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Sample records for expression multiple promoters

  1. Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

    Directory of Open Access Journals (Sweden)

    Rosner William

    2009-05-01

    Full Text Available Abstract Background Human sex hormone-binding globulin (SHBG regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (PL. The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (PT, and two minor transcripts. Results We report that transcriptional expression of the human SHBG gene is far more complex than previously described. PL and PT direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of PL and PT, and present evidence for a third SHBG gene promoter (PN within the neighboring FXR2 gene; PN regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N. Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. PL- PT- and PN-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells. Conclusion These results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

  2. Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues

    Directory of Open Access Journals (Sweden)

    Ahmed Al-Jazzar

    2016-12-01

    Full Text Available Mice harbouring a dentin matrix protein 1 (Dmp1 promoter-driven human diphtheria toxin (DT receptor (HDTR transgene (Tg have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2, was also found in many non-skeletal tissues in Tg mice; indicative of direct “off-target” DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.

  3. Multiple promoters drive tissue-specific expression of the human M2 muscarinic acetylcholine receptor gene

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Alena; Bruce, A. W.; Doležal, Vladimír; Tuček, Stanislav; Buckley, N. J.

    2004-01-01

    Roč. 91, č. 1 (2004), s. 88-98 ISSN 0022-3042 R&D Projects: GA AV ČR IAA5011306 Institutional research plan: CEZ:AV0Z5011922 Keywords : M2 muscarinic receptor * neuron-restrictive silence factor * promoter Subject RIV: ED - Physiology Impact factor: 4.824, year: 2004

  4. Recurrent TERT promoter mutations identified in a large-scale study of multiple tumor types are associated with increased TERT expression and telomerase activation

    Science.gov (United States)

    Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H.; Yang, Rui; Killela, Patrick J.; Liang, Junbo; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Wang, Si-Zhen; Jiao, Yu-Chen; Yan, Hai; Tao, Hou-Quan

    2015-01-01

    Background Several somatic mutation hotspots were recently identified in the TERT promoter region in human cancers. Large scale studies of these mutations in multiple tumor types are limited, in particular in Asian populations. This study aimed to: analyze TERT promoter mutations in multiple tumor types in a large Chinese patient cohort, investigate novel tumor types and assess the functional significance of the mutations. Methods TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumor types and 799 tumor tissues from Chinese cancer patients. Thymic epithelial tumors, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by RT-qPCR, telomerase activity by the TRAP assay, and promoter activity by the luciferase reporter assay. Results TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%), and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in GIST, thymic epithelial tumors, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. Conclusions TERT promoter mutations are frequent in multiple tumor types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumorigenesis, making them potential therapeutic targets. PMID:25843513

  5. Effects of gene orientation and use of multiple promoters on the expression of XYL1 and XYL2 in Saccharomyces cerevisiae

    Science.gov (United States)

    Ju Yun Bae; Jose Laplaza; Thomas W. Jeffries

    2008-01-01

    Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We...

  6. Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

    Science.gov (United States)

    Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

  7. EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells

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    Florian Wanke

    2017-01-01

    Full Text Available Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE, the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23 and interleukin-1 beta (IL-1β, maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.

  8. Phosphate solubilization and multiple plant growth promoting ...

    African Journals Online (AJOL)

    Phosphate solubilizing efficiencies of the strains were analyzed using different insoluble phosphorus sources and the results show that most isolates released a substantial amount of soluble phosphate from tricalcium phosphate, rock phosphate and bone meal. Screening for multiple plant growth promoting attributes ...

  9. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R

    2014-03-26

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  10. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R; Kawaji, Hideya; Rehli, Michael; Baillie, John Kenneth; De Hoon, Michiel Jl L; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V.; Lizio, Marina; Itoh, Masayoshi; Andersson, Robin; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R.; Jia, Hui; Joshi, Anagha Madhusudan; Jurman, Giuseppe; Kaczkowski, Bogumił; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Mungall, Christopher J.; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S.; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kawashima, Tsugumi; Meehan, Terrence F.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klinken, Svend Peter; Knox, Alan; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Schmeier, Sebastian; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T.; Laros, Jeroen F J; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Bertin, Nicolas; Lipovich, Leonard; MacKay-Sim, Alan; Manabe, Riichiroh; Mar, Jessica; Marchand, Benoî t; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison M.; Mizuno, Yosuke; De Morais, David A Lima; Jø rgensen, Mette Christine; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L.; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Dimont, Emmanuel; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; Van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Arner, Erik; Ohmiya, Hiroko; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Pain, Arnab; Passier, Robert C J J; Patrikakis, Margaret; Schmidl, Christian; Persson, Helena A.; Piazza, Silvano; Prendergast, James G D; Rackham, Owen J L; Ramilowski, Jordan A.; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Schaefer, Ulf; Rye, Morten Beck; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Medvedeva, Yulia; Schneider, Claudio H.; Schultes, Erik A.; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W.; Simon, Chris M.; Plessy, Charles; Sugiyama, Daisuke; Sugiyama, Takaaki; Suzuki, Masanori; Suzuki, Naoko; Swoboda, Rolf K.; 't Hoen, Peter Ac Chr; Tagami, Michihira; Tagami, Naokotakahashi; Takai, Jun; Tanaka, Hiroshi; Vitezic, Morana; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyoda, Hiroo; Toyoda, Tetsuro; Valen, Eivind; Van De Wetering, Marc L.; Van Den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Severin, Jessica M.; Vorontsov, Ilya E.; Wasserman, Wyeth W.; Watanabe, Shoko; Wells, Christine A.; Winteringham, Louise Natalie; Wolvetang, Ernst Jurgen; Wood, Emily J.; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Semple, Colin Am M; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Susan E.; Zhang, Peter; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M.; Suzuki, Harukazu; Daub, Carsten Olivier; Kawai, Jun; Ishizu, Yuri; Heutink, Peter; Hide, Winston; Freeman, Tom C.; Lenhard, Boris; Bajic, Vladimir B.; Taylor, Martin S.; Makeev, Vsevolod J.; Sandelin, Albin; Hume, David A.; Carninci, Piero; Young, Robert S.; Hayashizaki, Yoshihide Yoshihide; Francescatto, Margherita; Altschuler, Intikhab Alam; Albanese, Davide; Altschule, Gabriel M.; Arakawa, Takahiro; Archer, John A.C.; Arner, Peter; Babina, Magda; Rennie, Sarah; Balwierz, Piotr J.; Beckhouse, Anthony G.; Pradhan-Bhatt, Swati; Blake, Judith A.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James A.; Brombacher, Frank; Burroughs, Alexander Maxwell; Califano, Andrea C.; Cannistraci, Carlo; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C.; Dalla, Emiliano; Davis, Carrie Anne; Detmar, Michael J.; Diehl, Alexander D.; Dohi, Taeko; Drablø s, Finn; Edge, Albert SB B; Edinger, Matthias G.; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey R.; Fang, Hai; Farach-Carson, Mary Cindy; Faulkner, Geoffrey J.; Favorov, Alexander V.; Fisher, Malcolm E.; Frith, Martin C.; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furuno, Masaaki; Furusawa, Junichi; Geijtenbeek, Teunis Bh H; Gibson, Andrew P.; Gingeras, Thomas R.; Goldowitz, Dan; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Takehiro; Herlyn, Meenhard F.; Hitchens, Kelly J.; Sui, Shannan J Ho; Hofmann, Oliver M.; Hoof, Ilka; Hori, Fumi; Huminiecki, Łukasz B.

    2014-01-01

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  11. A novel BDNF gene promoter directs expression to skeletal muscle

    Directory of Open Access Journals (Sweden)

    Heinrich Gerhard

    2003-06-01

    Full Text Available Abstract Background Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. Results The cloning and analysis of three additional zebrafish (Danio rerio BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. Conclusion The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are

  12. Mining gene expression data of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Pi Guo

    Full Text Available Microarray produces a large amount of gene expression data, containing various biological implications. The challenge is to detect a panel of discriminative genes associated with disease. This study proposed a robust classification model for gene selection using gene expression data, and performed an analysis to identify disease-related genes using multiple sclerosis as an example.Gene expression profiles based on the transcriptome of peripheral blood mononuclear cells from a total of 44 samples from 26 multiple sclerosis patients and 18 individuals with other neurological diseases (control were analyzed. Feature selection algorithms including Support Vector Machine based on Recursive Feature Elimination, Receiver Operating Characteristic Curve, and Boruta algorithms were jointly performed to select candidate genes associating with multiple sclerosis. Multiple classification models categorized samples into two different groups based on the identified genes. Models' performance was evaluated using cross-validation methods, and an optimal classifier for gene selection was determined.An overlapping feature set was identified consisting of 8 genes that were differentially expressed between the two phenotype groups. The genes were significantly associated with the pathways of apoptosis and cytokine-cytokine receptor interaction. TNFSF10 was significantly associated with multiple sclerosis. A Support Vector Machine model was established based on the featured genes and gave a practical accuracy of ∼86%. This binary classification model also outperformed the other models in terms of Sensitivity, Specificity and F1 score.The combined analytical framework integrating feature ranking algorithms and Support Vector Machine model could be used for selecting genes for other diseases.

  13. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  14. Expression of multiple proteins in transgenic plants

    Science.gov (United States)

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  15. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

  16. Expressions Creating Confusion among Elements of Promotion Mix: Sales Promotion

    OpenAIRE

    Kelesbayev, Dinmukhamed; Kalykulov, Kuatbek; Yertayev, Yermek; Turlybekova, Altynay; Kamalov, Akhmet

    2017-01-01

    More than twenty years on marketing, research and publications have dealt with research problems based on marketing mix and have mostly examined marketing processes. However, the types or elements of the marketing mix that are important for both the enterprises and the consumers are not analyzed too deeply, and different expressions or concepts are used in the analyzed sources. For example, when we look at the literature on marketing, which is being used as a source in our universities, the c...

  17. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  18. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  19. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  20. Analysis of the promoters involved in enterocin AS-48 expression.

    Science.gov (United States)

    Cebrián, Rubén; Rodríguez-Ruano, Sonia; Martínez-Bueno, Manuel; Valdivia, Eva; Maqueda, Mercedes; Montalbán-López, Manuel

    2014-01-01

    The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

  1. Analysis of the promoters involved in enterocin AS-48 expression.

    Directory of Open Access Journals (Sweden)

    Rubén Cebrián

    Full Text Available The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2 and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2 promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2 promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

  2. MUS81 promotes common fragile site expression

    DEFF Research Database (Denmark)

    Ying, Songmin; Minocherhomji, Sheroy; Chan, Kok Lung

    2013-01-01

    Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the fait......Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair...... the faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81-EME1 localizes to CFS loci in early...

  3. Silicalite nanoparticles that promote transgene expression

    International Nuclear Information System (INIS)

    Pearce, Megan E; Mai, Hoang Q; Salem, Aliasger K; Lee, Namhoon; Larsen, Sarah C

    2008-01-01

    Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55 nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3 mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7 mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%

  4. Precise regulation of gene expression dynamics favors complex promoter architectures.

    Directory of Open Access Journals (Sweden)

    Dirk Müller

    2009-01-01

    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  5. A strategy for health promotion at multiple corporate sites.

    Science.gov (United States)

    Alexy, B; Eynon, D

    1991-02-01

    1. The logistical problems associated with delivery of health promotion programs at multiple corporate sites can be addressed through the use of health education packets. 2. The role of the nurse or health coordinator is critical in assisting and guiding the employee as the health plan is executed. 3. Problematic areas related to implementation of programs at remote sites can be alleviated through careful planning and extensive communication. 4. Advertising, input from employees and staff, and follow up are important in the success of a program.

  6. Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome.

    Science.gov (United States)

    Ren, He-Lin; Hu, Yuan; Guo, Ya-Jun; Li, Lu-Lin

    2016-06-01

    Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus (PlxyGV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, and compared with homologous late gene promoters of AcMNPV in Sf9 cells. In transient expression assays, all PlxyGV late promoters were activated in cells transfected with the individual reporter plasmids together with an AcMNPV bacmid. In infected cells, reporter gene expression levels with the promoters of PlxyGV e18 and AcMNPV vp39 and gp41 were significantly higher than those of the corresponding AcMNPV or PlxyGV promoters, which had fewer late promoter motifs. Observed expression levels were lower for the PlxyGV p6.9, pk1, gran, p10a, and p10b promoters than for the corresponding AcMNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the AcMNPV polh, p10, and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of PlxyGV gran, p10c, and pk1. The results of this study demonstrated that PlxyGV late gene promoters could be effectively activated by the RNA polymerase from AcMNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.

  7. Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss.

    Science.gov (United States)

    Zahoor, Muhammad; Westhrin, Marita; Aass, Kristin Roseth; Moen, Siv Helen; Misund, Kristine; Psonka-Antonczyk, Katarzyna Maria; Giliberto, Mariaserena; Buene, Glenn; Sundan, Anders; Waage, Anders; Sponaas, Anne-Marit; Standal, Therese

    2017-12-26

    Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α-dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients.

