta to escape in the event of single cell TR. Virtual cells (14P Banks) that are supported to provide cell spacing with interstitial materials to prevent side can failures that can produce cell to cell TR propagation. These features were successfully test in four separate TR run with the full scale DTA1 test article in February 2016. Successfully Completed Test Objectives - Four separate TR test runs with Full-Scale DTA1 housing with Two SuperBricks, Two SuperBrick Emulators All Tests resulted in "clean" gas with less than 6 C rise at Battery vent All Tests resulted in less than 2 C temperature rise on cold-plate outlet All Tests resulted in less than 6 psi pressure rise in the battery housing Test Run 1 -One neighbor cell TR, highest remaining neighbor 139 C. Ejecta shorted to bus caused prolonged additional heating, One shorted cell did experience TR after 12 minutes, remaining cells had adequate thermal margin Test Run 2 - No cell to cell propagation, highest neighbor cell 112 C; Test Run 3 - No cell to cell propagation, highest neighbor cell 96 C; Test Run 4 - No cell to cell propagation, highest neighbor cell 101 C; Primary TR testing

  10. Germ Cell Nuclear Factor (GCNF) Represses Oct4 Expression and Globally Modulates Gene Expression in Human Embryonic Stem (hES) Cells.

    Science.gov (United States)

    Wang, Hongran; Wang, Xiaohong; Xu, Xueping; Kyba, Michael; Cooney, Austin J

    2016-04-15

    Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.

  11. Expression of precerebellins in cultured rat calvaria osteoblast-like cells.

    Science.gov (United States)

    Rucinski, Marcin; Zok, Agnieszka; Guidolin, Diego; De Caro, Raffaele; Malendowicz, Ludwik K

    2008-10-01

    Cerebellin (CER), originally isolated from rat cerebellum, is a hexadecapeptide derived from the larger precursor called precerebellin 1 (Cbln1). At present 4 propeptides designated as Cbln1, Cbln2, Cbln3 and Cbln4 are recognized. They belong to precerebellin subfamily of the C1q family proteins. Precerebellins act as transneuronal regulators of synapse development and synaptic plasticity in various brain regions. Initially CER was thought to be a cerebellum specific peptide, however subsequent studies revealed its presence in other brain regions as well as in extraneuronal tissues. We investigated whether precerebellins are expressed and involved in regulation of cultured rat calvarial osteoblast-like (ROB) cells. Classic RT-PCR revealed the presence of Cbln1 and Cbln3 mRNA in fragments of rat calvaria, in freshly isolated ROB cells and in ROB cells cultured for 7, 14 and 21 days. Cbln2 and Cbln4 mRNA, on the other hand, could not be demonstrated in ROB cells but was found to be present in the brain. In freshly isolated ROB cells expression of Cbln1 gene was very low and gradually increased in relation to the duration of culture. Expression of Cbln3, on the other hand, was very low in fragments of rat calvaria, and increased notably after digestion with collagenase-I. The highest expression of this precerebellin was observed at day 14 of culture while at days 7 and 21 levels of expressions were notably lower. Neither Cbln2 nor Cbln4 was found to be expressed in the ROB cells. Neither CER nor des-Ser1-CER (10(-10)-10(-6)M) affect osteocalcin production and proliferation rate of studied cells. The above findings suggest that CER, which theoretically would be derived from Cbln1, modulate neither differentiated (osteocalcin secretion) nor basic (proliferation) functions of cultured rat osteoblast-like cells. The obtained data raise an intriguing hypothesis that precerebellins may be involved in regulating of spatial organization of osteoblastic niches in the bone.

  12. Expression of Caspase-3 in Laryngeal Squamous Cell Carcinoma and its Relationship with Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    YU Yuan-chen; ZHONG Zhao-kun; LI Ji-xia; YU Chuan-ting

    2015-01-01

    Objective:To investigate the expression of Caspase-3 in laryngeal squamous cell carcinoma (LSCC) and its relationship with cell apoptosis. Methods: The expression of Caspase-3 protein in 43 LSCC tissues and 21 vocal cord polyp tissues was detected using immunohistochemical SP method; the apoptosis of LSCC was measured by in situ end-labeling (TUNEL) assay, and the relationships between Caspase-3 expression and clinicopathological features as well as cell apoptosis in LSCC tissue were analyzed. Results:The positive rate of Caspase-3 expression in LSCC tissue was lower than in vocal cord polyp tissue dramatically, with statistical significance (51.2%vs. 85.7%,P=0.007). Caspase-3 expression in LSCC tissue was closely related to the tumor differentiated degrees, clinical staging and presence or absence of lymph node metastasis (P=0.009, 0.001, 0.018) instead of the gender, age and tumor size (P>0.05). The apoptosis index (AI) of Caspase-3 was (4.31±0.49)% in LSCC tissue, while (24.28±1.07)% in vocal cord polyp tissue. Significant difference was presented between two groups by comparison to the AI (P<0.001). Spearman correlation analysis displayed that Caspase-3 expression in LSCC tissue had a signiifcantly positive correlation with the number of positive TUNEL cells (r=0.435,P=0.000). Conclusion: Low expression of Caspase-3 protein might promote the tumorigenesis and progression by reducing the apoptosis of tumor cells, and detection to its protein can be considered as an important index for judging the differentiation, clinical staging, inifltration and metastasis of laryngeal carcinoma.

  13. Eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells.

    Science.gov (United States)

    Kim, Sun Suk; Oh, O-Jin; Min, Hye-Young; Park, Eun-Jung; Kim, Youngleem; Park, Hyen Joo; Nam Han, Yong; Lee, Sang Kook

    2003-06-01

    Inducible cyclooxygenase (COX-2) has been implicated in the processes of inflammation and carcinogenesis. Thus, the potential COX-2 inhibitors have been considered as anti-inflammatory or cancer chemopreventive agents. In this study, the methanolic extract of the cortex of Eugenia caryophyllata Thunberg (Myrtaceae) was found to potently inhibit the prostaglandin E(2) production in lipopolysaccharide (LPS)-activated mouse macrophage RAW264.7 cells (98.3% inhibition at the test concentration of 10 microg/ml). Further, hexane-soluble layer was the most active partition compared to ethyl acetate, n-butanol, and water-soluble parts. By bioassay-guided fractionation of hexane-soluble partition, eugenol was isolated and exhibited a significant inhibition of PGE(2) production (IC(50) = 0.37 microM). In addition, eugenol suppressed the cyclooxygenase-2 (COX-2) gene expression in LPS-stimulated mouse macrophage cells. On the line of COX-2 playing an important role in colon carcinogenesis further study was designed to investigate the effect of eugenol on the growth and COX-2 expression in HT-29 human colon cancer cells. Eugenol inhibited the proliferation of HT-29 cells and the mRNA expression of COX-2, but not COX-1. This result suggests that eugenol might be a plausible lead candidate for further developing the COX-2 inhibitor as an anti-inflammatory or cancer chemopreventive agent.

  14. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  15. TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

    Science.gov (United States)

    Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

    2014-01-24

    Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.

  16. Broadband back grating design for thin film solar cells

    KAUST Repository

    Janjua, Bilal

    2013-01-01

    In this paper, design based on tapered circular grating structure was studied, to provide broadband enhancement in thin film amorphous silicon solar cells. In comparison to planar structure an absorption enhancement of ~ 7% was realized.

  17. Design of a Fuel Cell Hybrid Electric Vehicle Drive System

    DEFF Research Database (Denmark)

    Schaltz, Erik

    Fuel cells achieve more and more attention due to their potential of replacing the traditional internal combustion engine (ICE) used in the area of transportation. In this PhD thesis a fuel cell shaft power pack (FCSPP) is designed and implemented in a small truck. The FCSPP replaces the original...... the efficiency. In this work a lead-acid battery, an ultracapacitor, or a combination of both are considered as energy storage devices. A FCSPP is designed for 10 different configurations of connecting the energy storage device(s) and fuel cell to a common bus, which comply with the 42V PowerNet standard. Each...... of the ten configurations is designed for different fuel cell power ratings. The FCSPP is designed in an iterative process where the power flow through the system is under the influence of a certain energy management strategy and charging strategy, which sufficiently divides the power between the units...

  18. PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity

    OpenAIRE

    Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

    2015-01-01

    Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of P...

  19. Gene Expression Profiling of Dendritic Cells in Different Physiological Stages under Cordyceps sinensis Treatment

    Science.gov (United States)

    Li, Chia-Yang; Chiang, Chi-Shiun; Cheng, Wei-Chung; Wang, Shu-Chi; Cheng, Hung-Tsu; Chen, Chaang-Ray; Shu, Wun-Yi; Tsai, Min-Lung; Hseu, Ruey-Shyang; Chang, Cheng-Wei; Huang, Chao-Ying; Fang, Shih-Hua; Hsu, Ian C.

    2012-01-01

    Cordyceps sinensis (CS) has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs), in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS), or LPS plus CS (LPS/CS) for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs), were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE) genes of the three different treatments (CS, LPS, and LPS/CS), which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects of a complex

  20. Gene expression profiling of dendritic cells in different physiological stages under Cordyceps sinensis treatment.

    Directory of Open Access Journals (Sweden)

    Chia-Yang Li

    Full Text Available Cordyceps sinensis (CS has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs, in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS, or LPS plus CS (LPS/CS for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs, were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE genes of the three different treatments (CS, LPS, and LPS/CS, which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects

  1. Design Methodology of the Structure of Postal Express Mail Networks of Aviation Channels in China

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    On the basis of the postal area center office system, the continuous space approximation is used to study the structure of postal express mail networks of aviation channels in China. Tradeoffs among sorting, handling, transportation, administrative and facilities costs are examined. The optimizing design methodology proposed in this paper can be used to analyze and design the postal express mail network. The objective is to minimize the total system cost.

  2. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  3. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  4. Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

    Science.gov (United States)

    Fehrmann, Steffen; Bottin-Duplus, Hélène; Leonidou, Andri; Mollereau, Esther; Barthelaix, Audrey; Wei, Wu; Steinmetz, Lars M; Yvert, Gaël

    2013-10-08

    Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

  5. Cytokine-Induced Cell Surface Expression of Adhesion Molecules in Vascular Endothelial Cells In vitro

    Institute of Scientific and Technical Information of China (English)

    陈红辉; 刘昌勤; 孙圣刚; 梅元武; 童萼塘

    2001-01-01

    Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF-α (1-250 U/ml) or IL-1β (0.1-50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF-α (100 U/ml) or IL-1β (10 U/ml), for 4-72 h, cell surface expression of adhesion molecules (ICAM-1 and VCAM-1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up-regulated by TNF-α, IL-1β in a concentration- and time-dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM-1 and on VCAM-1 expression. Cytokines might directly induce the expression of ICAM-1 and VCAM-1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.

  6. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  7. Design & development of innovative proton exchange membrane fuel cells

    OpenAIRE

    Carton, James

    2011-01-01

    The research undertaken in this thesis is concerned with the design and development of Proton Exchange Membrane (PEM) fuel cells and provides a body of information for continued PEM fuel cell development, which will ideally aid in the future commercialisation of these electrochemical devices. Through a combination of numerical analysis, computational fluid dynamic modelling and experimental work, effective flow plate designs, flow field configurations and materials are analysed and new inn...

  8. Rat visceral yolk sac cells: viability and expression of cell markers during maternal diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Aires, M.B. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Santos, J.R.A. [Departamento de Enfermagem, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Souza, K.S.; Farias, P.S. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Santos, A.C.V. [Departamento de Enfermagem, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Fioretto, E.T. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Maria, D.A. [Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP (Brazil)

    2015-07-10

    The function of the visceral yolk sac (VYS) is critical for embryo organogenesis until final fetal development in rats, and can be affected by conditions such as diabetes. In view of the importance of diabetes during pregnancy for maternal and neonatal health, the objective of this study was to assess fetal weight, VYS cell markers, and viability in female Wistar rats (200-250 g) with induced diabetes (alloxan, 37 mg/kg) on the 8th gestational day (gd 8). At gd 15, rats from control (n=5) and diabetic (n=5) groups were anesthetized and laparotomized to remove the uterine horns for weighing of fetuses and collecting the VYS. Flow cytometry was used for characterizing VYS cells, and for determining mitochondrial activity, cell proliferation, DNA ploidy, cell cycle phases, and caspase-3 activity. Fetal weight was reduced in the diabetic group. Expression of the cell markers CD34, VEGFR1, CD115, CD117, CD14, CCR2, CD90, CD44, STRO-1, OCT3/4, and Nanog was detected in VYS cells in both groups. In the diabetic group, significantly decreased expression of CD34 (P<0.05), CCR2 (P<0.001), and OCT3/4 (P<0.01), and significantly increased expression of CD90 (P<0.05), CD117 (P<0.01), and CD14 (P<0.05) were observed. VYS cells with inactive mitochondria, activated caspase-3, and low proliferation were present in the rats with diabetes. Severe hyperglycemia caused by maternal diabetes had negative effects on pregnancy, VYS cell viability, and the expression of cell markers.

  9. PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity

    Science.gov (United States)

    Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

    2016-01-01

    ABSTRACT Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8+ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c+ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8+ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1. PMID:27141339

  10. Glioblastoma formation from cell population depleted of Prominin1-expressing cells.

    Directory of Open Access Journals (Sweden)

    Kenji Nishide

    Full Text Available Prominin1 (Prom1, also known as CD133 in human has been widely used as a marker for cancer stem cells (CSCs, which self-renew and are tumorigenic, in malignant tumors including glioblastoma multiforme (GBM. However, there is other evidence showing that Prom1-negative cancer cells also form tumors in vivo. Thus it remains controversial whether Prom1 is a bona fide marker for CSCs. To verify if Prom1-expressing cells are essential for tumorigenesis, we established a mouse line, whose Prom1-expressing cells can be eliminated conditionally by a Cre-inducible DTA gene on the Prom1 locus together with a tamoxifen-inducible CreER(TM, and generated glioma-initiating cells (GICs-LD by overexpressing both the SV40 Large T antigen and an oncogenic H-Ras(L61 in neural stem cells of the mouse line. We show here that the tamoxifen-treated GICs-LD (GICs-DTA form tumor-spheres in culture and transplantable GBM in vivo. Thus, our studies demonstrate that Prom1-expressing cells are dispensable for gliomagenesis in this mouse model.

  11. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  12. Designer nanoparticle: nanobiotechnology tool for cell biology

    Science.gov (United States)

    Thimiri Govinda Raj, Deepak B.; Khan, Niamat Ali

    2016-09-01

    This article discusses the use of nanotechnology for subcellular compartment isolation and its application towards subcellular omics. This technology review significantly contributes to our understanding on use of nanotechnology for subcellular systems biology. Here we elaborate nanobiotechnology approach of using superparamagnetic nanoparticles (SPMNPs) optimized with different surface coatings for subcellular organelle isolation. Using pulse-chase approach, we review that SPMNPs interacted differently with the cell depending on its surface functionalization. The article focuses on the use of functionalized-SPMNPs as a nanobiotechnology tool to isolate high quality (both purity and yield) plasma membranes and endosomes or lysosomes. Such nanobiotechnology tool can be applied in generating subcellular compartment inventories. As a future perspective, this strategy could be applied in areas such as immunology, cancer and stem cell research.

  13. Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

    Science.gov (United States)

    Chan, Wing Hei; Gonsalvez, David G; Young, Heather M; Southard-Smith, E Michelle; Cane, Kylie N; Anderson, Colin R

    2016-02-01

    Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

  14. Embryo quality predictive models based on cumulus cells gene expression

    Directory of Open Access Journals (Sweden)

    Devjak R

    2016-06-01

    Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

  15. Abnormal expressions of proliferating cell nuclear antigen and P27 protein in brain glioma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Both proliferating cell nuclear antigen and P27 protein are important factors to regulate cell cycle. While, the combination of them can provide exactly objective markers to evaluate prognosis of patients with brain glioma needs to be further studied based on pathological level.OBJECTIVE: To observe the expressions of proliferating cell nuclear antigen and P27 protein in both injured and normal brain glioma tissues and analyze the effect of them on onset and development of brain glioma.DESIGN: Case contrast observation.SETTING: Department of Neurosurgery, the Second Affiliated Hospital of Xi'an Jiaotong University.PARTICIPANTS: A total of 63 patients with brain glioma were selected from Department of Neurosurgery,the Second Affiliated Hospital of Xi'an Jiaotong University from July 1996 to June 2000. There were 38 males and 25 females and their ages ranged from 23 to 71 years. Based on pathological classification and grading standards of brain glioma, patients were divided into grade Ⅰ - tⅡ (n =30) and grade Ⅲ - Ⅳ (n =33). All cases received one operation but no radiotherapy and chemiotherapy before operation. Sample tissues were collected from tumor parenchyma. Non-neoplastic brain tissues were collected from another 12 non-tumor subjects who received craniocerebral trauma infra-decompression and regarded as the control group. There were 10 males and 2 females and their ages ranged from 16 to 54 years. The experiment had got confirmed consent from local ethic committee and the collection was provided confirmed consent from patients and their relatives. All samples were restained with HE staining so as to diagnose as the brain glioma.While, all patients with brain glioma received radiotherapy after operation and their survival periods were followed up.METHODS: Primary lesion wax of brain glioma was cut into serial sections and stained with S-P immunohistochemical staining. Brown substance which was observed in tumor nucleus was regarded as the

  16. Effect of Exogenous Ghrelin on Cell Differentiation Antigen 40 Expression in Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Min ZHANG; Fang YUAN; Hui CHEN; Xingbiao QIU; Weiyi FANG