  8. Regulation of the syncytin-1 promoter in human astrocytes by multiple sclerosis-related cytokines

    International Nuclear Information System (INIS)

    Mameli, Giuseppe; Astone, Vito; Khalili, Kamel; Serra, Caterina; Sawaya, Bassel E.; Dolei, Antonina

    2007-01-01

    Syncytin-1 has a physiological role during early pregnancy, as mediator of trophoblast fusion into the syncytiotrophoblast layer, hence allowing embryo implantation. In addition, its expression in nerve tissue has been proposed to contribute to the pathogenesis of multiple sclerosis (MS). Syncytin-1 is the env glycoprotein of the ERVWE1 component of the W family of human endogenous retroviruses (HERV), located on chromosome 7q21-22, in a candidate region for genetic susceptibility to MS. The mechanisms of ERVWE1 regulation in nerve tissue remain to be identified. Since there are correlations between some cytokines and MS outcome, we examined the regulation of the syncytin-1 promoter by MS-related cytokines in human U-87MG astrocytic cells. Using transient transfection assays, we observed that the MS-detrimental cytokines TNFα, interferon-γ, interleukin-6, and interleukin-1 activate the ERVWE1 promoter, while the MS-protective interferon-β is inhibitory. The effects of cytokines are reduced by the deletion of the cellular enhancer domain of the promoter that contains binding sites for several transcription factors. In particular, we found that TNFα had the ability to activate the ERVWE1 promoter through an NF-κB-responsive element located within the enhancer domain of the promoter. Electrophoretic mobility shift and ChIP assays showed that TNFα enhances the binding of the p65 subunit of NF-κB, to its cognate site within the promoter. The effect of TNFα is abolished by siRNA directed against p65. Taken together, these results illustrate a role for p65 in regulating the ERVWE1 promoter and in TNFα-mediated induction of syncytin-1 in multiple sclerosis

  9. Mycobacterium tuberculosis PPE44 (Rv2770c) is involved in response to multiple stresses and promotes the macrophage expression of IL-12 p40 and IL-6 via the p38, ERK, and NF-κB signaling axis.

    Science.gov (United States)

    Yu, Zhaoxiao; Zhang, Chenhui; Zhou, Mingliang; Li, Qiming; Li, Hui; Duan, Wei; Li, Xue; Feng, Yonghong; Xie, Jianping

    2017-09-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H 2 O 2 , SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage. Copyright © 2017. Published by Elsevier B.V.

  10. Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Huaying; Li, Guiyuan; Zhang, Liming; Niu, Zhaoxia; Zhou, Ming; Peng, Cong; Li, Xiayu; Deng, Tan; Shi, Lei; Tan, Yixin

    2008-01-01

    Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

  11. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    Directory of Open Access Journals (Sweden)

    V Shilpa

    2014-01-01

    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  12. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

    Science.gov (United States)

    Cheung, Samantha K; Scott, Kristin

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

  13. Src Induces Podoplanin Expression to Promote Cell Migration*

    Science.gov (United States)

    Shen, Yongquan; Chen, Chen-Shan; Ichikawa, Hitoshi; Goldberg, Gary S.

    2010-01-01

    Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration. PMID:20123990

  14. Methylation of class II transactivator gene promoter IV is not associated with susceptibility to Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Lincoln Matthew R

    2008-07-01

    Full Text Available Abstract Background Multiple sclerosis (MS is a complex trait in which alleles at or near the class II loci HLA-DRB1 and HLA-DQB1 contribute significantly to genetic risk. The MHC class II transactivator (MHC2TA is the master controller of expression of class II genes, and methylation of the promoter of this gene has been previously been shown to alter its function. In this study we sought to assess whether or not methylation of the MHC2TA promoter pIV could contribute to MS disease aetiology. Methods In DNA from peripheral blood mononuclear cells from a sample of 50 monozygotic disease discordant MS twins the MHC2TA promoter IV was sequenced and analysed by methylation specific PCR. Results No methylation or sequence variation of the MHC2TA promoter pIV was found. Conclusion The results of this study cannot support the notion that methylation of the pIV promoter of MHC2TA contributes to MS disease risk, although tissue and timing specific epigenetic modifications cannot be ruled out.

  15. Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

    Science.gov (United States)

    Chintala, Shravan; Cheng, Mei; Zhang, Xiao

    2015-01-01

    The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and

  16. Genetic variation in the proximal promoter of ABC and SLC superfamilies: liver and kidney specific expression and promoter activity predict variation.

    Directory of Open Access Journals (Sweden)

    Stephanie E Hesselson

    2009-09-01

    Full Text Available Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC and Solute Carrier (SLC superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

  17. Evaluation of schistosome promoter expression for transgenesis and genetic analysis.

    Directory of Open Access Journals (Sweden)

    Shuang Liang

    Full Text Available Schistosome worms of the genus Schistosoma are the causative agents of schistosomiasis, a devastating parasitic disease affecting more than 240 million people worldwide. Schistosomes have complex life cycles, and have been challenging to manipulate genetically due to the dearth of molecular tools. Although the use of gene overexpression, gene knockouts or knockdowns are straight-forward genetic tools applied in many model systems, gene misexpression and genetic manipulation of schistosome genes in vivo has been exceptionally challenging, and plasmid based transfection inducing gene expression is limited. We recently reported the use of polyethyleneimine (PEI as a simple and effective method for schistosome transfection and gene expression. Here, we use PEI-mediated schistosome plasmid transgenesis to define and compare gene expression profiles from endogenous and nonendogenous promoters in the schistosomula stage of schistosomes that are potentially useful to misexpress (underexpress or overexpress gene product levels. In addition, we overexpress schistosome genes in vivo using a strong promoter and show plasmid-based misregulation of genes in schistosomes, producing a clear and distinct phenotype--death. These data focus on the schistosomula stage, but they foreshadow strong potential for genetic characterization of schistosome molecular pathways, and potential for use in overexpression screens and drug resistance studies in schistosomes using plasmid-based gene expression.

  18. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  19. Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

    OpenAIRE

    Ferreira, Joshua P.; Peacock, Ryan W. S.; Lawhorn, Ingrid E. B.; Wang, Clifford L.

    2011-01-01

    The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evalua...

  20. Aberrant microRNA expression in multiple myeloma

    DEFF Research Database (Denmark)

    Dimopoulos, Konstantinos; Gimsing, Peter; Grønbæk, Kirsten

    2013-01-01

    Multiple myeloma (MM) is a devastating disease with a complex biology, and in spite of improved survivability by novel treatment strategies over the last decade, MM is still incurable by current therapy. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post...

  1. CD147 Promotes CXCL1 Expression and Modulates Liver Fibrogenesis

    Directory of Open Access Journals (Sweden)

    Wen-Pu Shi

    2018-04-01

    Full Text Available Activated hepatic stellate cells (HSCs release pro-inflammatory and pro-fibrogenic factors. CXC chemokine-ligand-1 (CXCL1 is expressed on HSCs. We previously found that the CD147 is overexpressed in activated HSCs. In this study, we showed an important role of CD147 in promoting liver fibrosis by activating HSCs and upregulating expression of chemokines. Specifically, we found that CD147 specific deletion in HSCs mice alleviated CCl4-induced liver fibrosis and inhibited HSCs activation. Overexpression of CD147 upregulated the secretion of CXCL1. Meanwhile, CXCL1 promoted HSCs activation through autocrine. Treating with PI3K/AKT inhibitor could effectively suppress CD147-induced CXCL1 expression. Taken together, these findings suggest that CD147 regulates CXCL1 release in HSCs by PI3K/AKT signaling. Inhibition of CD147 attenuates CCl4-induced liver fibrosis and inflammation. Therefore, administration of targeting CD147 could be a promising therapeutic strategy in liver fibrosis.

  2. Strontium Promotes Cementoblasts Differentiation through Inhibiting Sclerostin Expression In Vitro

    Directory of Open Access Journals (Sweden)

    Xingfu Bao

    2014-01-01

    Full Text Available Cementogenesis, performed by cementoblasts, is important for the repair of root resorption caused by orthodontic treatment. Based on recent studies, strontium has been applied for osteoporosis treatment due to its positive effect on osteoblasts. Although promising, the effect of strontium on cementoblasts is still unclear. So the aim of this research was to clarify and investigate the effect of strontium on cementogenesis via employing cementoblasts as model. A series of experiments including MTT, alkaline phosphatase activity, gene analysis, alizarin red staining, and western blot were carried out to evaluate the proliferation and differentiation of cementoblasts. In addition, expression of sclerostin was checked to analyze the possible mechanism. Our results show that strontium inhibits the proliferation of cementoblasts with a dose dependent manner; however, it can promote the differentiation of cementoblasts via downregulating sclerostin expression. Taking together, strontium may facilitate cementogenesis and benefit the treatment of root resorption at a low dose.

  3. Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani.

    Science.gov (United States)

    Soysa, Radika; Tran, Khoa D; Ullman, Buddy; Yates, Phillip A

    2015-12-01

    We have designed a novel series of integrating ribosomal RNA promoter vectors with five incrementally different constitutive expression profiles, covering a 250-fold range. Differential expression was achieved by placing different combinations of synthetic or leishmanial DNA sequences upstream and downstream of the transgene coding sequence in order to modulate pre-mRNA processing efficiency and mRNA stability, respectively. All of the vectors have extensive multiple cloning sites, and versions are available for producing N- or C- terminal GFP fusions at each of the possible relative expression levels. In addition, the modular configuration of the vectors allows drug resistance cassettes and other components to be readily exchanged. In toto, these vectors should be useful additions to the toolkit available for molecular and genetic studies of Leishmania donovani. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  5. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  6. Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

    Directory of Open Access Journals (Sweden)

    Yan Coulombe

    2010-05-01

    Full Text Available The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

  7. Melanoma NOS1 expression promotes dysfunctional IFN signaling.

    Science.gov (United States)

    Liu, Qiuzhen; Tomei, Sara; Ascierto, Maria Libera; De Giorgi, Valeria; Bedognetti, Davide; Dai, Cuilian; Uccellini, Lorenzo; Spivey, Tara; Pos, Zoltan; Thomas, Jaime; Reinboth, Jennifer; Murtas, Daniela; Zhang, Qianbing; Chouchane, Lotfi; Weiss, Geoffrey R; Slingluff, Craig L; Lee, Peter P; Rosenberg, Steven A; Alter, Harvey; Yao, Kaitai; Wang, Ena; Marincola, Francesco M

    2014-05-01

    In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells.

  8. Glucose metabolism in pigs expressing human genes under an insulin promoter.

    Science.gov (United States)

    Wijkstrom, Martin; Bottino, Rita; Iwase, Hayoto; Hara, Hidetaka; Ekser, Burcin; van der Windt, Dirk; Long, Cassandra; Toledo, Frederico G S; Phelps, Carol J; Trucco, Massimo; Cooper, David K C; Ayares, David

    2015-01-01

    Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. This preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Okada

    Full Text Available In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4 is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

  10. The Effect of Fearful Expressions on Multiple Face Tracking

    Directory of Open Access Journals (Sweden)

    Hongjun Jin

    2015-07-01

    Full Text Available How does the visual system realize dynamic tracking? This topic has become popular within cognitive science in recent years. The classical theory argues that multiple object tracking is accomplished via pre-attention visual indexes as part of a cognitively impenetrable low-level visual system. The present research aimed to investigate whether and how tracking processes are influenced by facial expressions that convey abundant social information about one’s mental state and situated environment. The results showed that participants tracked fearful faces more effectively than neutral faces. However, this advantage was only present under the low-attentional load condition, and distractor face emotion did not impact tracking performance. These findings imply that visual tracking is not driven entirely by low-level vision and encapsulated by high-level representations; rather, that facial expressions, a kind of social information, are able to influence dynamic tracking. Furthermore, the effect of fearful expressions on multiple face tracking is mediated by the availability of attentional resources.

  11. Semiotic mediation: from multiplication properties to arithmetical expressions

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    Andrea Maffia

    2016-04-01

    Full Text Available Multiplication is introduced early in primary school, but its properties are usually introduced after the rote memorization of multiplicative facts. In this paper we present a teaching experiment aimed to early introducing arithmetical properties of multiplication. It is realized through an artefact built on the rectangle model for multiplication. Children activity is designed and analyzed using Theory of Semiotic Mediation. The development of the relational meaning of arithmetical expressions is shown through the enchaining of representations from signs related to the activity with the artefact to mathematical ones. In particular, the role of the teacher in the process of semiotic mediation results as crucial. Mediazione semiotica: dalle proprietà della moltiplicazione alle espressioni aritmeticheLa moltiplicazione viene presentata presto nella scuola primaria, ma le sue proprietà sono introdotte solo dopo che le cosiddette tabelline sono state memorizzate. Nell’articolo si presenta un teaching experiment volto a introdurre precocemente le proprietà della moltiplicazione per facilitare la memorizzazione di fatti moltiplicativi. L’esperimento è centrato sull’uso di un artefatto costruito sul modello rettangolare della moltiplicazione. L’attività degli studenti è progettata e analizzata nel quadro della Teoria della Mediazione Semiotica (TMS. Lo sviluppo del significato relazionale delle espressioni aritmetiche viene mostrato attraverso la concatenazione di rappresentazioni che vanno da segni strettamente legati all’attività con l’artefatto fino a segni matematici. In particolare, si evidenzia il ruolo dell’insegnante nello sviluppo del processo di mediazione semiotica.

  12. STAT6 expression in glioblastoma promotes invasive growth

    International Nuclear Information System (INIS)

    Merk, Barbara C; Owens, Jennifer L; Lopes, Maria-Beatriz S; Silva, Corinne M; Hussaini, Isa M

    2011-01-01

    different shRNA- silenced clones, but all had a reduction in 3 H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt [1] gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance. Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target

  13. Recurrent and multiple bladder tumors show conserved expression profiles

    International Nuclear Information System (INIS)

    Lindgren, David; Fioretos, Thoas; Månsson, Wiking; Höglund, Mattias; Gudjonsson, Sigurdur; Jee, Kowan Ja; Liedberg, Fredrik; Aits, Sonja; Andersson, Anna; Chebil, Gunilla; Borg, Åke; Knuutila, Sakari

    2008-01-01

    Urothelial carcinomas originate from the epithelial cells of the inner lining of the bladder and may appear as single or as multiple synchronous tumors. Patients with urothelial carcinomas frequently show recurrences after treatment making follow-up necessary. The leading hypothesis explaining the origin of meta- and synchronous tumors assumes a monoclonal origin. However, the genetic relationship among consecutive tumors has been shown to be complex in as much as the genetic evolution does not adhere to the chronological appearance of the metachronous tumors. Consequently, genetically less evolved tumors may appear chronologically later than genetically related but more evolved tumors. Forty-nine meta- or synchronous urothelial tumors from 22 patients were analyzed using expression profiling, conventional CGH, LOH, and mutation analyses. We show by CGH that partial chromosomal losses in the initial tumors may not be present in the recurring tumors, by LOH that different haplotypes may be lost and that detected regions of LOH may be smaller in recurring tumors, and that mutations present in the initial tumor may not be present in the recurring ones. In contrast we show that despite apparent genomic differences, the recurrent and multiple bladder tumors from the same patients display remarkably similar expression profiles. Our findings show that even though the vast majority of the analyzed meta- and synchronous tumors from the same patients are not likely to have originated directly from the preceding tumor they still show remarkably similar expressions profiles. The presented data suggests that an expression profile is established early in tumor development and that this profile is stable and maintained in recurring tumors

  14. Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk.

    Science.gov (United States)

    Nolan, David; Castley, Alison; Tschochner, Monika; James, Ian; Qiu, Wei; Sayer, David; Christiansen, Frank T; Witt, Campbell; Mastaglia, Frank; Carroll, William; Kermode, Allan

    2012-08-07

    The identification of a vitamin D-responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1-associated MS risk. We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles (p = 10(-12)) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles (p < 10(-18)). HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D-dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the "nonresponsive" VDRE motif were associated with significantly reduced risk of MS.

  15. STAT6 expression in glioblastoma promotes invasive growth

    Directory of Open Access Journals (Sweden)

    Silva Corinne M

    2011-05-01

    -type. There was some variation among the different shRNA- silenced clones, but all had a reduction in 3H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1 and urokinase Plasminogen activator (uPA as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P Conclusions Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

  16. Multiple distinct stimuli increase measured nucleosome occupancy around human promoters.

    Directory of Open Access Journals (Sweden)

    Chuong D Pham

    Full Text Available Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning ∼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over ∼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.

  17. SATB1 tethers multiple gene loci to reprogram expression profiledriving breast cancer metastasis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hye-Jung; Kohwi, Yoshinori; Kohwi-Shigematsu, Terumi

    2006-07-13

    Global changes in gene expression occur during tumor progression, as indicated by expression profiling of metastatic tumors. How this occurs is poorly understood. SATB1 functions as a genome organizer by folding chromatin via tethering multiple genomic loci and recruiting chromatin remodeling enzymes to regulate chromatin structure and expression of a large number of genes. Here we show that SATB1 is expressed at high levels in aggressive breast cancer cells, and is undetectable in non-malignant breast epithelial cells. Importantly, RNAi-mediated removal of SATB1 from highly-aggressive MDA-MB-231 cells altered the expression levels of over 1200 genes, restored breast-like acinar polarity in three-dimensional cultures, and prevented the metastastic phenotype in vivo. Conversely, overexpression of SATB1 in the less-aggressive breast cancer cell line Hs578T altered the gene expression profile and increased metastasis dramatically in vivo. Thus, SATB1 is a global regulator of gene expression in breast cancer cells, directly regulating crucial metastasis-associated genes, including ERRB2 (HER2/NEU), TGF-{beta}1, matrix metalloproteinase 3, and metastasin. The identification of SATB1 as a protein that re-programs chromatin organization and transcription profiles to promote breast cancer metastasis suggests a new model for metastasis and may provide means of therapeutic intervention.

  18. Altered gene-expression profile in rat plasma and promoted body ...

    African Journals Online (AJOL)

    Altered gene-expression profile in rat plasma and promoted body and brain development ... The study was aimed to explore how the prenatal EE impacts affect the ... positively promote the body and nervous system development of offspring, ...

  19. Behavioral training promotes multiple adaptive processes following acute hearing loss.

    Science.gov (United States)

    Keating, Peter; Rosenior-Patten, Onayomi; Dahmen, Johannes C; Bell, Olivia; King, Andrew J

    2016-03-23

    The brain possesses a remarkable capacity to compensate for changes in inputs resulting from a range of sensory impairments. Developmental studies of sound localization have shown that adaptation to asymmetric hearing loss can be achieved either by reinterpreting altered spatial cues or by relying more on those cues that remain intact. Adaptation to monaural deprivation in adulthood is also possible, but appears to lack such flexibility. Here we show, however, that appropriate behavioral training enables monaurally-deprived adult humans to exploit both of these adaptive processes. Moreover, cortical recordings in ferrets reared with asymmetric hearing loss suggest that these forms of plasticity have distinct neural substrates. An ability to adapt to asymmetric hearing loss using multiple adaptive processes is therefore shared by different species and may persist throughout the lifespan. This highlights the fundamental flexibility of neural systems, and may also point toward novel therapeutic strategies for treating sensory disorders.

  20. Multiple upstream modules regulate zebrafish myf5 expression

    Directory of Open Access Journals (Sweden)

    Weng Chih-Wei

    2007-01-01

    that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish.

  1. Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

    Science.gov (United States)

    Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

    2011-12-01

    The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

  2. Promoting pneumococcal immunizations among rural Medicare beneficiaries using multiple strategies.

    Science.gov (United States)

    Johnson, Elizabeth A; Harwell, Todd S; Donahue, Peg M; Weisner, M'liss A; McInerney, Michael J; Holzman, Greg S; Helgerson, Steven D

    2003-01-01

    Vaccine-preventable diseases among adults are major contributing causes of morbidity and mortality in the United States. However, adult immunizations continue to be underutilized in both urban and rural areas. To evaluate the effectiveness of a community-wide education campaign and mailed reminders promoting pneumococcal immunizations to rural Medicare beneficiaries. We implemented a community-wide education campaign, and mailed reminders were sent to Medicare beneficiaries in 1 media market in Montana to increase pneumococcal immunizations. In a second distinct media market, mailed reminders only were sent to beneficiaries. The proportion of respondents aged 65 years and older aware of pneumococcal immunizations increased significantly from baseline to follow-up among respondents both in the education-plus-reminder (63% to 78%, P = 0.04) and the reminder-only (64% to 74%, P = 0.05) markets. Overall from 1998 to 1999, there was a 3.7-percentage-point increase in pneumococcal immunization claims for Medicare beneficiaries in the education-plus-reminder market and a 1.5-percentage-point increase in the reminder-only market. Medicare beneficiaries sent reminders in the education-plus-reminder market compared to those in the reminder-only market were more likely to have a claim for pneumococcal immunization in 1999 (odds ratio 1.18, 95% confidence interval 1.08 to 1.28). The results suggest that these quality improvement strategies (community education plus reminders and reminders alone) modestly increased pneumococcal immunization awareness and pneumococcal immunization among rural adults. Mailed reminder exposure was associated with an increased prevalence of pneumococcal immunizations between 1998 and 1999 and was augmented somewhat by the education campaign.

  3. Illegitimate WNT signaling promotes proliferation of multiple myeloma cells

    Science.gov (United States)

    Derksen, Patrick W. B.; Tjin, Esther; Meijer, Helen P.; Klok, Melanie D.; Mac Gillavry, Harold D.; van Oers, Marinus H. J.; Lokhorst, Henk M.; Bloem, Andries C.; Clevers, Hans; Nusse, Roel; van der Neut, Ronald; Spaargaren, Marcel; Pals, Steven T.

    2004-01-01

    The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also influenced by signals from the environment. In multiple myeloma (MM), the factors and signals coming from the bone marrow microenvironment are possibly even essential for the growth of the tumor cells. As targets for intervention, these signals may be equally important as mutated oncogenes. Given their oncogenic potential, WNT signals form a class of paracrine growth factors that could act to influence MM cell growth. In this paper, we report that MM cells have hallmarks of active WNT signaling, whereas the cells have not undergone detectable mutations in WNT signaling genes such as adenomatous polyposis coli and β-catenin (CTNNB1). We show that the malignant MM plasma cells overexpress β-catenin, including its N-terminally unphosphorylated form, suggesting active β-catenin/T cell factor-mediated transcription. Further accumulation and nuclear localization of β-catenin, and/or increased cell proliferation, was achieved by stimulation of WNT signaling with either Wnt3a, LiCl, or the constitutively active S33Y mutant of β-catenin. In contrast, by blocking WNT signaling by dominant-negative T cell factor, we can interfere with the growth of MM cells. We therefore suggest that MM cells are dependent on an active WNT signal, which may have important implications for the management of this incurable form of cancer. PMID:15067127

  4. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  5. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

    Directory of Open Access Journals (Sweden)

    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  6. The analysis of correlation between IL-1B gene expression and genotyping in multiple sclerosis patients.

    Science.gov (United States)

    Heidary, Masoumeh; Rakhshi, Nahid; Pahlevan Kakhki, Majid; Behmanesh, Mehrdad; Sanati, Mohammad Hossein; Sanadgol, Nima; Kamaladini, Hossein; Nikravesh, Abbas

    2014-08-15

    IL-1B is released by monocytes, astrocytes and brain endothelial cells and seems to be involved in inflammatory reactions of the central nervous system (CNS) in multiple sclerosis (MS). This study aims to evaluate the expression level of IL-1B mRNA in peripheral blood mononuclear cells (PBMCs), genotype the rs16944 SNP and find out the role of this SNP on the expression level of IL-1B in MS patients. We found that the expression level of IL-1B in MS patients increased 3.336 times more than controls in PBMCs but the rs16944 SNP in the promoter region of IL-1B did not affect the expression level of this gene and there was not association of this SNP with MS in the examined population. Also, our data did not reveal any correlation between normalized expressions of IL-1B gene with age of participants, age of onset, and disease duration. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    Science.gov (United States)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  8. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Rachel M. Woodfint

    2017-01-01

    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  9. C/EBPβ Mediates Growth Hormone-Regulated Expression of Multiple Target Genes

    Science.gov (United States)

    Cui, Tracy X.; Lin, Grace; LaPensee, Christopher R.; Calinescu, Anda-Alexandra; Rathore, Maanjot; Streeter, Cale; Piwien-Pilipuk, Graciela; Lanning, Nathan; Jin, Hui; Carter-Su, Christin; Qin, Zhaohui S.

    2011-01-01

    Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein β (C/EBPβ). This study examines the role of C/EBPβ in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPβ depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPβ led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPβ mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPβ at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPβ and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPβ. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPβ, and recruitment of p300. Overall, these studies suggest that C/EBPβ, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. PMID:21292824

  10. ΔNp63α induces the expression of FAT2 and Slug to promote tumor invasion

    Science.gov (United States)

    Dang, Tuyen T.; Westcott, Jill M.; Maine, Erin A.; Kanchwala, Mohammed; Xing, Chao; Pearson, Gray W.

    2016-01-01

    Tumor invasion can be induced by changes in gene expression that alter cell phenotype. The transcription factor ΔNp63α promotes basal-like breast cancer (BLBC) migration by inducing the expression of the mesenchymal genes Slug and Axl, which confers cells with a hybrid epithelial/mesenchymal state. However, the extent of the ΔNp63α regulated genes that support invasive behavior is not known. Here, using gene expression analysis, ChIP-seq, and functional testing, we find that ΔNp63α promotes BLBC motility by inducing the expression of the atypical cadherin FAT2, the vesicular binding protein SNCA, the carbonic anhydrase CA12, the lipid binding protein CPNE8 and the kinase NEK1, along with Slug and Axl. Notably, lung squamous cell carcinoma migration also required ΔNp63α dependent FAT2 and Slug expression, demonstrating that ΔNp63α promotes migration in multiple tumor types by inducing mesenchymal and non-mesenchymal genes. ΔNp63α activation of FAT2 and Slug influenced E-cadherin localization to cell-cell contacts, which can restrict spontaneous cell movement. Moreover, live-imaging of spheroids in organotypic culture demonstrated that ΔNp63α, FAT2 and Slug were essential for the extension of cellular protrusions that initiate collective invasion. Importantly, ΔNp63α is co-expressed with FAT2 and Slug in patient tumors and the elevated expression of ΔNp63α, FAT2 and Slug correlated with poor patient outcome. Together, these results reveal how ΔNp63α promotes cell migration by directly inducing the expression of a cohort of genes with distinct cellular functions and suggest that FAT2 is a new regulator of collective invasion that may influence patient outcome. PMID:27081041

  11. Beyond Promotion-Based Store Switching: Antecedents and Consequences of Systematic Multiple-Store Shopping

    OpenAIRE

    Gijsbrechts, E.; Campo, K.; Nisol, P.

    2005-01-01

    In this paper, we demonstrate that single-purpose multiple store shopping is not only driven by opportunistic, promotion-based motivations, but may also be part of a longer term shopping planning process based on stable store characteristics.Starting from a utility-maximizing shopping behavior model, we find that consumers systematically visit multiple stores to take advantage of two types of store complementarity.With 'fixed cost complementarity', consumers alternate visits to highly preferr...

  12. OCT4 increases BIRC5 and CCND1 expression and promotes cancer progression in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Cao, Lu; Wu, Mengchao; Zhang, Ying; Su, Changqing; Li, Chunguang; Shen, Shuwen; Yan, Yan; Ji, Weidan; Wang, Jinghan; Qian, Haihua; Jiang, Xiaoqing; Li, Zhigang

    2013-01-01

    OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized. By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated. Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice. OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment

  13. Oxidized LDL Promotes Apoptosis and Expression of Pro ...

    African Journals Online (AJOL)

    Accumulation of lipid within non-adipose tissues can induce inflammation by promoting macrophage infiltration and activation. Oxidized lipoproteins (oxLDL) have been known to induce cellular dysfunction in resident macrophages through pro-inflammatory and pro-apoptotic properties. However research into the ...

  14. Beyond Promotion-Based Store Switching : Antecedents and Consequences of Systematic Multiple-Store Shopping

    NARCIS (Netherlands)

    Gijsbrechts, E.; Campo, K.; Nisol, P.

    2005-01-01

    In this paper, we demonstrate that single-purpose multiple store shopping is not only driven by opportunistic, promotion-based motivations, but may also be part of a longer term shopping planning process based on stable store characteristics.Starting from a utility-maximizing shopping behavior

  15. Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

    Science.gov (United States)

    Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

    2016-06-01

    Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter.