    2007-01-01

    Ghrelin is a brain-gut peptide that serves as a natural ligand for growth hormone secretagogue receptor (GHSR). It also exists abundantly in the cardiovascular system. In order to evaluate the possible role of ghrelin in the development of atherosclerosis, the effect of ghrelin on the expression of cell differentiation antigen 40 (CD40) were studied. Human umbilical vein endothelial cell (HUVEC) line-ECV 304 was pretreated with different concentrations of ghrelin, des-acyl ghrelin or [d-Lys]-GHRP-6 (a ghrelin receptor antagonist), and then induced with tumor necrosis factor-α (TNF-α) and interferon γ (IFN-γ). The mRNA levels of CD40 were analyzed by reverse transcription-polymerase chain reaction, and the expressions of CD40 protein in the cells were measured by flow cytometry (FCM) and Western blotting. The results showed that exogenous ghrelin could significantly inhibit TNF-α/IFN-γinduced CD40 expression in HUVEC cells in a concentration-dependent manner. When treated with 1000 ng/ml of ghrelin, the mRNA level of CD40 in the cells was decreased by approximately 77%, but when treated with both 1000 ng/ml of ghrelin and 1000 ng/ml of [d-Lys]-GHRP-6, the mRNA level of CD40 in the cells was decreased by only 42%,suggesting that [d-Lys]-GHRP-6 could counteract the inhibitory effect of ghrelin in these cells. However,CD40 expression was not inhibited by des-acyl ghrelin at 1000 ng/ml. The results in protein expression analysis detected by FCM and Western blotting further confirmed these results. Our results suggested that in the cardiovascular system, ghrelin not only has an anti-inflammatory effect, but also has a significant immunoregulatory effect that may be mediated through the GHSR-1 a receptor.

  17. Effect of Interaction between Chromatin Loops on Cell-to-Cell Variability in Gene Expression.

    Directory of Open Access Journals (Sweden)

    Tuoqi Liu

    2016-05-01

    Full Text Available According to recent experimental evidence, the interaction between chromatin loops, which can be characterized by three factors-connection pattern, distance between regulatory elements, and communication form, play an important role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effect that addresses the question of how changes in these factors affect variability at the expression level in a systematic rather than case-by-case fashion. Here we make such an effort, based on a mechanic model that maps three fundamental patterns for two interacting DNA loops into a 4-state model of stochastic transcription. We first show that in contrast to side-by-side loops, nested loops enhance mRNA expression and reduce expression noise whereas alternating loops have just opposite effects. Then, we compare effects of facilitated tracking and direct looping on gene expression. We find that the former performs better than the latter in controlling mean expression and in tuning expression noise, but this control or tuning is distance-dependent, remarkable for moderate loop lengths, and there is a limit loop length such that the difference in effect between two communication forms almost disappears. Our analysis and results justify the facilitated chromatin-looping hypothesis.

  18. Conceptual layout design of CFETR Hot Cell Facility

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Zheng, E-mail: gongz@mail.ustc.edu.cn [University of Science and Technology of China, Hefei 230026 (China); Institute of Plasma Physics, Chinese Academy of Sciences, Hefei (China); Qi, Minzhong, E-mail: qiminzhong@ipp.ac.cn [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei (China); Cheng, Yong, E-mail: chengyong@ipp.ac.cn [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei (China); Song, Yuntao, E-mail: songyt@ipp.ac.cn [University of Science and Technology of China, Hefei 230026 (China); Institute of Plasma Physics, Chinese Academy of Sciences, Hefei (China)

    2015-11-15

    Highlights: • This article proposed a conceptual layout design for CFETR. • The design principles are to support efficient maintenance to ensure the realization of high duty time. • The preliminary maintenance process and logistics are described in detail. • Life cycle management, maneuverability, risk and safety are in the consideration of design. - Abstract: CFETR (China Fusion Engineering Test Reactor) is new generation of Tokomak device beyond EAST in China. An overview of hot cell layout design for CFETR has been proposed by ASIPP&USTC. Hot Cell, as major auxiliary facility, not only plays a pivotal role in supporting maintenance to meet the requirements of high duty time 0.3–0.5 but also supports installation and decommissioning. Almost all of the Tokomak devices are lateral handling internal components like ITER and JET, but CFETR maintain the blanket module from 4 vertical ports, which is quite a big challenge for the hot cell layout design. The activated in-vessel components and several diagnosis instruments will be repaired and refurbished in the Hot Cell Facility, so the appropriate layout is very important to the Hot Cell Facility to ensure the high duty time, it is divided into different parts equipped with a variety of RH equipment and diagnosis devices based on the functional requirements. The layout of the Hot Cell Facility should make maintenance process more efficient and reliable, and easy to service and rescue when a sudden events taking place, that is the capital importance issue considered in design.

  19. Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

    Science.gov (United States)

    Kim, Hyun; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2012-01-01

    The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

  20. Effects of different ingredients of zedoary on gene expression of HSC-T6 cells

    Institute of Scientific and Technical Information of China (English)

    Yuan Jiang; Ze-Song Li; Fu-Sheng Jiang; Xin Deng; Cong-Shun Yao; Guang Nie

    2005-01-01

    AIM: To investigate the effects of four different ingredients of zedoary (Curcuma aromatica oil, Curcumol,β-elemence, and Curcumin) on the gene expressions of hepatic stellate cells (HSCs), and to explore the molecular mechanism of zedoary against hepatic fibrosis at gene network level.METHODS: We detected the mRNA sequences of 50 liver fibrosis-related genes in GenBank and designed oligonucleotide probes. We synthesized oligonucleotides with PE8909 DNA synthesizing instrument, and carried out oligonucleotide microarray with OGR-04 dropping instrument and aldehyded glass chip. Cultured HSC-T6cells were treated with different concentrations of Colchicine,Curcuma aromatica oil, Curcumol, β-elemence, and Curcumin. According to the experiment of cell toxicity,we took the appropriate concentrations of medicines that resulted in over 50% of cell survival as experiment concentrations. We collected the cells at 1, 6, 12, and 24 h, and extracted total RNA with TRIzol reagent, then labeled cDNAs with Cy3-dUTP and Cy5-dUTP. These labeled cDNAs were hybridized to an oligonucleotide microarray which was washed several times and scanned by scanner GenePix 4000B. Different gene expressions of HSC-T6 cells were analyzed by ImaGene 4.2 software.RESULTS: After HSC-T6 cells were cultured in a medium containing 6.25 μg/mL Colchicine for 12 h, expression of TIMP-1 decreased 2.2-folds. After HSC-T6 cells were cultured in a medium containing 78.125 μg/mL of Curcuma aromatica oil for 24 h, the expression of TIMP-2and IL-6 decreased 2.3- and 2.2-folds, respectively.Moreover, after HSC-T6 cells were cultured in a medium containing 1.5625 μg/mL of Curcumol for 12 h, the expression of TGFβ1 and P450a decreased 2.3- and 2.1-folds, respectively.CONCLUSION: Our results may show the possible molecular mechanism of Curcuma aromatica oil and Curcumol against hepatic fibrosis.

  1. Growth hormone increases vascular cell adhesion molecule 1 expression

    DEFF Research Database (Denmark)

    Hansen, Troels Krarup; Fisker, Sanne; Dall, Rolf

    2004-01-01

    We investigated the impact of GH administration on endothelial adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, in vivo and in vitro. Soluble VCAM-1, E-selectin, and C-reactive protein concentrations were measured before and after treatment in 25 healthy subjects...... and 25 adult GH-deficient (GHD) patients randomized to GH treatment or placebo. Furthermore, we studied the direct effect of GH and IGF-I and serum from GH-treated subjects on basal and TNF alpha-stimulated expression of VCAM-1 and E-selectin on cultured human umbilical vein endothelial cells. Baseline...... levels of VCAM-1, but not E-selectin, were significantly lower in GHD patients than in healthy subjects (362 +/- 15 microg/liter vs. 516 +/- 21 microg/liter, P treatment, compared with placebo [net difference between groups 151.8 microg/liter (95...

  2. Effects of cadmium on cell proliferation, apoptosis, and proto-oncogene expression in zebrafish liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying Ying; Zhu, Jin Yong; Chan, King Ming, E-mail: kingchan@cuhk.edu.hk

    2014-12-15

    Highlights: • Cd stimulated ZFL cell proliferation with decreasing apoptotic cell numbers. • Cd down regulated p53 and RAD51. • Cd up regulated immediate early cancer genes of GADD45 and growth factors. • Cd promoted tumorigenic effects in ZFL cells. - Abstract: Cadmium (Cd) is one of the major transitional metal that has toxic effects in aquatic organisms and their associated ecosystem; however, its hepatic toxicity and carcinogenicity are not very well characterized. We used a zebrafish liver (ZFL) cell line as a model to investigate the mechanism of Cd-induced toxicity on hepatocytes. Our results showed that Cd can be effectively accumulated in ZFL cells in our exposure experiments. Cell cytotoxicity assays and flow cytometer measurements revealed that Cd{sup 2+} stimulated ZFL cell proliferation with decreasing apoptotic cell numbers indicating potentially tumorigenic effects of Cd in ZFL cells. Gene expression profiles also indicated that Cd downregulated oncogenes p53 and rad51 and upregulated immediate response oncogenes, growth arrest and DNA damage-inducible (gadd45) genes, and growth factors. We also found dramatic changes in the gene expression of c-jun and igf1rb at different exposure time points, supporting the notion that potentially tumorigenic of Cd-is involved in the activation of immediate early genes or genes related to apoptosis in cancer promotion.

  3. Effects of cell-cycle-dependent expression on random fluctuations in protein levels.

    Science.gov (United States)

    Soltani, Mohammad; Singh, Abhyudai

    2016-12-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

  4. Transgenic expression of the human growth hormone minigene promotes pancreatic β-cell proliferation.

    Science.gov (United States)

    Baan, Mieke; Kibbe, Carly R; Bushkofsky, Justin R; Harris, Ted W; Sherman, Dawn S; Davis, Dawn Belt

    2015-10-01

    Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls.

  5. Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization.

    Science.gov (United States)

    Zhang, Wei; Ding, Tianbing; Zhang, Jian; Su, Jin; Yu, Jiangtian; Li, Jipeng; Li, Fuyang; Wang, Chunmei; Liu, Nannan; Liu, Xinping; Ma, Wenyu; Yao, Libo

    2007-09-01

    Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.

  6. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1.

    Science.gov (United States)

    Liu, Hua; Kennard, Simone; Lilly, Brenda

    2009-02-27

    Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types. In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the gamma-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression.

  7. Expression and function of mixed lineage kinases in dendritic cells.

    Science.gov (United States)

    Handley, Matthew E; Rasaiyaah, Jane; Barnett, James; Thakker, Manish; Pollara, Gabriele; Katz, David R; Chain, Benjamin M

    2007-08-01

    Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.

  8. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture.

    Science.gov (United States)

    Farcet, J P; Gourdin, M F; Testa, U; Andre, C; Jouault, H; Reyes, F

    1983-01-01

    Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

  9. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongtao [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China); Gao, Peng [Department of Internal Medicine, University of Iowa, Iowa City, IA 52242 (United States); Zheng, Jie, E-mail: jiezheng54@126.com [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China)

    2014-09-05

    Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.

  10. Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Julià, Eva; Montalban, Xavier; Al-Zayat, Hammad;

    2006-01-01

    OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T...... cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring...

  11. SEMICONDUCTOR DEVICES: Optimization of grid design for solar cells

    Science.gov (United States)

    Wen, Liu; Yueqiang, Li; Jianjun, Chen; Yanling, Chen; Xiaodong, Wang; Fuhua, Yang

    2010-01-01

    By theoretical simulation of two grid patterns that are often used in concentrator solar cells, we give a detailed and comprehensive analysis of the influence of the metal grid dimension and various losses directly associated with it during optimization of grid design. Furthermore, we also perform the simulation under different concentrator factors, making the optimization of the front contact grid for solar cells complete.

  12. Analysis of cell growth and gene expression of porcine adipose tissue-derived mesenchymal stem cells as nuclear donor cell.

    Science.gov (United States)

    Oh, Hyun Ju; Park, Jung Eun; Park, Eun Jung; Kim, Min Jung; Kim, Geon A; Rhee, Sang Ho; Lim, Sang Hyun; Kang, Sung Keun; Lee, Byeong Chun

    2014-12-01

    In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming-related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4-year-old female miniature pig. The ASC expressed cell-surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation-inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.

  13. Degradation of amyloid beta by human induced pluripotent stem cell-derived macrophages expressing Neprilysin-2

    Directory of Open Access Journals (Sweden)

    Koutaro Takamatsu

    2014-11-01

    Full Text Available The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS cell-derived macrophage-like cells for Alzheimer's disease (AD. In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2, which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2 in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.

  14. Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Luis Steven Servín-González

    2015-06-01

    Full Text Available Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2, which regulated mitogen-activated protein kinases (MAPK, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design.

  15. Adult human liver mesenchymal progenitor cells express phenylalanine hydroxylase.

    Science.gov (United States)

    Baruteau, Julien; Nyabi, Omar; Najimi, Mustapha; Fauvart, Maarten; Sokal, Etienne

    2014-09-01

    Phenylketonuria (PKU) is one of the most prevalent inherited metabolic diseases and is accountable for a severe encephalopathy by progressive intoxication of the brain by phenylalanine. This results from an ineffective L-phenylalanine hydroxylase enzyme (PAH) due to a mutated phenylalanine hydroxylase (PAH) gene. Neonatal screening programs allow an early dietetic treatment with restrictive phenylalanine intake. This diet prevents most of the neuropsychological disabilities but remains challenging for lifelong compliance. Adult-derived human liver progenitor cells (ADHLPC) are a pool of precursors that can differentiate into hepatocytes. We aim to study PAH expression and PAH activity in a differenciated ADHLPC. ADHLPC were isolated from human hepatocyte primary culture of two different donors and differenciated under specific culture conditions. We demonstrated the high expression of PAH and a large increase of PAH activity in differenciated LPC. The age of the donor, the cellular viability after liver digestion and cryopreservation affects PAH activity. ADHLPC might therefore be considered as a suitable source for cell therapy in PKU.

  16. Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Lars Boenicke; Bradley D. Howard; Ilka Vogel; Hoiger Kalthoff

    2003-01-01

    AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

  17. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Science.gov (United States)

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna

    2016-01-01

    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  18. BBU design of linear induction accelerator cells for radiography application

    Energy Technology Data Exchange (ETDEWEB)

    Shang, C.C.; Chen, Y.J.; Gaporaso, G.J.; Houck, T.L.; Molau, N.E.; Focklen, J.; Gregory, S.

    1997-05-06

    There is an ongoing effort to develop accelerating modules for high-current electron accelerators for advanced radiography application. Accelerating modules with low beam-cavity coupling impedances along with gap designs with acceptable field stresses comprise a set of fundamental design criteria. We examine improved cell designs which have been developed for accelerator application in several radiographic operating regimes. We evaluate interaction impedances, analyze the effects of beam structure coupling on beam dynamics (beam break-up instability and corkscrew motion). We also provide estimates of coupling through interesting new high-gradient insulators and evaluate their potential future application in induction cells.

  19. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

    Directory of Open Access Journals (Sweden)

    Nilay J Lakhkar

    2015-11-01

    Full Text Available In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5 that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

  20. Ectopic expression of B-lymphoid kinase in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Krejsgaard, Thorbjørn; Vetter-Kauczok, Claudia S; Woetmann, Anders;

    2009-01-01

    B-lymphoid kinase (Blk) is exclusively expressed in B cells and thymocytes. Interestingly, transgenic expression of a constitutively active form of Blk in the T-cell lineage of mice results in the development of T-lymphoid lymphomas. Here, we demonstrate nuclear factor-kappa B (NF......-kappaB)-mediated ectopic expression of Blk in malignant T-cell lines established from patients with cutaneous T-cell lymphoma (CTCL). Importantly, Blk is also expressed in situ in lesional tissue specimens from 26 of 31 patients with CTCL. Already in early disease the majority of epidermotropic T cells express Blk...... phosphorylated in malignant CTCL cell lines and spontaneously active in kinase assays. Furthermore, targeting Blk activity and expression by Src kinase inhibitors and small interfering RNA (siRNA) inhibit the proliferation of the malignant T cells. In conclusion, this is the first report of Blk expression...

  1. Design Principles for Nickel/Hydrogen Cells and Batteries

    Science.gov (United States)

    Thaller, Lawrence H.; Manzo, Michelle A.; Gonzalez-Sanabria, Olga D.

    1987-01-01

    Individual-pressure-vessel (IPV) nickel/hydrogen cells and bipolar batteries developed for use as energy-storage subsystems for satelite applications. Design principles applied draw upon extensive background in separator technology, alkaline-fuel-cell technology and several alkaline-cell technology areas. Principals are rather straightforward applications of capillary-force formalisms, coupled with slowly developing data base resulting from careful post-test analyses. Based on preconceived assumptions relative to how devices work and how to be designed so they display longer cycle lives at deep discharge.

  2. Functional expression of human leukocyte elastase (HLE)/medullasin in eukaryotic cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Shimizu, H; Naruto, M

    1990-03-30

    We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.

  3. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Harjeet Singh

    Full Text Available Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28 that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC in the presence of interleukin (IL-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT. We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

  4. FcRn expression, ligands binding properties and its regulation in human immune cells and hepatocytes

    OpenAIRE

    2007-01-01

    ABSTRACT Expression and diverse functions of MHC class I related neonatal Fc receptor in different tissues is continually reported. To contribute to the understanding of how the receptor functions according to cell type, we investigated the expression and ligands binding properties of FcRn in human immune cells and hepatocytes. Here, we report that heterodimeric FcRn is expressed in these cells as evidenced by RT-PCR, Western immunoblottting and flow cytometry. The receptor expression i...

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

    Science.gov (United States)

    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

  6. Increased T cell expression of CD154 (CD40-ligand) in multiple sclerosis

    DEFF Research Database (Denmark)

    Jensen, J; Krakauer, M; Sellebjerg, F

    2001-01-01

    CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients with sec......CD154 (CD40-ligand, gp39), expressed on activated T cells, is crucial in T cell-dependent immune responses and may be involved in the pathogenesis of multiple sclerosis (MS). We studied cerebro-spinal fluid and peripheral blood T cell expression of CD154 in MS by flow cytometry. Patients...