  16. EBF factors drive expression of multiple classes of target genes governing neuronal development.

    Science.gov (United States)

    Green, Yangsook S; Vetter, Monica L

    2011-04-30

    Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

  17. EBF factors drive expression of multiple classes of target genes governing neuronal development

    Directory of Open Access Journals (Sweden)

    Vetter Monica L

    2011-04-01

    Full Text Available Abstract Background Early B cell factor (EBF family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. Results We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. Conclusions The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

  18. Short-term fasting promotes insulin expression in rat hypothalamus.

    Science.gov (United States)

    Dakic, Tamara B; Jevdjovic, Tanja V; Peric, Mina I; Bjelobaba, Ivana M; Markelic, Milica B; Milutinovic, Bojana S; Lakic, Iva V; Jasnic, Nebojsa I; Djordjevic, Jelena D; Vujovic, Predrag Z

    2017-07-01

    In the hypothalamus, insulin takes on many roles involved in energy homoeostasis. Therefore, the aim of this study was to examine hypothalamic insulin expression during the initial phase of the metabolic response to fasting. Hypothalamic insulin content was assessed by both radioimmunoassay and Western blot. The relative expression of insulin mRNA was examined by qPCR. Immunofluorescence and immunohistochemistry were used to determine the distribution of insulin immunopositivity in the hypothalamus. After 6-h fasting, both glucose and insulin levels were decreased in serum but not in the cerebrospinal fluid. Our study showed for the first time that, while the concentration of circulating glucose and insulin decreased, both insulin mRNA expression and insulin content in the hypothalamic parenchyma were increased after short-term fasting. Increased insulin immunopositivity was detected specifically in the neurons of the hypothalamic periventricular nucleus and in the ependymal cells of fasting animals. These novel findings point to the complexity of mechanisms regulating insulin expression in the CNS in general and in the hypothalamus in particular. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  19. Expression of MiR-9 promotes proliferation, migration and ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of miR-9 on the proliferation, differentiation and migration of human neural stem cells (NSCs). Methods: The expression of miR-9 was investigated by quantitative real-time polymerase chain reaction (RT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK8) assay, while cell ...

  20. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  1. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

  2. Comparative study of four interleukin 17 cytokines of tongue sole Cynoglossus semilaevis: Genomic structure, expression pattern, and promoter activity.

    Science.gov (United States)

    Chi, Heng; Sun, Li

    2015-11-01

    The interleukin (IL)-17 cytokine family participates in the regulation of many cellular functions. In the present study, we analyzed the genomic structure, expression, and promoter activity of four IL-17 members from the teleost fish tongue sole (Cynoglossus semilaevis), i.e. CsIL-17C CsIL-17D, CsIL-17F, and IL-17F like (IL-17Fl). We found that CsIL-17C, CsIL-17D, CsIL-17F, and CsIL-17Fl share 21.2%-28.6% overall sequence identities among themselves and 31.5%-71.2% overall sequence identities with their counterparts in other teleost. All four CsIL-17 members possess an IL-17 domain and four conserved cysteine residues. Phylogenetic analysis classified the four CsIL-17 members into three clusters. Under normal physiological conditions, the four CsIL-17 expressed in multiple tissues, especially non-immune tissues. Bacterial infection upregulated the expression of all four CsIL-17, while viral infection upregulated the expression of CsIL-17D and CsIL-17Fl but downregulated the expression of CsIL-17C and CsIL-17F. The 1.2 kb 5'-flanking regions of the four CsIL-17 exhibited apparent promoter activity and contain a number of putative transcription factor-binding sites. Furthermore, the promoter activities of CsIL-17C, CsIL-17D, and CsIL-17F, but not CsIL-17Fl, were modulated to significant extents by lipopolysaccharide, PolyI:C, and PMA. This study provides the first evidence that in teleost, different IL-17 members differ in expression pattern and promoter activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Engineered promoters enable constant gene expression at any copy number in bacteria.

    Science.gov (United States)

    Segall-Shapiro, Thomas H; Sontag, Eduardo D; Voigt, Christopher A

    2018-04-01

    The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

  4. Rational polypharmacology: systematically identifying and engaging multiple drug targets to promote axon growth

    Science.gov (United States)

    Al-Ali, Hassan; Lee, Do-Hun; Danzi, Matt C.; Nassif, Houssam; Gautam, Prson; Wennerberg, Krister; Zuercher, Bill; Drewry, David H.; Lee, Jae K.; Lemmon, Vance P.; Bixby, John L.

    2016-01-01

    Mammalian Central Nervous System (CNS) neurons regrow their axons poorly following injury, resulting in irreversible functional losses. Identifying therapeutics that encourage CNS axon repair has been difficult, in part because multiple etiologies underlie this regenerative failure. This suggests a particular need for drugs that engage multiple molecular targets. Although multi-target drugs are generally more effective than highly selective alternatives, we lack systematic methods for discovering such drugs. Target-based screening is an efficient technique for identifying potent modulators of individual targets. In contrast, phenotypic screening can identify drugs with multiple targets; however, these targets remain unknown. To address this gap, we combined the two drug discovery approaches using machine learning and information theory. We screened compounds in a phenotypic assay with primary CNS neurons and also in a panel of kinase enzyme assays. We used learning algorithms to relate the compounds’ kinase inhibition profiles to their influence on neurite outgrowth. This allowed us to identify kinases that may serve as targets for promoting neurite outgrowth, as well as others whose targeting should be avoided. We found that compounds that inhibit multiple targets (polypharmacology) promote robust neurite outgrowth in vitro. One compound with exemplary polypharmacology, was found to promote axon growth in a rodent spinal cord injury model. A more general applicability of our approach is suggested by its ability to deconvolve known targets for a breast cancer cell line, as well as targets recently shown to mediate drug resistance. PMID:26056718

  5. Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis

    NARCIS (Netherlands)

    Geurts, J. J. G.; Wolswijk, G.; Bö, L.; van der Valk, P.; Polman, C. H.; Troost, D.; Aronica, E.

    2003-01-01

    Recent evidence supports a role for glutamate receptors in the pathophysiology of multiple sclerosis. In the present study, we have focused specifically on the expression of metabotropic glutamate receptors (mGluRs) in multiple sclerosis brain tissue. The expression of group I (mGluR1alpha and

  6. Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

    Science.gov (United States)

    Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

    2016-03-10

    Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

  7. Klf5 deletion promotes Pten deletion-initiated luminal-type mouse prostate tumors through multiple oncogenic signaling pathways.

    Science.gov (United States)

    Xing, Changsheng; Ci, Xinpei; Sun, Xiaodong; Fu, Xiaoying; Zhang, Zhiqian; Dong, Eric N; Hao, Zhao-Zhe; Dong, Jin-Tang

    2014-11-01

    Krüppel-like factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer-mutation/deletion of Pten and deletion of Klf5.

  8. PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Peihao; Guo, Honggang [Department of Hematology, Navy General Hospital, Beijing 100048 (China); Li, Guangchao [School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006 (China); Han, Siqi [Department of Medical Oncology, Jinling Hospital, Nanjing 210002 (China); Luo, Fei [Department of Stomatology, Jinling Hospital, Nanjing 210002 (China); Liu, Yi, E-mail: liuyi2033@163.com [Department of Hematology, Navy General Hospital, Beijing 100048 (China)

    2015-03-06

    Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expression of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.

  9. Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

    Science.gov (United States)

    Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

    2006-03-01

    Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. Copyright 2005 Wiley-Liss, Inc.

  10. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  11. Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

    Directory of Open Access Journals (Sweden)

    Hastie C James

    2006-01-01

    Full Text Available Abstract Background Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

  12. Role of promoter element in c-mpl gene expression induced by TPO.

    Science.gov (United States)

    Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

    2013-01-01

    Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role for the development of megakaryocyte and considered to regulate megakaryocytopoiesis. Previously we reported that TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter and the expression of the c-mpl gene is modulated by transcription through a protein kinase C (PKC)-dependent pathway in the megakaryoblastic cells. In this research, to elucidate the required elements in c-mpl promoter, the promoter activity of the deletion constructs and site-directed mutagenesis were measured by a transient transfection assay system. Destruction of -77GATA in c-mpl promoter decreased the activity by 22.8%. Our study elucidated that -77GATA involved in TPO-induced c-mpl gene expression in a human megakaryoblastic cell line, CMK.

  13. GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

    Science.gov (United States)

    Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

    2016-01-01

    With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs.

  14. Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

    Science.gov (United States)

    Jové, Thomas; Da Re, Sandra; Tabesse, Aurore; Gassama-Sow, Amy; Ploy, Marie-Cécile

    2017-01-01

    Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the P intI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

  15. Expression of Mucin-1 in multiple myeloma and its precursors

    DEFF Research Database (Denmark)

    Andrulis, Mindaugas; Ellert, Elena; Mandel, Ulla

    2014-01-01

    AIMS: Recent reports suggest a possible role for extracellular (MUC1N) and transmembrane (MUC1C) subunits of Mucin 1 (MUC1) in the pathogenesis of multiple myeloma (MM). Nuclear translocation of MUC1C is involved in activation of various oncogenic signalling pathways and both MUC1 subunits...

  16. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

    Science.gov (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y.

    2016-01-01

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning two orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  17. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.

    2013-02-19

    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

  18. A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

    Science.gov (United States)

    Smith, A F; Bigsby, R M; Word, R A; Herring, B P

    1998-05-01

    A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

  19. An examination of the Apo-1/Fas promoter Mva I polymorphism in Japanese patients with multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Yabe Ichiro

    2002-08-01

    Full Text Available Abstract Background The Apo-1/Fas (CD95 molecule is an apoptosis-signaling cell surface receptor belonging to the tumor necrosis factor (TNF receptor family. Both Fas and Fas ligand (FasL are expressed in activated mature T cells, and prolonged cell activation induces susceptibility to Fas-mediated apoptosis. The Apo-1/Fas gene is located in a chromosomal region that shows linkage in multiple sclerosis (MS genome screens, and studies indicate that there is aberrant expression of the Apo-1/Fas molecule in MS. Methods Mva I polymorphism on the Apo-1/Fas promoter gene was detected by PCR-RFLP from the DNA of 114 Japanese patients with conventional MS and 121 healthy controls. We investigated the association of the Mva I polymorphism in Japanese MS patients using a case-control association study design. Results We found no evidence that the polymorphism contributes to susceptibility to MS. Furthermore, there was no association between Apo-1/Fas gene polymorphisms and clinical course (relapsing-remitting course or secondary-progressive course. No significant association was observed between Apo-1/Fas gene polymorphisms and the age at disease onset. Conclusions Overall, our findings suggest that Apo-1/Fas promoter gene polymorphisms are not conclusively related to susceptibility to MS or the clinical characteristics of Japanese patients with MS.

  20. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    International Nuclear Information System (INIS)

    Asting, Annika Gustafsson; Carén, Helena; Andersson, Marianne; Lönnroth, Christina; Lagerstedt, Kristina; Lundholm, Kent

    2011-01-01

    Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue

  1. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    Directory of Open Access Journals (Sweden)

    Lagerstedt Kristina

    2011-06-01

    Full Text Available Abstract Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4 showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3 were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

  2. Multiple Functional Variants in cis Modulate PDYN Expression.

    Science.gov (United States)

    Babbitt, Courtney C; Silverman, Jesse S; Haygood, Ralph; Reininga, Jennifer M; Rockman, Matthew V; Wray, Gregory A

    2010-02-01

    Understanding genetic variation and its functional consequences within cis-regulatory regions remains an important challenge in human genetics and evolution. Here, we present a fine-scale functional analysis of segregating variation within the cis-regulatory region of prodynorphin, a gene that encodes an endogenous opioid precursor with roles in cognition and disease. In order to characterize the functional consequences of segregating variation in cis in a region under balancing selection in different human populations, we examined associations between specific polymorphisms and gene expression in vivo and in vitro. We identified five polymorphisms within the 5' flanking region that affect transcript abundance: a 68-bp repeat recognized in prior studies, as well as two microsatellites and two single nucleotide polymorphisms not previously implicated as functional variants. The impact of these variants on transcription differs by brain region, sex, and cell type, implying interactions between cis genotype and the differentiated state of cells. The effects of individual variants on expression level are not additive in some combinations, implying epistatic interactions between nearby variants. These data reveal an unexpectedly complex relationship between segregating genetic variation and its expression-trait consequences and highlights the importance of close functional scrutiny of natural genetic variation within even relatively well-studied cis-regulatory regions.

  3. An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex

    Directory of Open Access Journals (Sweden)

    Bernd eKuhn

    2012-07-01

    Full Text Available Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN led to high levels of expression (50-100 µM in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs (≤10 μM, yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA, a second rAAV containing the bidirectional TET promoter (Ptetbi could drive strong D3cpv expression in PCs (10-300 µM, enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.

  4. A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues.

    Directory of Open Access Journals (Sweden)

    Athma A Pai

    2011-02-01

    Full Text Available The modification of DNA by methylation is an important epigenetic mechanism that affects the spatial and temporal regulation of gene expression. Methylation patterns have been described in many contexts within and across a range of species. However, the extent to which changes in methylation might underlie inter-species differences in gene regulation, in particular between humans and other primates, has not yet been studied. To this end, we studied DNA methylation patterns in livers, hearts, and kidneys from multiple humans and chimpanzees, using tissue samples for which genome-wide gene expression data were also available. Using the multi-species gene expression and methylation data for 7,723 genes, we were able to study the role of promoter DNA methylation in the evolution of gene regulation across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. However, we also found a large number of gene expression differences between species that might be explained, at least in part, by corresponding differences in methylation levels. In particular, we estimate that, in the tissues we studied, inter-species differences in promoter methylation might underlie as much as 12%-18% of differences in gene expression levels between humans and chimpanzees.