  7. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis.

    Science.gov (United States)

    Tai, Mei-Hui; Chang, Chia-Cheng; Kiupel, Matti; Webster, Joshua D; Olson, L Karl; Trosko, James E

    2005-02-01

    The Oct3/4 gene, a POU family transcription factor, has been noted as being specifically expressed in embryonic stem cells and in tumor cells but not in cells of differentiated tissues. With the ability to isolate adult human stem cells it became possible to test for the expression of Oct3/4 gene in adult stem cells and to test the stem cell theory of carcinogenesis. Using antibodies and PCR primers we tested human breast, liver, pancreas, kidney, mesenchyme and gastric stem cells, the cancer cell lines HeLa and MCF-7 and human, dog and rat tumors for Oct4 expression. The results indicate that adult human stem cells, immortalized non-tumorigenic cells and tumor cells and cell lines, but not differentiated cells, express Oct4. Oct4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that adult stem cells maintain expression of Oct4, consistent with the stem cell hypothesis of carcinogenesis.

  8. Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

    Science.gov (United States)

    Palma, C S; Tannous, M A; Malta, T M; Russo, E M S; Covas, D T; Picanço-Castro, V

    2013-04-02

    Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and βCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

  9. Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

    Science.gov (United States)

    Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

    2016-01-01

    Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.

  10. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  11. The Expression of p53 and Cox-2 in Basal Cell Carcinoma, Squamous Cell Carcinoma and Actinic Keratosis Cases

    Directory of Open Access Journals (Sweden)

    Ülker KARAGECE YALÇIN

    2012-05-01

    Full Text Available Objective: The aim of this study was to investigate p53 and COX-2 expressions in basal cell carcinoma, squamous cell carcinoma and actinic keratoses, and to determine a possible relationship.Material and Method: 50 basal cell carcinoma, 45 squamous cell carcinoma and 45 actinic keratosis cases were evaluated. The type of tumor in basal cell carcinoma and tumor differentiation in squamous cell carcinoma were noted and the paraffin block that best represented the tumor was chosen. Immunostaining by p53 and COX-2 was performed on sections of the paraffin blocks.Results: p53 expression was observed in 98% of basal cell carcinoma, 88.9% of squamous cell carcinoma and all actinic keratosis cases. p53 expression was also noted in non-dysplastic appearing epithelium in actinic keratosis cases. COX-2 expression was seen in 90, 100 and 88.9% of the basal cell carcinoma, squamous cell carcinoma and actinic keratosis groups, respectively. Skin appendages, inflammatory cells and vascular structures were also stained by COX-2 besides tumor tissue. COX-2 expression increased by the p53 expression increase in basal cell carcinoma and squamous cell carcinoma. p53 and COX-2 expressions were not related in terms of tumor type in the BCC and were not related in terms of differentiation in SCC.Conclusion: The existence of p53 expression in actinic keratosis cases has supported the idea that p53 plays a role in the early steps of carcinogenesis in skin cancers. The fact that the expression of COX-2 increases in line with the increase of p53 expression in basal cell carcinoma and squamous cell carcinoma cases indicates that COX-2 expression may be affected by p53

  12. Design and performance of a prototype fuel cell powered vehicle

    Energy Technology Data Exchange (ETDEWEB)

    Lehman, P.A.; Chamberlin, C.E. [Humboldt State Univ., Arcata, CA (United States)

    1996-12-31

    The Schatz Energy Research Center (SERC) is now engaged in the Palm Desert Renewable Hydrogen Transportation System Project. The Project involves a consortium which includes the City of Palm Desert, SERC, the U.S. Department of Energy, the South Coast Air Quality Management District, and Sandia and Lawrence Livermore National Laboratories. Its goal to develop a clean and sustainable transportation system for a community will be accomplished by producing a fleet of fuel cell vehicles, installing a refueling infrastructure utilizing hydrogen generated from solar and wind power, and developing and staffing a fuel cell service and diagnostic center. We will describe details of the project and performance goals for the fuel cell vehicles and associated peripheral systems. In the past year during the first stage in the project, SERC has designed and built a prototype fuel cell powered personal utility vehicle (PUV). These steps included: (1) Designing, building, and testing a 4.0 kW proton exchange membrane (PEM) fuel cell as a power plant for the PUV. (2) Designing, building and testing peripherals including the air delivery, fuel storage/delivery, refueling, water circulation, cooling, and electrical systems. (3) Devising a control algorithm for the fuel cell power plant in the PUV. (4) Designing and building a test bench in which running conditions in the PUV could be simulated and the fuel cell and its peripheral systems tested. (5) Installing an onboard computer and associated electronics into the PUV (6) Assembling and road testing the PUV.

  13. Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Ru DUAN; Nan HU; Hai-yan LIU; Jia LI; Hai-fang GUO; Can LIU; Li LIU; Xiao-dong LIU

    2012-01-01

    Aim:To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.Methods:Caco-2 cells were exposed to NO donors for designated times.P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting,respectively.Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits,respectively.Results:Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners.A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS.In contrast,a long-term (24 h) exposure stimulated the P-gp function and expression.The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP.The other NO donors SIN-1 and SNAP showed similar effects.Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function.On the other hand,free radical scavengers ascorbate,glutathione and uric acid (2 mmol/L for each),PKC inhibitor chelerythrine (5 μmol/L),PI3K/Akt inhibitor wortmannin (1 pmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP,but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.Conclusion:NO donors time-dependently regulate P-gp function and expression in Caco-2 cells:short-term exposure impairs P-gp function and expression,whereas long-term exposure stimulates P-gp function and expression.The regulation occurs via a NO-independent mechanism.

  14. A rapid and efficient method to express target genes in mammalian cells by baculovirus

    Institute of Scientific and Technical Information of China (English)

    Tong Cheng; Chen-Yu Xu; Ying-Bin Wang; Min Chen; Ting Wu; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses.METHODS: Two recombinant baculoviruses (BacV-CMVEGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells.RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells.CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign

  15. Differential Gene Expression in Thrombomodulin (TM; CD141)+ and TM− Dendritic Cell Subsets

    OpenAIRE

    Masaaki Toda; Zhifei Shao; Yamaguchi, Ken D.; Takehiro Takagi; Corina N D'Alessandro-Gabazza; Osamu Taguchi; Hugh Salamon; Leung, Lawrence L. K.; Gabazza, Esteban C.; John Morser

    2013-01-01

    Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived de...

  16. Wogonoside induces growth inhibition and cell cycle arrest via promoting the expression and binding activity of GATA-1 in chronic myelogenous leukemia cells.

    Science.gov (United States)

    Li, Hui; Hui, Hui; Xu, Jingyan; Yang, Hao; Zhang, Xiaoxiao; Liu, Xiao; Zhou, Yuxin; Li, Zhiyu; Guo, Qinglong; Lu, Na

    2016-06-01

    GATA-1, a zinc finger transcription factor, has been demonstrated to play a key role in the progression of leukemia. In this study, we investigate the effects of wogonoside, a naturally bioactive flavonoid derived from Scutellaria baicalensis Georgi, on cell growth and cell cycle in chronic myeloid leukemia (CML) cells, and uncover its underlying mechanisms. The experimental design comprised CML cell lines K562, imatinib-resistant K562 (K562r) cells, and primary CML cells, treated in vitro or in vivo, respectively, with wogonoside; growth and cell cycle were then evaluated. We found that wogonoside could induce growth inhibition and G0/G1 cell cycle arrest in both normal and K562r cells. Wogonoside promotes the expression of GATA-1 and facilitates the binding to methyl ethyl ketone (MEK) and p21 promoter, thus inhibiting MEK/extracellular signal-regulated kinase signaling and cell cycle checkpoint proteins, including CDK2, CDK4, cyclin A, and cyclin D1, and increasing p21 expression. Furthermore, in vivo studies showed that administration of wogonoside decreased CML cells and prolonged survival in NOD/SCID mice with CML cell xenografts. In conclusion, these results clearly revealed the inhibitory effect of wogonoside on the growth in CML cells and suggested that wogonoside may act as a promising drug for the treatment of imatinib-resistant CML.

  17. Basket and basal-duct cells in domestic animals: different cytokeratin expression and shape.

    Science.gov (United States)

    Zedda, M; Farina, V

    1996-12-01

    Cytokeratins (CKs) are a multigenic family of proteins constituting intermediate filaments in epithelia, indicated in humans by the numbers 1-20. Different cell-types can be immunocytochemically identified on the grounds of their CK expression. This investigation was designed to study CK expression of basket cells (BCs) and basal-duct cells (BDCs) in some domestic animals. Frozen sections of mammary and major salivary glands from cows, sheep, pigs and rabbits were treated using the immunofluorescent method, using as monoclonal antibodies clones CK-E3, CKB1, KS-1A3, and LDS-68, respectively, revealing the human CKs 17, 14, 13, 7. BCs surrounding acini and BDCs were stained by CK 17 antibody only in the rabbit. CK 14 was detectable in both cell types in cows, sheep and pigs, except in the case of bovine salivary BCs. CK 13 was revealed in BCs and BDCs of all mammary glands and also rabbit salivary glands. In the salivary glands of the other species, only BDCs were stained. CK 7 gave unreliable results in all the species and cell types examined. Interestingly, in the rabbit, also BDCs are basket-like in shape. The antibodies employed showed different staining depending on species and gland. On the grounds of immunoreactivity and shape, BCs and BDCs can be considered the same cell type in the rabbit. In the other species, they appear to be different, since BDCs may express additional CKs and are triangular-shaped, whereas BCs are truly basket-like. It is worth noting that clone KS-1A3 in the rabbit and CKB1 in the sheep and pig can be considered markers of the basket/ basal system.

  18. Identification of a potential ovarian cancer stem cell gene expression profile from advanced stage papillary serous ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Vinod Vathipadiekal

    Full Text Available Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP and main population (MP, and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5% genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.

  19. Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.

    Science.gov (United States)

    Vijayachandran, Lakshmi Sumitra; Viola, Cristina; Garzoni, Frederic; Trowitzsch, Simon; Bieniossek, Christoph; Chaillet, Maxime; Schaffitzel, Christiane; Busso, Didier; Romier, Christophe; Poterszman, Arnaud; Richmond, Timothy J; Berger, Imre

    2011-08-01

    Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

  20. Center for Intelligent Fuel Cell Materials Design

    Energy Technology Data Exchange (ETDEWEB)

    Santurri, P.R., (Chemsultants International); Hartmann-Thompson, C.; Keinath, S.E. (Michigan Molecular Inst.)

    2008-08-26

    The goal of this work was to develop a composite proton exchange membrane utilizing 1) readily available, low cost materials 2) readily modified and 3) easily processed to meet the chemical, mechanical and electrical requirements of high temperature PEM fuel cells. One of the primary goals was to produce a conducting polymer that met the criteria for strength, binding capability for additives, chemical stability, dimensional stability and good conductivity. In addition compatible, specialty nanoparticles were synthesized to provide water management and enhanced conductivity. The combination of these components in a multilayered, composite PEM has demonstrated improved conductivity at high temperatures and low humidity over commercially available polymers. The research reported in this final document has greatly increased the knowledge base related to post sulfonation of chemically and mechanically stable engineered polymers (Radel). Both electrical and strength factors for the degree of post sulfonation far exceed previous data, indicating the potential use of these materials in suitable proton exchange membrane architectures for the development of fuel cells. In addition compatible, hydrophilic, conductive nano-structures have been synthesized and incorporated into unique proton exchange membrane architectures. The use of post sulfonation for the engineered polymer and nano-particle provide cost effective techniques to produce the required components of a proton exchange membrane. The development of a multilayer proton exchange membrane as described in our work has produced a highly stable membrane at 170°C with conductivities exceeding commercially available proton exchange membranes at high temperatures and low humidity. The components and architecture of the proton exchange membrane discussed will provide low cost components for the portable market and potentially the transportation market. The development of unique components and membrane architecture

  1. Expression of soluble triggering receptor expression on myeloid cells-1 in pleural effusion

    Institute of Scientific and Technical Information of China (English)

    HUANG Lu-ying; SHI Huan-zhong; LIANG Qiu-li; WU Yan-bin; QIN Xue-jun; CHEN Yi-qiang

    2008-01-01

    Background Tdggedng receptors expressed on myeloid cells(TREM)proteins are a family of cell surface receptors expressed broadly by cells of the myeloid lineage.The aim of this study was to investigate the clinical significance of soluble TREM-1(sTREM-1)in pleural effusions,and to determine the effects of pneumonia on pleural sTREM-1 concentrations.Methods PleuraI fluid was collected from 109 patients who presented to the respiratory institute (35 with malignant pleural effusion,31 with tuberculous pleural effusion,21 with bacteriaI pleural effusion,and 22 with transudate).The concentrations of sTREM-1,tumor necrosis factor-o(TNF-α)and interleukin-1β(IL-1β)were determined jn effusion and serum samples by enzyme Iinked immunosorbent assay(ELISA).Results The concentrations of sTREM-1 in bacterial pleural effusion were significantly higher than those in malignant.tuberculous,and transudative groups(all P<0.001).An sTREM-1 cutoff value of 768.1 ng/L had a sensitivity of 86%and a specificity of 93%.Pleural sTREM-1 Ievels were positively correlated with Ievels of TNF-α and IL-1β.Patients with complicating bacterial pneumonia did not have elevated concentration of STREM-1 jn pleural effusion when compared with patients without pneumonia.Conclusions Determination of pleural sTREM-1 may improve the ability of clinicians to differentiate pleural effusion patients of bacterial origin from those with other etiologies.The occurrence of bacterial pneumonia did not affect pleural sTREM-1 concentrations.

  2. Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

    Directory of Open Access Journals (Sweden)

    Baschieri Selene

    2007-02-01

    Full Text Available Abstract Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa and a truncated form (p25, 25 kDa. Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

  3. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    Science.gov (United States)

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

  4. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

    Directory of Open Access Journals (Sweden)

    Kanako Miyabayashi

    Full Text Available Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

  5. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  6. EXPRESSION OF PHOSPHO-(-CATENIN AND ITS SIGNIFICANCE IN NON-SMALL CELL LUNG CANCER

    Institute of Scientific and Technical Information of China (English)

    林东; 王恩华; 张志坤; 徐洪涛; 李庆昌; 邱雪杉

    2003-01-01

    Objective: To investigate phospho-(-catenin expression in non-small cell lung cancer (NSCLC) and to study the relationship between phospho-(-catenin expression and some clinical pathological factors. Methods: The expression of phospho-(-catenin in 67 primary NSCLC cases detected immunohistochemically. Results: phospho- (-catenin was not expressed in normal bronchial mucous cell and showed cytoplasmic and nuclear expression in NSCLC cell. Total positive expression rate reached 62.7%, and positive expression rate of nucleus was 38.8%. The positive expression rate (87.5%) and nuclear expression rate of adenocarcinoma (62.5%) were apparently higher than those of squamous cell cancer (40.0% and 17.1%) (P<0.01). Expression of phospho-(-catenin had no relationship to differentiation degree and lymphatic metastasis. The postoperative survival time is not related to phospho-(- catenin expression. (Log-rank test, P=0.9198; P=0.6274). COX model analysis showed that tumor stage and differentiation are independent risk factors to prognosis (P=0.001; P=0.020). Conclusion: NSCLC cells show positive expression of phospho-(-catenin, phospho-(-catenin nuclear expression is relevant to histological types. There is no difference in postoperative survival time between patients with phospho-(-catenin positive expression and patients with negative expression, expression of phospho-(- catenin is not independent risk factor to prognosis.

  7. Heterogeneity of aberrant immunoglobulin expression in cancer cells

    Institute of Scientific and Technical Information of China (English)

    Duosha Hu; Ya Cao; Zhi Duan; Ming Li; Yiqun Jiang; Haidan Liu; Hui Zheng; Lili Li; Ann M Bode; Zigang Dong

    2011-01-01

    Accumulating evidence has shown that immunoglobulin (Ig) is 'unexpectedly' expressed by epithelial cancer cells and that it can promote tumor growth.The main purpose of this study was to explore the components of the cancerous Ig and its possible function.The presence of cancerous Ig in the Golgi apparatus was confirmed by immunofluorescence,indirectly suggesting that the cancerous Ig was processed and packaged in cancer cells.Western blot analysis and ELISA results indicated that cancer cells produced membrane Ig and secreted Ig into the supernatant fraction.The cancerous Ig consists of an α heavy chain and a κ light chain.Finally,by analyzing the Ig components pulled down by protein A beads,the cancerous Ig was found to be structurally distinct from normal Ig.The cancerous Ig was truncated or aberrant.Although the underlying mechanism that causes the abnormalities has not been determined,our current discoveries strengthen our previous findings and promise fruitful future explorations.

  8. Expression of Telomerase Activity in Gastric Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To study the relationship between telomerase activity and biological behavior in human gastric cells and appraise the clinical significance of detecting telomerase activity. Methods The telomerase activity in 47 gastric cancer tissue samples,their matched nomal tissues,7 gastric ulcer and 2 gastric cancer cell lines was detected using a PCR-based non-radioisotopic telomeric repeat amplification protocol(TRAP) assay. Results None of the 47 samples from normal gastric tissues expressed telomerase activity.The 41 of 47 cases of gastric cancer presented telomerase activity with an 87.2% positive rate (P<0.001). 2/2 gastric cancer cell lines and 0/7 gastric ulcer line were also positive for telmerase activity.The activity of telomerase was associated with the pathological differentiation of gastric cancer. Conclusion Telomerase activity may be related to the biological behavior of gastric cancer and can help in assessing the malignant poten-tial of gastric cancer.Telomerase activity will be a good diagnostic marker for the detection of gastric cancer.