  5. AKT increases VEGF expression in tumor cells by transactivating the proximal VEGF promoter

    International Nuclear Information System (INIS)

    Pore, N.; Bernhard, E.J.; Shu, H.-K.; Li, B.; O'Rourke, D.M.; Maity, A.; Haas-Kogan, D.

    2003-01-01

    Vascular endothelial growth factor (VEGF) is overexpressed in many cancers including glioblastomas and may contribute to their growth. Epidermal growth factor receptor (EGFR) amplification and loss of PTEN, commonly found in glioblastomas leading to increase phosphatidylinositol-3-kinase (PI3K) activity and VEGF expression. In the current study we show that AKT, which is downstream of PI3K, regulates VEGF expression. U87MG human glioblastoma cells lack wildtype PTEN and express high levels of phosphorylated AKT. Over expression of AKT either by stable expression in immortalized human astrocytes or by transduction with adenovirus containing activated myristoylated AKT in SF188 glioblastoma cells increases VEGF expression. Moreover the elevation of angiogenesis by constitutively expressed AKT is further confirmed by in vivo matrigel plug assay in nude mice. The upregulation of VEGF by AKT is mediated through a region in the proximal promoter located between -88 and -70 (+1 is transcription start site). In transient transfection activity of a luciferase reporter containing the -88/+54 region of the VEGF promoter is increased by cotransfection with myristoylated AKT and downregulated by a dominant negative AKT expression vector. Mutation of the putative Sp1 binding sites located in the -88/-70 region we show that AKT acts through Sp1 to transactivate the VEGF promoter. Cotransfection of the VEGF promoter reporter with both Sp1 and myristoylated AKT expression vectors increases promoter activity to a greater extent than either Sp1 or Akt by itself. In vivo phosphate labeling of proteins reveals that AKT leads to increased Sp1 phosphorylation. Gel shift assays using a radio labeled probe corresponding to nucleotides -88 through -66 in the promoter show increased binding with nuclear extracts from cells transduced with adenovirus expressing myristoylated AKT. In conclusion, our results suggest that loss of PTEN leads to increased VEGF expression by increasing AKT

  6. Protein arginine methyltransferase 5 regulates multiple signaling pathways to promote lung cancer cell proliferation

    International Nuclear Information System (INIS)

    Sheng, Xiumei; Wang, Zhengxin

    2016-01-01

    Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of symmetrical dimethylation of arginine residues in proteins. WD repeat domain 77 (WDR77), also known as p44, MEP50, or WD45, forms a stoichiometric complex with PRMT5. The PRMT5/p44 complex is required for cellular proliferation of lung and prostate epithelial cells during earlier stages of development and is re-activated during prostate and lung tumorigenesis. The molecular mechanisms by which PRMT5 and p44 promote cellular proliferation are unknown. Expression of PRMT5 and p44 in lung and prostate cancer cells was silenced and their target genes were identified. The regulation of target genes was validated in various cancer cells during lung development and tumorigenesis. Altered expression of target genes was achieved by ectopic cDNA expression and shRNA-mediated silencing. PRMT5 and p44 regulate expression of a specific set of genes encoding growth and anti-growth factors, including receptor tyrosine kinases and antiproliferative proteins. Genes whose expression was suppressed by PRMT5 and p44 encoded anti-growth factors and inhibited cell growth when ectopically expressed. In contrast, genes whose expression was enhanced by PRMT5 and p44 encoded growth factors and increased cell growth when expressed. Altered expression of target genes is associated with re-activation of PRMT5 and p44 during lung tumorigenesis. Our data provide the molecular basis by which PRMT5 and p44 regulate cell growth and lay a foundation for further investigation of their role in lung tumor initiation. The online version of this article (doi:10.1186/s12885-016-2632-3) contains supplementary material, which is available to authorized users

  7. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    International Nuclear Information System (INIS)

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P.; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-01-01

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  8. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  9. 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...

  10. Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors

    Directory of Open Access Journals (Sweden)

    Altenbuchner Josef

    2011-10-01

    Full Text Available Abstract Background Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis. Results Regulation of the promoters of mtlAFD operon (PmtlA and mtlR (PmtlR encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the σA like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR. However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D mutant. Conclusions The mtl operon promoter (PmtlA is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on

  11. Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

    Science.gov (United States)

    Khan, Ahamed; Shrestha, Ankita; Bhuyan, Kashyap; Maiti, Indu B; Dey, Nrisingha

    2018-01-01

    The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

  12. Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

    Science.gov (United States)

    Chen, Hui; Huang, Rui; Zhang, Y-H Percival

    2017-06-01

    The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

  13. Multiple Suboptimal Solutions for Prediction Rules in Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Osamu Komori

    2013-01-01

    Full Text Available This paper discusses mathematical and statistical aspects in analysis methods applied to microarray gene expressions. We focus on pattern recognition to extract informative features embedded in the data for prediction of phenotypes. It has been pointed out that there are severely difficult problems due to the unbalance in the number of observed genes compared with the number of observed subjects. We make a reanalysis of microarray gene expression published data to detect many other gene sets with almost the same performance. We conclude in the current stage that it is not possible to extract only informative genes with high performance in the all observed genes. We investigate the reason why this difficulty still exists even though there are actively proposed analysis methods and learning algorithms in statistical machine learning approaches. We focus on the mutual coherence or the absolute value of the Pearson correlations between two genes and describe the distributions of the correlation for the selected set of genes and the total set. We show that the problem of finding informative genes in high dimensional data is ill-posed and that the difficulty is closely related with the mutual coherence.

  14. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

    Science.gov (United States)

    Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

  15. Identifying Growth Conditions for Nicotiana benthimiana Resulting in Predictable Gene Expression of Promoter-Gus Fusion

    Science.gov (United States)

    Sandoval, V.; Barton, K.; Longhurst, A.

    2012-12-01

    Revoluta (Rev) is a transcription factor that establishes leaf polarity inArabidopsis thaliana. Through previous work in Dr. Barton's Lab, it is known that Revoluta binds to the ZPR3 promoter, thus activating the ZPR3 gene product inArabidopsis thaliana. Using this knowledge, two separate DNA constructs were made, one carrying revgene and in the other, the ZPR3 promoter fussed with the GUS gene. When inoculated in Nicotiana benthimiana (tobacco), the pMDC32 plasmid produces the Rev protein. Rev binds to the ZPR3 promoter thereby activating the transcription of the GUS gene, which can only be expressed in the presence of Rev. When GUS protein comes in contact with X-Gluc it produce the blue stain seen (See Figure 1). In the past, variability has been seen of GUS expression on tobacco therefore we hypothesized that changing the growing conditions and leaf age might improve how well it's expressed.

  16. MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

    Science.gov (United States)

    Xu, Meixiang; Cross, Courtney E; Speidel, Jordan T; Abdel-Rahman, Sherif Z

    2016-10-01

    The O 6 -methylguanine-DNA methyltransferase (MGMT) protein removes O 6 -alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity. In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

  17. c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells

    International Nuclear Information System (INIS)

    Chen, Yinghua; Xu, Jinhua; Borowicz, Stanley; Collins, Cindy; Huo, Dezheng; Olopade, Olufunmilayo I

    2011-01-01

    The BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc. c-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay. Depletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein. Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity. The distal BRCA1 promoter region is associated with c

  18. The application of powerful promoters to enhance gene expression in industrial microorganisms.

    Science.gov (United States)

    Zhou, Shenghu; Du, Guocheng; Kang, Zhen; Li, Jianghua; Chen, Jian; Li, Huazhong; Zhou, Jingwen

    2017-02-01

    Production of useful chemicals by industrial microorganisms has been attracting more and more attention. Microorganisms screened from their natural environment usually suffer from low productivity, low stress resistance, and accumulation of by-products. In order to overcome these disadvantages, rational engineering of microorganisms to achieve specific industrial goals has become routine. Rapid development of metabolic engineering and synthetic biology strategies provide novel methods to improve the performance of industrial microorganisms. Rational regulation of gene expression by specific promoters is essential to engineer industrial microorganisms for high-efficiency production of target chemicals. Identification, modification, and application of suitable promoters could provide powerful switches at the transcriptional level for fine-tuning of a single gene or a group of genes, which are essential for the reconstruction of pathways. In this review, the characteristics of promoters from eukaryotic, prokaryotic, and archaea microorganisms are briefly introduced. Identification of promoters based on both traditional biochemical and systems biology routes are summarized. Besides rational modification, de novo design of promoters to achieve gradient, dynamic, and logic gate regulation are also introduced. Furthermore, flexible application of static and dynamic promoters for the rational engineering of industrial microorganisms is highlighted. From the perspective of powerful promoters in industrial microorganisms, this review will provide an extensive description of how to regulate gene expression in industrial microorganisms to achieve more useful goals.

  19. Four inducible promoters for controlled gene expression in the oleaginous yeast Rhodotorula toruloides

    Directory of Open Access Journals (Sweden)

    Alexander Michael Bedford Johns

    2016-10-01

    Full Text Available Rhodotorula (Rhodosporidium toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70 % of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3 and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.

  20. Multiple post-transcriptional regulatory mechanisms in ferritin gene expression

    International Nuclear Information System (INIS)

    Mattia, E.; Den Blaauwen, J.; Van Renswoude, J.; Ashwell, G.

    1989-01-01

    The authors have investigated the mechanisms involved in the regulation of ferritin biosynthesis in K562 human erythroleukemia cells during prolonged exposure to iron. They show that, upon addition of hemin (an efficient iron donor) to the cell culture, the rate of ferritin biosynthesis reaches a maximum after a few hours and then decreases. During a 24-hr incubation with the iron donor the concentrations of total ferritin heavy (H) and light (L) subunit mRNAs rise 2- to 5-fold and 2- to 3-fold, respectively, over the control values, while the amount of the protein increases 10- to 30-fold. The hemin-induced increment in ferritin subunit mRNA is not prevented by deferoxamine, suggesting that it is not directly mediated by chelatable iron. In vitro nuclear transcription analyses performed on nuclei isolated from control cells and cells grown in the presence of hemin indicate that the rates of synthesis of H- and L-subunit mRNAs remain constant. They conclude that iron-induced ferritin biosynthesis is governed by multiple post-transcriptional regulatory mechanisms. They propose that exposure of cells to iron leads to stabilization of ferritin mRNAs, in addition to activation and translation of stored H-and L-subunit mRNAs

  1. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  2. CCR5 in Multiple Sclerosis : expression, regulation, and modulation by statins

    NARCIS (Netherlands)

    Kuipers, Hedwich Fardau

    2007-01-01

    Activation of microglia, the macrophages of the central nervous system, is a key element in multiple sclerosis (MS) lesion development and is characterized by enhanced expression of both classes of major histocompatibility complex (MHC) molecules. This enhanced expression results from increased

  3. 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...

  4. Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

    Science.gov (United States)

    Amirhaeri, S; Wohlrab, F; Wells, R D

    1995-02-17

    The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

  5. Osteoprotegerin expression in triple-negative breast cancer cells promotes metastasis

    International Nuclear Information System (INIS)

    Weichhaus, Michael; Segaran, Prabu; Renaud, Ashleigh; Geerts, Dirk; Connelly, Linda

    2014-01-01

    Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells

  6. Tissue specific promoters improve the localization of radiation-inducible gene expression

    International Nuclear Information System (INIS)

    Hallahan, Dennis; Kataoka, Yasushi; Kuchibhotla, Jaya; Virudachalam, Subbu; Weichselbaum, Ralph

    1996-01-01

    Purpose: Site-specific activation of gene expression can be achieved by the use of a promoter that is induced by physical agents such as x-rays. The purpose of the present study was to determine whether site-specific activation of gene therapy can also be achieved within the vascular endothelium by use of radiation-inducible promoters. We studied induction of promoter-reporter gene constructs using previously identified radiation-promoters from c-jun, c-fos, Egr-1, ICAM-1, ELAM-1 after transfection into in the vascular endothelium. Methods: The following radiation-inducible genetic constructs were created: The ELAM-1 promoter fragment was cloned into pOGH to obtain the pE-sel(-587 +35)GH reporter construct. The ICAM-1 promoter fragment (-1162/+1) was cloned upstream of the CAT coding region of the pCAT-plasmid (Promega) after removal of the SV40 promoter by Bgl2/Stu1 digestion to create the pBS-CAT plasmid. The 132 to +170 bp segment of the 5' untranslated region of the c-jun promoter was cloned to the CAT reporter gene to create the -132/+170 cjun-CAT. The Egr-1 promoter fragment (-425/+75) was cloned upstream of the CAT coding region to create the pE425-CAT plasmid. Tandem repeats of the AP-1 binding site were cloned upstream of the CAT coding region (3 xTRE-CAT). Tandem repeats of the Egr binding site (EBS) were cloned upstream of the CAT coding region (EBS-CAT). Human vascular endothelial cells from both large vessel and small vessel origin (HUVEC and HMEC), as well as human tumor cell lines were transfected with plasmids -132/+170 cjun-CAT, pE425-CAT, 3 xTRE-CAT, EBS-CAT, pE-sel-GH and pBS-CAT by use of liposomes. Humor tumor cell lines included SQ20B (squamous), RIT3 (sarcoma), and HL525 (leukemia). Each plasmid was cotransfected with a plasmid containing a CMV promoter linked to the LacZ gene (1 μg). Transfected cells were treated with mock irradiation or x-rays. Cell extracts were assayed for reporter gene expression. Results: Radiation-induced gene

  7. ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

    International Nuclear Information System (INIS)

    Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H.; Morton, Derrick J.; Patel, Divya; Joshi, Jugal; Upadhyay, Sunil; Chaudhary, Jaideep

    2016-01-01

    Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. - Highlights: • ID4 expression induces AR expression in PC3 cells, which generally lack AR. • ID4 expression increased apoptosis and decreased cell proliferation and invasion. • Overexpression of ID4 reduces tumor growth of subcutaneous xenografts in vivo. • ID4 induces p21 and FKBP51 expression- co-factors of AR tumor suppressor activity.