  9. Cell-based capacitance sensor for analysis of EGFR expression on cell membrane

    Science.gov (United States)

    Shin, Dong-Myeong; Shin, Yong-Cheol; Ha, Ji Hye; Lee, Jong-Ho; Han, Dong-Wook; Kim, Jong-Man; Kim, Hyung Kook; Hwang, Yoon-Hwae

    2013-02-01

    Cancer cells have many kinds of cancer biomarkers. Among them, the epidermal growth factor (EGF) receptors can show a possibility for a cancer marker because the over-expression of EGF receptor is related with fibrous, colorectal, cervical and gastric tumorigenesis. We fabricated the capacitance sensor with a gap area of 50 μm × 200 μm by using photolithography and lift-off method. Using the capacitance sensor, we investigated the time dependent capacitance changes of different kinds of fibrous cells, such as HT1080 fibrosarcoma, L-929 fibroblast cell line and nHDF dermal fibroblast primary cell. We found that when we put the EGF, the capacitance decreased due to the immobilization of EGF to EGF receptor on the cell membrane. The quantitative determination of EGF receptor level for various fibrous cells was carried out and the results showed good correlation with conventional method. Based on our results, we suggest that the capacitance sensor can measure the expression level of the EGF receptor on cell membrane and be a good candidate as a cancer diagnosis.

  10. Effect of promoter architecture on the cell-to-cell variability in gene expression.

    Directory of Open Access Journals (Sweden)

    Alvaro Sanchez

    2011-03-01

    Full Text Available According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.

  11. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  12. Balancing Expression and Structure in Game Design: Developing Computational Participation Using Studio-Based Design Pedagogy

    Science.gov (United States)

    DeVane, Ben; Steward, Cody; Tran, Kelly M.

    2016-01-01

    This article reports on a project that used a game-creation tool to introduce middle-school students ages 10 to 13 to problem-solving strategies similar to those in computer science through the lens of studio-based design arts. Drawing on historic paradigms in design pedagogy and contemporary educational approaches in the digital arts to teach…

  13. Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells.

    Science.gov (United States)

    Erbe, Amy K; Wang, Wei; Gallenberger, Mikayla; Hank, Jacquelyn A; Sondel, Paul M

    2016-01-01

    Natural killer (NK) cells are one of the main effector immune cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Upon recognition of cell-bound IgG antibodies, which occurs through Fc gamma receptors (FCGRs) expressed on the cell surface of NK cells, NK cells become activated and lyse target tumor or infected cells. The FCGRs, FCGR3A and FCGR2C, expressed on the surface of NK cells have single nucleotide polymorphisms (SNPs) that result in differential activity of NK cells. In addition to SNP genetic variation within each of these genes, the FCGRs are subject to copy number variation (CNV), which leads to variable protein expression levels on the cell surface. Studies have found that FCGR genotype for FCGR3A and FCGR2C is associated with variation in the response to immunotherapy.Due to high sequence homology within FCGR3 and FCGR2 families, there are difficulties associated with genotyping these specific receptors related to cross-amplification of non-targeted FCGRs. To improve specificity for both FCGR3A and FCGR2C, Rnase-H (RH) primers were designed to amplify specifically FCGR3A (while not co-amplifying FCGR3B) and FCGR2C (while not co-amplifying FCGR2B). In addition, fluorescently labeled locked nucleic acid (LNA) probes provide additional precision for determination of the SNPs within both FCGR3A and FCGR2C. For CNV determination, separate fluorescently labeled probes for FCGR3A, and for FCGR2C, can be used with the same RH primers for each gene. These probes can be combined in the same well with control primers/probe for a known diploid gene and used to calculate the copy number of both FCGR3A and FCGR2C. Here we provide new detailed methodology that allows for the specific amplification of these FCGRs in a single PCR reaction, allowing for genotyping of both the SNPs and CNVs using real-time PCR.

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

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

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

  15. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhumur Ghosh

    Full Text Available Human induced pluripotent stem cells (hiPSCs generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs, as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  16. Forced expression of the Oct-4 gene influences differentiation of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists,forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.

  17. Design and fabrication of wraparound contact silicon solar cells

    Science.gov (United States)

    Scott-Monck, J. A.; Stella, P. M.; Avery, J. E.

    1972-01-01

    Both dielectric insulation and etched junction contact techniques were evaluated for use in wraparound contact cell fabrication. Since a suitable process for depositing the dielectrics was not achieved, the latter approach was taken. The relationship between loss of back contact and power degradation due to increased series resistance was established and used to design a simple contact configuration for 10 ohm-cm etched wraparound junction contact N/P cells. A slightly deeper junction significantly improved cell curve shape and the associated loss of current was regained by using thinner contact grid fingers. One thousand cells with efficiencies greater than 10.5% were fabricated to demonstrate the process.

  18. Screening the stage-specific expression gene from different germ cells of rat

    Institute of Scientific and Technical Information of China (English)

    贾孟春; 娄利霞; 裴开颜; 赵龙梅; 石心泉

    2002-01-01

    Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene spermatocytes and round spermatids from adult rats by sedimentation velocity at unit gravity, using 2%-4% BSA gradient in DMEM/F12 medium. A mRNA differential display method was used for screening the stage-specific expression gene. Results: Nineteen differentially expressed cDNA fragments were obtained. After excluding the false positive cDNA fragments by dot blot, 13 cDNAs were selected to clone and sequence. To obtain longer cDNAs, six ESTs were used to screen the rat testis λ-zap II cDNA library. Two longer cDNA fragments, designated as LY21 and LM66, were obtained. The analysis with DNAMAN software indicated that LY21 had a long open reading frame coding 372 amino acids while LM66 had no long open reading frame. LY21 were highly homologous with hnRNP H1. To observe the expression patterns of LY21 gene in the testicular cells, we performed in situ hybridization on testis sections from adult rats. The LY21 gene expression was found in the spermatogonia and primary spermatocytes.Conclusion: This study indicated that LY21 gene was associated with spermatogenesis. Further studies will be needed to explore the function of LY21.

  19. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    Science.gov (United States)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  20. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells

    Science.gov (United States)

    Ito, Daisuke

    2017-01-01

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1− and SSEA-1+ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines. PMID:27456773

  1. Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Rong GAO; Bang-hao ZHU; Shi-bo TANG; Jiang-feng WANG; Jun REN

    2008-01-01

    Aim: This study was designed to examine the effect of scutellarein on high glu-cose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). Methods: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. Results: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1 × 10-11-1 × 10-5 mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. Conclusion: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.

  2. Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins.

    Science.gov (United States)

    Shen, Kai-Kai; Liu, Tian-Yu; Xu, Chong; Ji, Li-Li; Wang, Zheng-Tao

    2009-09-01

    The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0-50 micromol x L(-1)) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0-30 micromol x L(-1)) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.

  3. Neuropilin-1 expression characterizes T follicular helper (Tfh) cells activated during B cell differentiation in human secondary lymphoid organs.

    Science.gov (United States)

    Renand, Amédée; Milpied, Pierre; Rossignol, Julien; Bruneau, Julie; Lemonnier, François; Dussiot, Michael; Coulon, Séverine; Hermine, Olivier

    2013-01-01

    T follicular helper (Tfh) cells play an essential role in the development of antigen-specific B cell immunity. Tfh cells regulate the differentiation and survival of activated B cells outside and inside germinal centers (GC) of secondary lymphoid organs. They act through cognate contacts with antigen-presenting B cells, but there is no current marker to specifically identify those Tfh cells which productively interact with B cells. Here we show that neuropilin 1 (Nrp1), a cell surface receptor, is selectively expressed by a subset of Tfh cells in human secondary lymphoid organs. Nrp1 expression on Tfh cells correlates with B cell differentiation in vivo and in vitro, is transient, and can be induced upon co-culture with autologous memory B cells in a cell contact-dependent manner. Comparative analysis of ex vivo Nrp1(+) and Nrp1(-) Tfh cells reveals gene expression modulation during activation. Finally, Nrp1 is expressed by malignant Tfh-like cells in a severe case of angioimmunoblastic T-cell lymphoma (AITL) associated with elevated terminal B cell differentiation. Thus, Nrp1 is a specific marker of Tfh cells cognate activation in humans, which may prove useful as a prognostic factor and a therapeutic target in neoplastic diseases associated with Tfh cells activity.

  4. Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

    Science.gov (United States)

    González, Fermín E; Ortiz, Carolina; Reyes, Montserrat; Dutzan, Nicolás; Patel, Vyomesh; Pereda, Cristián; Gleisner, Maria A; López, Mercedes N; Gutkind, J Silvio; Salazar-Onfray, Flavio

    2014-07-01

    We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.