  8. ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H.; Morton, Derrick J.; Patel, Divya; Joshi, Jugal; Upadhyay, Sunil; Chaudhary, Jaideep, E-mail: jchaudhary@cau.edu

    2016-09-09

    Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. - Highlights: • ID4 expression induces AR expression in PC3 cells, which generally lack AR. • ID4 expression increased apoptosis and decreased cell proliferation and invasion. • Overexpression of ID4 reduces tumor growth of subcutaneous xenografts in vivo. • ID4 induces p21 and FKBP51 expression- co-factors of AR tumor suppressor activity.

  9. EphrinB1 expression is dysregulated and promotes oncogenic signaling in medulloblastoma.

    Science.gov (United States)

    McKinney, Nicole; Yuan, Liangping; Zhang, Hongying; Liu, Jingbo; Cho, Yoon-Jae; Rushing, Elisabeth; Schniederjan, Matthew; MacDonald, Tobey J

    2015-01-01

    Eph receptors and ephrin ligands are master regulators of oncogenic signaling required for proliferation, migration, and metastasis. Yet, Eph/ephrin expression and activity in medulloblastoma (MB), the most common malignant brain tumor of childhood, remains poorly defined. We hypothesized that Eph/ephrins are differentially expressed by sonic hedgehog (SHH) and non-SHH MB and that specific members contribute to the aggressive phenotype. Affymetrix gene expression profiling of 29 childhood MB, separated into SHH (N = 11) and non-SHH (N = 18), was performed followed by protein validation of selected Eph/ephrins in another 60 MB and two MB cell lines (DAOY, D556). Functional assays were performed using MB cells overexpressing or deleted for selected ephrins. We found EPHB4 and EFNA4 almost exclusively expressed by SHH MB, whereas EPHA2, EPHA8, EFNA1 and EFNA3 are predominantly expressed by non-SHH MB. The remaining family members, except EFNB1, are ubiquitously expressed by over 70-90 % MB, irrespective of subgroup. EFNB1 is the only member differentially expressed by 28 % of SHH and non-SHH MB. Corresponding protein expression for EphB/ephrinB1 and B2 was validated in MB. Only ephrinB2 was also detected in fetal cerebellum, indicating that EphB/ephrinB1 expression is MB-specific. EphrinB1 immunopositivity localizes to tumor cells within MB with the highest proliferative index. EphrinB1 overexpression promotes EphB activation, alters F-actin distribution and morphology, decreases adhesion, and significantly promotes proliferation. Either silencing or overexpression of ephrinB1 impairs migration. These results indicate that EphrinB1 is uniquely dysregulated in MB and promotes oncogenic responses in MB cells, implicating ephrinB1 as a potential target.

  10. An integrated expression atlas of miRNAs and their promoters in human and mouse

    DEFF Research Database (Denmark)

    de Rie, Derek; Abugessaisa, Imad; Alam, Tanvir

    2017-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libr...

  11. Multiple sclerosis patients need and want information on exercise promotion from healthcare providers: a qualitative study.

    Science.gov (United States)

    Learmonth, Yvonne C; Adamson, Brynn C; Balto, Julia M; Chiu, Chung-Yi; Molina-Guzman, Isabel; Finlayson, Marcia; Riskin, Barry J; Motl, Robert W

    2017-08-01

    There is growing recognition of the benefits and safety of exercise and its importance in the comprehensive care of persons with multiple sclerosis (MS), yet uptake is low. We explored the needs and wants of patients with MS regarding exercise promotion through healthcare providers. Participants were adults with MS who had mild-or-moderate disability and a range of exercise levels. All participants lived in the Midwest of the United States. Fifty semi-structured interviews were conducted and analysed using thematic analysis. Two themes emerged, namely interactions between patients and healthcare providers and needs and wants of patients. Analysis of participant accounts illustrate that current exercise promotion by healthcare providers does not meet patient needs and wants. The identified needs and wants of persons with MS involved (i) information and knowledge on the benefits of exercise and exercise prescription, (ii) materials to allow home and community exercise and (iii) tools for initiating and maintaining exercise behaviour. Patients with MS frequently interact with healthcare providers and are generally unsatisfied with exercise promotion during interactions. Healthcare providers can address the low uptake of exercise among persons with MS by acting upon the identified unmet needs involving materials, knowledge and behaviour change strategies for exercise. © 2016 The Authors Health Expectations Published by John Wiley & Sons Ltd.

  12. An APOA1 promoter polymorphism is associated with cognitive performance in patients with multiple sclerosis.

    Science.gov (United States)

    Koutsis, G; Panas, M; Giogkaraki, E; Karadima, G; Sfagos, C; Vassilopoulos, D

    2009-02-01

    Elevated ApoA1 levels have been associated with decreased dementia risk. The A-allele of the APOA1 -75G/A promoter polymorphism has been associated with elevated ApoA1 levels. We sought to investigate the effect of the APOA1 -75G/A promoter polymorphism on cognitive performance in patients with multiple sclerosis (MS). A total of 138 patients with MS and 43 controls were studied and underwent neuropsychological assessment with Rao's Brief Repeatable Battery and the Stroop test. All patients were genotyped for APOA1. APOA1 A-allele carriers displayed superior overall cognitive performance compared with non-carriers (P 0.008) and had a three-fold decrease in the relative risk of overall cognitive impairment (OR 0.29, 95% CI 0.11-0.74). Regarding performance on individual cognitive domains, although APOA1 A-allele carriers performed better than non-carriers on all tests, this was significant only for semantic verbal fluency and the Stroop interference task (P 0.036 and 0.018, respectively). We found an association of the APOA1 -75G/A promoter polymorphism with cognitive performance in MS. This effect was most prominent on semantic verbal fluency and the Stroop interference task.

  13. Differential expression of estrogen receptor α and β isoforms in multiple and solitary leiomyomas

    International Nuclear Information System (INIS)

    Shao, Ruyue; Fang, Liaoqiong; Xing, Ruoxi; Xiong, Yu; Fang, Liaoqiong; Wang, Zhibiao

    2015-01-01

    Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E_2) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E_2 concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes. - Highlights: • In both multiple

  14. Differential expression of estrogen receptor α and β isoforms in multiple and solitary leiomyomas

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Ruyue; Fang, Liaoqiong [State Key Laboratory of Ultrasound Engineering in Medicine Co-Founded by Chongqing and The Ministry of Science and Technology, Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016 (China); Xing, Ruoxi [Institute of Life Science, Chongqing Medical University, Chongqing 400016 (China); Xiong, Yu [Department of Obstetrics and Gynecology, Chongqing Hifu Hospital, Chongqing 401121 (China); Fang, Liaoqiong, E-mail: lqfang06@163.com [State Key Laboratory of Ultrasound Engineering in Medicine Co-Founded by Chongqing and The Ministry of Science and Technology, Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016 (China); Wang, Zhibiao, E-mail: wangzb@cqmu.edu.cn [State Key Laboratory of Ultrasound Engineering in Medicine Co-Founded by Chongqing and The Ministry of Science and Technology, Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering, Chongqing Medical University, Chongqing 400016 (China)

    2015-12-04

    Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E{sub 2}) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E{sub 2} concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes. - Highlights: • In both

  15. Glucocorticoid receptor gene expression and promoter CpG modifications throughout the human brain.

    Science.gov (United States)

    Cao-Lei, Lei; Suwansirikul, Songkiet; Jutavijittum, Prapan; Mériaux, Sophie B; Turner, Jonathan D; Muller, Claude P

    2013-11-01

    Glucocorticoids and the glucocorticoid (GR) and mineralocorticoid (MR) receptors have been implicated in many processes, particularly in negative feedback regulation of the hypothalamic-pituitary-adrenal axis. Epigenetically programmed GR alternative promoter usage underlies transcriptional control of GR levels, generation of GR 3' splice variants, and the overall GC response in the brain. No detailed analysis of GR first exons or GR transcript variants throughout the human brain has been reported. Therefore we investigated post mortem tissues from 28 brain regions of 5 individuals. GR first exons were expressed throughout the healthy human brain with no region-specific usage patterns. First exon levels were highly inter-correlated suggesting that they are co-regulated. GR 3' splice variants (GRα and GR-P) were equally distributed in all regions, and GRβ expression was always low. GR/MR ratios showed significant differences between the 28 tissues with the highest ratio in the pituitary gland. Modification levels of individual CpG dinucleotides, including 5-mC and 5-hmC, in promoters 1D, 1E, 1F, and 1H were low, and diffusely clustered; despite significant heterogeneity between the donors. In agreement with this clustering, sum modification levels rather than individual CpG modifications correlated with GR expression. Two-way ANOVA showed that this sum modification was both promoter and brain region specific, but that there was however no promoter*tissue interaction. The heterogeneity between donors may however hide such an interaction. In both promoters 1F and 1H modification levels correlated with GRα expression suggesting that 5-mC and 5-hmC play an important role in fine tuning GR expression levels throughout the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.

    Directory of Open Access Journals (Sweden)

    Stephanie Jemielity

    2013-03-01

    Full Text Available Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4 specifically bind phosphatidylserine (PS. TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies.

  17. MicroRNA-1185 Promotes Arterial Stiffness though Modulating VCAM-1 and E-Selectin Expression

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    Haoyuan Deng

    2017-04-01

    Full Text Available Background/Aims: Atherosclerosis is the primary cause of cardiovascular ischaemic events; arterial stiffness is a characteristic of the atherosclerotic process. MicroRNAs (miRNAs have been revealed as crucial modulators of atherosclerosis. However, the role of arterial stiffness-related miRNAs in the atherosclerotic process is still unclear. Methods: Four hundred six participants from Northern China were enrolled in this study. Circulating miR-1185 and adhesion molecule levels were measured. Multiple linear regression models were used to evaluate the association of miR-1185 levels with brachial-ankle pulse wave velocity (baPWV and adhesion molecule levels. A mediation analysis was also performed to examine the mediating effect. Cell adhesion molecule levels were measured in primary human umbilical vein endothelial cells (pHUVECs and human umbilical vein smooth cells (HUVSMCs transfected with miR-1185 or co-transfected with a miR-1185 inhibitor. Results: miR-1185 was independently correlated with arterial stiffness. A positive relationship between miR-1185 and vascular cell adhesion molecule-1 (VCAM-1 and E-selectin levels was observed. VCAM- 1 and E-selectin partially mediated the correlation between miR-1185 and arterial stiffness. miR-1185 induced a significant increase in the VCAM-1 and E-selectin levels in pHUVECs and HUVSMCs in vitro. According to our mechanistic analysis, VCAM-1 and E-selectin mediated miR-1185-induced arterial stiffening. Conclusions: miR-1185 modulated the expression of VCAM-1 and E-selectin to promote arterial stiffening, suggesting that miR-1185 plays a crucial role in the development of atherosclerosis and may serve as a novel therapeutic target for atherosclerosis.

  18. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

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    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  19. Gene expression analysis of interferon-beta treatment in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F.; Datta, P.; Larsen, J.

    2008-01-01

    by treatment with IFN-beta. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-beta. After the first injection of IFN-beta, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni......Treatment with interferon-beta (IFN-beta) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-beta. However, to date no molecules have been identified that are stably induced...

  20. TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors.

    Science.gov (United States)

    Cheong, Jit Kong; Gunaratnam, Lakshman; Zang, Zhi Jiang; Yang, Christopher M; Sun, Xiaoming; Nasr, Susan L; Sim, Khe Guan; Peh, Bee Keow; Rashid, Suhaimi Bin Abdul; Bonventre, Joseph V; Salto-Tellez, Manuel; Hsu, Stephen I

    2009-01-20

    Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2). Oncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target. Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro. This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.

  1. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

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    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  2. FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression.

    Science.gov (United States)

    Ikpegbu, Ekele; Basta, Lena; Clements, Dylan N; Fleming, Robert; Vincent, Tonia L; Buttle, David J; Pitsillides, Andrew A; Staines, Katherine A; Farquharson, Colin

    2018-07-01

    E11/podoplanin is critical in the early stages of osteoblast-to-osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblasts to FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p 70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease. © 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  3. FGF‐2 promotes osteocyte differentiation through increased E11/podoplanin expression

    Science.gov (United States)

    Ikpegbu, Ekele; Basta, Lena; Clements, Dylan N.; Fleming, Robert; Vincent, Tonia L.; Buttle, David J.; Pitsillides, Andrew A.; Farquharson, Colin

    2018-01-01

    E11/podoplanin is critical in the early stages of osteoblast‐to‐osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF‐2 on E11‐mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast‐like cells and murine primary osteoblasts to FGF‐2 (10 ng/ml) increased E11 mRNA and protein expression (p 70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF‐2‐related changes in E11 expression and dendrite formation. FGF‐2 strongly activated the ERK signaling pathway in osteoblast‐like cells but inhibition of this pathway did not block the ability of FGF‐2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF‐2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF‐2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease. PMID:29215722

  4. Elevated expression of CD93 promotes angiogenesis and tumor growth in nasopharyngeal carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Lili [Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province (China); Tang, Mingming [Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Cancer Hospital of Nantong University, Nantong, 226361, Jiangsu (China); Zhang, Qicheng; You, Bo; Shan, Ying; Shi, Si; Li, Li [Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province (China); Hu, Songqun, E-mail: hsq@ntu.edu.cn [Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province (China); You, Yiwen, E-mail: youyiwen_nantong@163.com [Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province (China)

    2016-08-05

    CD93, also known as the complement component C1q receptor (C1qRp), has been reported to promote the progression of some cancer types. However, the expression and physiological significance of CD93 in nasopharyngeal carcinoma (NPC) remain largely elusive. In this study, we first examined the expression of CD93 in NPC and experimentally manipulated its expression. We observed that vascular CD93 expression is elevated in NPC and is correlated with T classification, N classification, distant metastasis, clinical stage and poor prognosis (all P < 0.05). In addition, overexpression of CD93 promoted angiogenesis in vitro. What’s more, we found that CD93 was highly expressed in NPC tissues and cells, and the regulation of CD93 on cell proliferation was determined by cell counting kit (CCK)-8 assay and cell cycle analyses. Our findings provide unique insight into the pathogenesis of NPC and underscore the need to explore novel therapeutic targets such as CD93 to improve NPC treatment. -- Highlights: •This is the first research about the relationship between CD93 and nasopharyngeal carcinoma. •We explored the prognostic significance of vascular CD93 expression in nasopharyngeal carcinoma. •We researched on angiogenesis and cell proliferation of nasopharyngeal carcinoma and how CD93 affected them.

  5. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Science.gov (United States)

    Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun

    2015-01-01

    Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  6. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    International Nuclear Information System (INIS)

    Nasab, E. E.; Nasab, E. E.; Hashemi, M.; Rafighdoost, F.

    2016-01-01

    Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent non neoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent non neoplastic tissue (OR=2.30, 95% CI=0.95-5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P=0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients’ clinical characteristics (P>0.05).

  7. Negative effect of the 5'-untranslated leader sequence on Ac transposon promoter expression.

    Science.gov (United States)

    Scortecci, K C; Raina, R; Fedoroff, N V; Van Sluys, M A

    1999-08-01

    Transposable elements are used in heterologous plant hosts to clone genes by insertional mutagenesis. The Activator (Ac) transposable element has been cloned from maize, and introduced into a variety of plants. However, differences in regulation and transposition frequency have been observed between different host plants. The cause of this variability is still unknown. To better understand the activity of the Ac element, we analyzed the Ac promoter region and its 5'-untranslated leader sequence (5' UTL). Transient assays in tobacco NT1 suspension cells showed that the Ac promoter is a weak promoter and its activity was localized by deletion analyses. The data presented here indicate that the core of the Ac promoter is contained within 153 bp fragment upstream to transcription start sites. An important inhibitory effect (80%) due to the presence of the 5' UTL was found on the expression of LUC reporter gene. Here we demonstrate that the presence of the 5' UTL in the constructs reduces the expression driven by either strong or weak promoters.

  8. Surface Expression of TGFβ Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer.

    Science.gov (United States)

    Metelli, Alessandra; Wu, Bill X; Fugle, Caroline W; Rachidi, Saleh; Sun, Shaoli; Zhang, Yongliang; Wu, Jennifer; Tomlinson, Stephen; Howe, Philip H; Yang, Yi; Garrett-Mayer, Elizabeth; Liu, Bei; Li, Zihai

    2016-12-15

    GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGFβ, which is expressed naturally by platelets and regulatory T cells (Treg). Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here, we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGFβ in the tumor microenvironment. We found that human breast, lung, and colon cancers expressed GARP aberrantly. In genetic studies in normal mammary gland epithelial and carcinoma cells, GARP expression increased TGFβ bioactivity and promoted malignant transformation in immunodeficient mice. In breast carcinoma-bearing mice that were immunocompetent, GARP overexpression promoted Foxp3 + Treg activity, which in turn contributed to enhancing cancer progression and metastasis. Notably, administration of a GARP-specific mAb limited metastasis in an orthotopic model of human breast cancer. Overall, these results define the oncogenic effects of the GARP-TGFβ axis in the tumor microenvironment and suggest mechanisms that might be exploited for diagnostic and therapeutic purposes. Cancer Res; 76(24); 7106-17. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. Induction of NFATc2 expression by interleukin 6 promotes T helper type 2 differentiation.

    Science.gov (United States)

    Diehl, Sean; Chow, Chi-Wing; Weiss, Linda; Palmetshofer, Alois; Twardzik, Thomas; Rounds, Laura; Serfling, Edgar; Davis, Roger J; Anguita, Juan; Rincón, Mercedes

    2002-07-01

    Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6-mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6-driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.

  10. PIF4 Promotes Expression of LNG1 and LNG2 to Induce Thermomorphogenic Growth in Arabidopsis

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    Geonhee Hwang

    2017-07-01

    Full Text Available Arabidopsis plants adapt to high ambient temperature by a suite of morphological changes including elongation of hypocotyls and petioles and leaf hyponastic growth. These morphological changes are collectively called thermomorphogenesis and are believed to increase leaf cooling capacity by enhancing transpiration efficiency, thereby increasing tolerance to heat stress. The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4 has been identified as a major regulator of thermomorphogenic growth. Here, we show that PIF4 promotes the expression of two homologous genes LONGIFOLIA1 (LNG1 and LONGIFOLIA2 (LNG2 that have been reported to regulate leaf morphology. ChIP-Seq analyses and ChIP assays showed that PIF4 directly binds to the promoters of both LNG1 and LNG2. The expression of LNG1 and LNG2 is induced by high temperature in wild type plants. However, the high temperature activation of LNG1 and LNG2 is compromised in the pif4 mutant, indicating that PIF4 directly regulates LNG1 and LNG2 expression in response to high ambient temperatures. We further show that the activities of LNGs support thermomorphogenic growth. The expression of auxin biosynthetic and responsive genes is decreased in the lng quadruple mutant, implying that LNGs promote thermomorphogenic growth by activating the auxin pathway. Together, our results demonstrate that LNG1 and LNG2 are directly regulated by PIF4 and are new components for the regulation of thermomorphogenesis.

  11. p8 inhibits the growth of human pancreatic cancer cells and its expression is induced through pathways involved in growth inhibition and repressed by factors promoting cell growth

    Directory of Open Access Journals (Sweden)

    Vasseur Sophie

    2003-11-01

    Full Text Available Abstract Background p8 is a stress-induced protein with multiple functions and biochemically related to the architectural factor HMG-I/Y. We analyzed the expression and function of p8 in pancreatic cancer-derived cells. Methods Expression of p8 was silenced in the human pancreatic cancer cell lines Panc-1 and BxPc-3 by infection with a retrovirus expressing p8 RNA in the antisense orientation. Cell growth was measured in control and p8-silenced cells. Influence on p8 expression of the induction of intracellular pathways promoting cellular growth or growth arrest was monitored. Results p8-silenced cells grew more rapidly than control cells transfected with the empty retrovirus. Activation of the Ras→Raf→MEK→ERK and JNK intracellular pathways down-regulated p8 expression. In addition, the MEK1/2 inhibitor U0126 and the JNK inhibitor SP600125 up-regulates expression of p8. Conversely, p38 or TGFβ-1 induced p8 expression whereas the specific p38 inhibitor SB203580 down-regulated p8 expression. Finally, TGFβ-1 induction was in part mediated through p38. Conclusions p8 inhibits the growth of human pancreatic cancer cells. p8 expression is induced through pathways involved in growth inhibition and repressed by factors that promote cell growth. These results suggest that p8 belongs to a pathway regulating the growth of pancreatic cancer cells.

  12. The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

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    Duensing Stefan

    2011-05-01

    Full Text Available Abstract Background Infection with high-risk human papillomaviruses (HPVs such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication. The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4 protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

  13. Novel strong tissue specific promoter for gene expression in human germ cells

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    Kuzmin Denis

    2010-08-01

    Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

  14. Promotion of Self-Transcendence in a Multiple Sclerosis Peer Support Groups

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    Tahereh Ashktorab

    2014-05-01

    Full Text Available Background: Self-transcendence can organize the challenges of multiple sclerosis patients to achieve and maintain a constant state of well-being and sense of integrity in the disease process. As a research based on self-transcendence didn't done in Iran, the present study was conducted to evaluate the effectiveness of peer groups on promoting selftranscendence level in multiple sclerosis (MS patients. Materials and Methods: This study is a before and after quasi-experimental study that was conducted on 33 patients with confirmed MS participated in three peer support groups: 10 men in male group, 11 women in female group and 12 men and women in mixed group. Eight weekly sessions and each session was 2 hours were held. Data collection tool was Self-Transcendence Scale (STS with 15 item and Cronbach's coefficient was 0.68 that after modifying, it increased to 0.81. Patients completed self administered questionnaires pre- and post of sessions. Results: Results showed that peer support groups promote the self-transcendence (p=0.001 with increases in mean self-transcendence scores in all 3 groups (men group: 0.008, women group 0.005 and mixed group: 0.003. Comparing scores before and after intervention demonstrated that self-transcendence increased equally in all groups. Conclusion: The results showed an improving in self-transcendence in peer support group participants at the end of the intervention. The results can be used in areas of nursing education and management. It is proposed that the self-transcendence assessment to be done in other chronic disease in order to evaluate its efficiency.

  15. Emerging importance of helicases in plant stress tolerance: characterization of Oryza sativa repair helicase XPB2 promoter and its functional validation in tobacco under multiple stresses

    Directory of Open Access Journals (Sweden)

    Shailendra eRaikwar

    2015-12-01

    Full Text Available Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. DNA hHelicases, the unique molecular motors, are emerged as potentialprospective molecules to engineer stress tolerance in plants and are involved in a variety of DNA nucleic acid metabolismc processes including DNA repair. The DNA repair helicase, OsXPB2 is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress The analysis of promoter sequence from plant genome is important in understanding the gene regulation. Hereconditions. Here, we report the in silico analysis of novel stress inducible promoter of rice Oryza sativa OsXPB2 (OsXPB2. gene is reported. The in vivo validation of functionality/activity of novel stress inducible promoter of rice OsXPB2 gene promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. Our resultsThe present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration or cold and hormone (Auxin, ABA or MeJA induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA or ABA responsive, respectively. Functional analysis was done by Agrobacterium-transient assays using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present

  16. Lack of death receptor 4 (DR4) expression through gene promoter methylation in gastric carcinoma.

    Science.gov (United States)

    Lee, Kyung Hwa; Lim, Sang Woo; Kim, Ho Gun; Kim, Dong Yi; Ryu, Seong Yeob; Joo, Jae Kyun; Kim, Jung Chul; Lee, Jae Hyuk

    2009-07-01

    To determine the underlying mechanism for the differential expression, the extent of promoter methylation in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-related genes acting downstream of TRAIL was examined in early and advanced gastric carcinomas. The extent of promoter methylation in the DR4, DR5, DcR1, DcR2, and CASP8 genes was quantified using bisulfite modification and methylation-specific polymerase chain reaction. The promoters for DcR1, DcR2, and CASP8 were largely unmethylated in early gastric carcinoma, advanced gastric carcinoma, and controls, with no significant difference among them. Protein levels of DR4, DcR1, and DcR2 as revealed by immunohistochemistry correlated with the extent of the respective promoter methylation (P < 0.05 in all cases). Hypomethylation, rather than hypermethylation, of the DR4 promoter was noted in invasive gastric malignancies, with statistical significance (P = 0.003). The promoter methylation status of TRAIL receptors in gastric carcinoma may have clinical implications for improving therapeutic strategies in patients with gastric carcinoma.

  17. Light-regulated promoters for tunable, temporal, and affordable control of fungal gene expression.

    Science.gov (United States)

    Fuller, Kevin K; Dunlap, Jay C; Loros, Jennifer J

    2018-05-01

    Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.

  18. Cloning of the chrysanthemum UEP1 promoter and comparative expression in leaves and ray and disc florets of Dendranthema grandiflora

    NARCIS (Netherlands)

    Annadana, S.; Beekwilder, M.J.; Kuipers, G.; Visser, P.B.; Outchkourov, N.; Pereira, A.; Udayakumar, M.; Jongsma, M.A.

    2002-01-01

    To attain high transgene expression in petal tissue of ray florets of chrysanthemum an endogenous ubiquitin extension protein (UEP1) promoter was cloned and tested with the β-glucuronidase (GUS) reporter gene. Expression levels were compared with four heterologous promoters: chalcone synthase

  19. Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression.

    Science.gov (United States)

    Zu, Xuyu; Ma, Jun; Liu, Hongxia; Liu, Feng; Tan, Chunyan; Yu, Lingling; Wang, Jue; Xie, Zhenhua; Cao, Deliang; Jiang, Yuyang

    2011-03-10

    Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms. Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene. Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P Pokemon induces survivin expression by binding to the GT boxes in its promoter. Pokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy.

  20. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    International Nuclear Information System (INIS)

    Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing

    2012-01-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  1. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wei [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Guifang [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Cai, Xiaojun [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Yang, Jing, E-mail: yangjingliu@yahoo.com.cn [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  2. Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis

    DEFF Research Database (Denmark)

    Pedersen, Annemarie Aarup; Pedersen, Tanja X; Junker, Nanna

    2016-01-01

    transplanted with bone marrow from mice with HIF-1α deficiency in the myeloid cells or control bone marrow. The HIF-1α deficiency in myeloid cells reduced atherosclerosis in aorta of the Ldlr(-/-) recipient mice by ≈72% (P=0.006).In vitro, HIF-1α-deficient macrophages displayed decreased differentiation...... to proinflammatory M1 macrophages and reduced expression of inflammatory genes. HIF-1α deficiency also affected glucose uptake, apoptosis, and migratory abilities of the macrophages. CONCLUSIONS: HIF-1α expression in macrophages affects their intrinsic inflammatory profile and promotes development of atherosclerosis....