  5. Poor prognostic clinicopathologic features correlate with VEGF expression but not with PTEN expression in squamous cell carcinoma of the larynx

    Directory of Open Access Journals (Sweden)

    Karagoz Filiz

    2010-06-01

    Full Text Available Abstract Background The aim of this study was to assess the relationship between expression of vascular endothelial growth factor (VEGF and phosphatase and tensin homolog deleted in chromosome ten (PTEN, angiogenesis and clinicopathological parameters of squamous cell carcinoma of the larynx. Methods We examined immunohistochemical expression of VEGF and PTEN and CD34 for microvessel density (MVD in sections of formalin-fixed, paraffin embedded tissue blocks of 140 patients with squamous cell carcinoma of the larynx. The intensity of VEGF and PTEN staining and the proportion of cells staining were scored. Results The tumor grade was not significantly related to PTEN expression, but it was to VEGF expression (p = 0.400; p = 0.015, respectively. While there was no significant relationship between PTEN expression and tumor size and cartilage invasion (p = 0.311, p = 0.128, there was a significant relationship between the severity of VEGF expression and tumor size (p = 0.006 and lymph node metastasis (p = 0.048 but not cartilage invasion (p = 0.129. MVD was significantly higher in high-grade tumors (p = 0.003 but had no significant relationship between MVD, lymph node metastasis, and cartilage invasion (p = 0.815, p = 0.204. There was also no significant relationship between PTEN and VEGF expression (p = 0.161 and between PTEN and VEGF expression and the MVD (p = 0.120 and p = 0.175, respectively. Conclusions Increased VEGF expression may play an important role in the outcome of squamous cell carcinoma of the larynx. PTEN expression was not related to VEGF expression and clinicopathological features of squamous cell carcinoma of the larynx.

  6. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

  7. Influence of scaffold design on 3D printed cell constructs.

    Science.gov (United States)

    Souness, Auryn; Zamboni, Fernanda; Walker, Gavin M; Collins, Maurice N

    2017-02-14

    Additive manufacturing is currently receiving significant attention in the field of tissue engineering and biomaterial science. The development of precise, affordable 3D printing technologies has provided a new platform for novel research to be undertaken in 3D scaffold design and fabrication. In the past, a number of 3D scaffold designs have been fabricated to investigate the potential of a 3D printed scaffold as a construct which could support cellular life. These studies have shown promising results; however, few studies have utilized a low-cost desktop 3D printing technology as a potential rapid manufacturing route for different scaffold designs. Here six scaffold designs were manufactured using a Fused deposition modeling, a "bottom-up" solid freeform fabrication approach, to determine optimal scaffold architecture for three-dimensional cell growth. The scaffolds, produced from PLA, are coated using pullulan and hyaluronic acid to assess the coating influence on cell proliferation and metabolic rate. Scaffolds are characterized both pre- and postprocessing using water uptake analysis, mechanical testing, and morphological evaluation to study the inter-relationships between the printing process, scaffold design, and scaffold properties. It was found that there were key differences between each scaffold design in terms of porosity, diffusivity, swellability, and compressive strength. An optimal design was chosen based on these physical measurements which were then weighted in accordance to design importance based on literature and utilizing a design matrix technique. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.

  8. Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, D.C.; Craigmile, S.; Campbell, J.D.M. [Roslin Inst., Midlothian (United Kingdom)] [and others

    1996-09-01

    Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.

  9. Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs)

    Science.gov (United States)

    Mazza, Emilia Maria Cristina; Zoso, Alessia; Mandruzzato, Susanna; Bronte, Vincenzo; Serafini, Paolo; Inverardi, Luca; Bicciato, Silvio

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), which are differentiated from umbilical cord blood (UCB) precursors (Zoso et al., 2014). This MDSC subset promotes regulatory T-cell expansion and induces normoglycemia in a xenogeneic model of type 1 diabetes. Here we describe in details the experimental design and the bioinformatics analyses of the gene expression dataset used to investigate the molecular mechanisms at the base of MDSC tolerogenic and suppressive properties. We also provide an R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset. Raw and pre-processed data are available at Gene Expression Omnibus under accession GSE52376. PMID:26484135

  10. Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs

    Directory of Open Access Journals (Sweden)

    Emilia Maria Cristina Mazza

    2014-12-01

    Full Text Available Myeloid-derived suppressor cells (MDSCs have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC, which are differentiated from umbilical cord blood (UCB precursors (Zoso et al., 2014. This MDSC subset promotes regulatory T-cell expansion and induces normoglycemia in a xenogeneic model of type 1 diabetes. Here we describe in details the experimental design and the bioinformatics analyses of the gene expression dataset used to investigate the molecular mechanisms at the base of MDSC tolerogenic and suppressive properties. We also provide an R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset. Raw and pre-processed data are available at Gene Expression Omnibus under accession GSE52376.

  11. Bacterial xylanase expression in mammalian cells and transgenic mice.

    Science.gov (United States)

    Fontes, C M; Ali, S; Gilbert, H J; Hazlewood, G P; Hirst, B H; Hall, J

    1999-06-11

    The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

  12. Gene expression in epithelial cells in response to pneumovirus infection

    Directory of Open Access Journals (Sweden)

    Rosenberg Helene F

    2001-05-01

    Full Text Available Abstract Respiratory syncytial virus (RSV and pneumonia virus of mice (PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

  13. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  14. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely.

    Science.gov (United States)

    Zhou, Beiyan; Wang, Stephanie; Mayr, Christine; Bartel, David P; Lodish, Harvey F

    2007-04-24

    MicroRNAs (miRNAs) are a family of approximately 22-nt noncoding RNAs that can posttranscriptionally regulate gene expression. Several miRNAs are specifically expressed in hematopoietic cells. Here we show that one such miRNA, miR-150, is mainly expressed in the lymph nodes and spleen and is highly up-regulated during the development of mature T and B cells; expression of miR-150 is sharply up-regulated at the immature B cell stage. Overexpression of miR-150 in hematopoietic stem cells, followed by bone marrow transplantation, had little effect on the formation of either mature CD8- and CD4-positive T cells or granulocytes or macrophages, but the formation of mature B cells was greatly impaired. Furthermore, premature expression of miR-150 blocked the transition from the pro-B to the pre-B stage. Our results indicate that miR-150 most likely down-regulates mRNAs that are important for pre- and pro-B cell formation or function, and its ectopic expression in these cells blocks further development of B cells.

  15. Inferring single-cell gene expression mechanisms using stochastic simulation

    Science.gov (United States)

    Daigle, Bernie J.; Soltani, Mohammad; Petzold, Linda R.; Singh, Abhyudai

    2015-01-01

    Motivation: Stochastic promoter switching between transcriptionally active (ON) and inactive (OFF) states is a major source of noise in gene expression. It is often implicitly assumed that transitions between promoter states are memoryless, i.e. promoters spend an exponentially distributed time interval in each of the two states. However, increasing evidence suggests that promoter ON/OFF times can be non-exponential, hinting at more complex transcriptional regulatory architectures. Given the essential role of gene expression in all cellular functions, efficient computational techniques for characterizing promoter architectures are critically needed. Results: We have developed a novel model reduction for promoters with arbitrary numbers of ON and OFF states, allowing us to approximate complex promoter switching behavior with Weibull-distributed ON/OFF times. Using this model reduction, we created bursty Monte Carlo expectation-maximization with modified cross-entropy method (‘bursty MCEM2’), an efficient parameter estimation and model selection technique for inferring the number and configuration of promoter states from single-cell gene expression data. Application of bursty MCEM2 to data from the endogenous mouse glutaminase promoter reveals nearly deterministic promoter OFF times, consistent with a multi-step activation mechanism consisting of 10 or more inactive states. Our novel approach to modeling promoter fluctuations together with bursty MCEM2 provides powerful tools for characterizing transcriptional bursting across genes under different environmental conditions. Availability and implementation: R source code implementing bursty MCEM2 is available upon request. Contact: absingh@udel.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573914

  16. Gene expression disparity in giant cell tumor of bone

    Institute of Scientific and Technical Information of China (English)

    Xiaohua PAN; Shuhua YANG; Deming XIAO; Yong DAI; Lili REN

    2009-01-01

    The aim of this paper was to study the differential gene expression of giant cell tumor of bone (GCTB) by gene chip technology. Total RNA of 8 fresh GCTB specimens (Jaffe Ⅰ:6 cases, Ⅱ: 1 case, Ⅲ: 1 case; Campanacci Ⅰ:6 cases, Ⅱ:1 case, Ⅲ:1 case; Enneking Staging G0T1-2M0, 5 cases, G1T1-2M0: 2 cases, G1T2M0: 1 case) and 4 normal bony callus specimens (the control group) were extracted and purified to get mRNA and then reverse transcribed to complementary DNA, respectively. Microarray screening with a set of 8064 human cDNA genes was conducted to analyze the difference among the samples and the control. The hybridization signals were scanned. The gene expression disparity between the GCTB samples and normal bony callus was significantly different (P<0.01), and the disparity of over 5-fold was found in 47 genes in the GCTB specimens, with 25 genes up-regulated and 22 down-regulated including the extracellular matrix and transforming-related genes, oncogene and its homolog genes, cytokine and its receptor genes. Specific gene spectrum associated with GCTB can be identified by cDNA microarray, which will be the foundation of progressive etiology elucidation, diagnosis and treatment of GCTB.

  17. Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

    Directory of Open Access Journals (Sweden)

    Ponti Donatella

    2011-03-01

    Full Text Available Abstract Background Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1 predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. Materials and methods Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. Results We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. Conclusion Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.