  3. Paradoxical expression of INK4c in proliferative multiple myeloma tumors: bi-allelic deletion vs increased expression

    Directory of Open Access Journals (Sweden)

    Hanamura Ichiro

    2006-10-01

    Full Text Available Abstract Background A high proliferative capacity of tumor cells usually is associated with shortened patient survival. Disruption of the RB pathway, which is critically involved in regulating the G1 to S cell cycle transition, is a frequent target of oncogenic events that are thought to contribute to increased proliferation during tumor progression. Previously, we determined that p18INK4c, an essential gene for normal plasma cell differentiation, was bi-allelically deleted in five of sixteen multiple myeloma (MM cell lines. The present study was undertaken to investigate a possible role of p18INK4c in increased proliferation of myeloma tumors as they progress. Results Thirteen of 40 (33% human myeloma cell lines do not express normal p18INK4c, with bi-allelic deletion of p18 in twelve, and expression of a mutated p18 fragment in one. Bi-allelic deletion of p18, which appears to be a late progression event, has a prevalence of about 2% in 261 multiple myeloma (MM tumors, but the prevalence is 6 to10% in the 50 tumors with a high expression-based proliferation index. Paradoxically, 24 of 40 (60% MM cell lines, and 30 of 50 (60% MM tumors with a high proliferation index express an increased level of p18 RNA compared to normal bone marrow plasma cells, whereas this occurs in only five of the 151 (3% MM tumors with a low proliferation index. Tumor progression is often accompanied by increased p18 expression and an increased proliferation index. Retroviral-mediated expression of exogenous p18 results in marked growth inhibition in three MM cell lines that express little or no endogenous p18, but has no effect in another MM cell line that already expresses a high level of p18. Conclusion Paradoxically, although loss of p18 appears to contribute to increased proliferation of nearly 10% of MM tumors, most MM cell lines and proliferative MM tumors have increased expression of p18. Apart from a small fraction of cell lines and tumors that have inactivated

  4. Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

    Science.gov (United States)

    Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

    2017-07-25

    Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of

  5. Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

    Directory of Open Access Journals (Sweden)

    Andrew D Beggs

    2013-05-01

    Full Text Available Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

  6. Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia.

    Science.gov (United States)

    Oram, S H; Thoms, J; Sive, J I; Calero-Nieto, F J; Kinston, S J; Schütte, J; Knezevic, K; Lock, R B; Pimanda, J E; Göttgens, B

    2013-06-01

    LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.

  7. CXCL17 expression by tumor cells recruits CD11b+Gr1 high F4/80- cells and promotes tumor progression.

    Directory of Open Access Journals (Sweden)

    Aya Matsui

    Full Text Available BACKGROUND: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1, recruits immature myeloid-derived cells and enhances early tumor progression. METHODOLOGY/PRINCIPAL FINDINGS: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+Gr1(+ myeloid-derived cells at tumor sites in mice and promoted CD31(+ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+Gr1(highF4/80(- cells (≈ 90% with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+Gr1(+ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

  8. Bayesian models and meta analysis for multiple tissue gene expression data following corticosteroid administration

    Directory of Open Access Journals (Sweden)

    Kelemen Arpad

    2008-08-01

    Full Text Available Abstract Background This paper addresses key biological problems and statistical issues in the analysis of large gene expression data sets that describe systemic temporal response cascades to therapeutic doses in multiple tissues such as liver, skeletal muscle, and kidney from the same animals. Affymetrix time course gene expression data U34A are obtained from three different tissues including kidney, liver and muscle. Our goal is not only to find the concordance of gene in different tissues, identify the common differentially expressed genes over time and also examine the reproducibility of the findings by integrating the results through meta analysis from multiple tissues in order to gain a significant increase in the power of detecting differentially expressed genes over time and to find the differential differences of three tissues responding to the drug. Results and conclusion Bayesian categorical model for estimating the proportion of the 'call' are used for pre-screening genes. Hierarchical Bayesian Mixture Model is further developed for the identifications of differentially expressed genes across time and dynamic clusters. Deviance information criterion is applied to determine the number of components for model comparisons and selections. Bayesian mixture model produces the gene-specific posterior probability of differential/non-differential expression and the 95% credible interval, which is the basis for our further Bayesian meta-inference. Meta-analysis is performed in order to identify commonly expressed genes from multiple tissues that may serve as ideal targets for novel treatment strategies and to integrate the results across separate studies. We have found the common expressed genes in the three tissues. However, the up/down/no regulations of these common genes are different at different time points. Moreover, the most differentially expressed genes were found in the liver, then in kidney, and then in muscle.

  9. HMGA2 promotes adipogenesis by activating C/EBPβ-mediated expression of PPARγ

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Yang; Shen, Wanjing; Ma, Lili; Zhao, Ming; Zheng, Jiachen [Diabetes Center, and Zhejiang Provincial Key Laboratory of Pathophysiology, Institute of Biochemistry and Molecular Biology, School of Medicine, Ningbo University, Ningbo 315211 (China); Bu, Shizhong, E-mail: bushizhong@nbu.edu.cn [Diabetes Center, and Zhejiang Provincial Key Laboratory of Pathophysiology, Institute of Biochemistry and Molecular Biology, School of Medicine, Ningbo University, Ningbo 315211 (China); Hino, Shinjiro [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 860-0811 (Japan); Nakao, Mitsuyoshi, E-mail: mnakao@gpo.kumamoto-u.ac.jp [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 860-0811 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development, Tokyo (Japan)

    2016-04-15

    Adipogenesis is orchestrated by a highly ordered network of transcription factors including peroxisome-proliferator activated receptor-gamma (PPARγ) and CCAAT-enhancer binding protein (C/EBP) family proteins. High mobility group protein AT-hook 2 (HMGA2), an architectural transcription factor, has been reported to play an essential role in preadipocyte proliferation, and its overexpression has been implicated in obesity in mice and humans. However, the direct role of HMGA2 in regulating the gene expression program during adipogenesis is not known. Here, we demonstrate that HMGA2 is required for C/EBPβ-mediated expression of PPARγ, and thus promotes adipogenic differentiation. We observed a transient but marked increase of Hmga2 transcript at an early phase of differentiation of mouse 3T3-L1 preadipocytes. Importantly, Hmga2 knockdown greatly impaired adipocyte formation, while its overexpression promoted the formation of mature adipocytes. We found that HMGA2 colocalized with C/EBPβ in the nucleus and was required for the recruitment of C/EBPβ to its binding element at the Pparγ2 promoter. Accordingly, HMGA2 and C/EBPβ cooperatively enhanced the Pparγ2 promoter activity. Our results indicate that HMGA2 is an essential constituent of the adipogenic transcription factor network, and thus its function may be affected during the course of obesity. - Highlights: • Overexpression of HMGA2 has been implicated in obesity in mice and humans. • HMGA2 is required for adipocyte formation. • HMGA2 colocalizes with C/EBPβ and is required for C/EBPβ recruitment to Pparγ2 promoter. • HMGA2 and C/EBPβ cooperatively enhance the Pparγ2 promoter activity.

  10. HMGA2 promotes adipogenesis by activating C/EBPβ-mediated expression of PPARγ

    International Nuclear Information System (INIS)

    Xi, Yang; Shen, Wanjing; Ma, Lili; Zhao, Ming; Zheng, Jiachen; Bu, Shizhong; Hino, Shinjiro; Nakao, Mitsuyoshi

    2016-01-01

    Adipogenesis is orchestrated by a highly ordered network of transcription factors including peroxisome-proliferator activated receptor-gamma (PPARγ) and CCAAT-enhancer binding protein (C/EBP) family proteins. High mobility group protein AT-hook 2 (HMGA2), an architectural transcription factor, has been reported to play an essential role in preadipocyte proliferation, and its overexpression has been implicated in obesity in mice and humans. However, the direct role of HMGA2 in regulating the gene expression program during adipogenesis is not known. Here, we demonstrate that HMGA2 is required for C/EBPβ-mediated expression of PPARγ, and thus promotes adipogenic differentiation. We observed a transient but marked increase of Hmga2 transcript at an early phase of differentiation of mouse 3T3-L1 preadipocytes. Importantly, Hmga2 knockdown greatly impaired adipocyte formation, while its overexpression promoted the formation of mature adipocytes. We found that HMGA2 colocalized with C/EBPβ in the nucleus and was required for the recruitment of C/EBPβ to its binding element at the Pparγ2 promoter. Accordingly, HMGA2 and C/EBPβ cooperatively enhanced the Pparγ2 promoter activity. Our results indicate that HMGA2 is an essential constituent of the adipogenic transcription factor network, and thus its function may be affected during the course of obesity. - Highlights: • Overexpression of HMGA2 has been implicated in obesity in mice and humans. • HMGA2 is required for adipocyte formation. • HMGA2 colocalizes with C/EBPβ and is required for C/EBPβ recruitment to Pparγ2 promoter. • HMGA2 and C/EBPβ cooperatively enhance the Pparγ2 promoter activity.

  11. Expression in Arabidopsis of a nucellus-specific promoter from watermelon (Citrullus lanatus).

    Science.gov (United States)

    Dwivedi, Krishna K; Roche, Dominique; Carman, John G

    2010-11-01

    Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Pei-Yi Wu

    Full Text Available Neuroblastoma (NB is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

  13. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    BACKGROUND: Interferon (IFN)-beta therapy in multiple sclerosis (MS) has been suggested to promote a deviation from T lymphocyte production of pathogenic Th1 cytokines to less detrimental Th2 cytokines, but this is still controversial. We studied patterns of in vivo blood mononuclear cell (MNC...... of any Th1 or Th2 cytokines. The largest changes in cytokine mRNA levels occurred early (~9-12 h) after an IFN-beta injection. CONCLUSION: We found no evidence of a Th1- or Th2-mRNA-promoting effect of IFN-beta therapy. The therapeutic effect of IFN-beta is more likely attributable to the induction...

  14. Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter

    Directory of Open Access Journals (Sweden)

    Wolfgang Boecker

    2004-04-01

    Full Text Available Background: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. Methods: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc reporter gene (Ad.4 × MLC250.Luc. Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 × MLC.Luc or control vectors. For in vivo testing, Ad.4 × MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. Results: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells was 20.4 versus 0.9 (Ad.4 × MLC250.Luc vs. Ad.CMV. In vivo: Ad.4 × MLC250.Luc significantly reduced luc activity in liver (38.4-fold, lung (16.1-fold, and kidney (21.8-fold versus Ad.CMV (p = .01; whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 × MLC.Luc versus 1:1.4 (Ad.CMV.Luc. Discussion: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.

  15. Analyzing Multiple-Probe Microarray: Estimation and Application of Gene Expression Indexes

    KAUST Repository

    Maadooliat, Mehdi

    2012-07-26

    Gene expression index estimation is an essential step in analyzing multiple probe microarray data. Various modeling methods have been proposed in this area. Amidst all, a popular method proposed in Li and Wong (2001) is based on a multiplicative model, which is similar to the additive model discussed in Irizarry et al. (2003a) at the logarithm scale. Along this line, Hu et al. (2006) proposed data transformation to improve expression index estimation based on an ad hoc entropy criteria and naive grid search approach. In this work, we re-examined this problem using a new profile likelihood-based transformation estimation approach that is more statistically elegant and computationally efficient. We demonstrate the applicability of the proposed method using a benchmark Affymetrix U95A spiked-in experiment. Moreover, We introduced a new multivariate expression index and used the empirical study to shows its promise in terms of improving model fitting and power of detecting differential expression over the commonly used univariate expression index. As the other important content of the work, we discussed two generally encountered practical issues in application of gene expression index: normalization and summary statistic used for detecting differential expression. Our empirical study shows somewhat different findings from the MAQC project (MAQC, 2006).

  16. Biased Iglambda expression in hypermutated IgD multiple myelomas does not result from receptor revision

    NARCIS (Netherlands)

    van der Burg, M.; Bende, R. J.; Aarts, W. M.; Langerak, A. W.; van Dongen, J. J. M.; van Noesel, C. J. M.

    2002-01-01

    Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal

  17. Gene expression profiling for molecular classification of multiple myeloma in newly diagnosed patients

    NARCIS (Netherlands)

    Broyl, Annemiek; Hose, Dirk; Lokhorst, Henk; de Knegt, Yvonne; Peeters, Justine; Jauch, Anna; Bertsch, Uta; Buijs, Arjan; Stevens-Kroef, Marian; Beverloo, H. Berna; Vellenga, Edo; Zweegman, Sonja; Kersten, Marie-Josée; van der Holt, Bronno; el Jarari, Laila; Mulligan, George; Goldschmidt, Hartmut; van Duin, Mark; Sonneveld, Pieter

    2010-01-01

    To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138(+) plasma cells of 320 newly diagnosed myeloma patients included in the Dutch-Belgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6

  18. Glatiramer acetate treatment normalizes deregulated microRNA expression in relapsing remitting multiple sclerosis.

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    Anne Waschbisch

    Full Text Available The expression of selected microRNAs (miRNAs known to be involved in the regulation of immune responses was analyzed in 74 patients with relapsing remitting multiple sclerosis (RRMS and 32 healthy controls. Four miRNAs (miR-326, miR-155, miR-146a, miR-142-3p were aberrantly expressed in peripheral blood mononuclear cells from RRMS patients compared to controls. Although expression of these selected miRNAs did not differ between treatment-naïve (n = 36 and interferon-beta treated RRMS patients (n = 18, expression of miR-146a and miR-142-3p was significantly lower in glatiramer acetate (GA treated RRMS patients (n = 20 suggesting that GA, at least in part, restores the expression of deregulated miRNAs in MS.

  19. Visual Comparison of Multiple Gene Expression Datasets in a Genomic Context

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    Borowski Krzysztof

    2008-06-01

    Full Text Available The need for novel methods of visualizing microarray data is growing. New perspectives are beneficial to finding patterns in expression data. The Bluejay genome browser provides an integrative way of visualizing gene expression datasets in a genomic context. We have now developed the functionality to display multiple microarray datasets simultaneously in Bluejay, in order to provide researchers with a comprehensive view of their datasets linked to a graphical representation of gene function. This will enable biologists to obtain valuable insights on expression patterns, by allowing them to analyze the expression values in relation to the gene locations as well as to compare expression profiles of related genomes or of di erent experiments for the same genome.

  20. Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

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    Viana Antonio AB

    2011-11-01

    Full Text Available Abstract Background Cotton (Gossypium spp. is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2 family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2 and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2 and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

  1. Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

    Science.gov (United States)

    Viana, Antonio A B; Fragoso, Rodrigo R; Guimarães, Luciane M; Pontes, Naiara; Oliveira-Neto, Osmundo B; Artico, Sinara; Nardeli, Sarah M; Alves-Ferreira, Marcio; Batista, João A N; Silva, Maria C M; Grossi-de-Sa, Maria F

    2011-11-24

    Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

  2. Cell-Specific PEAR1 Methylation Studies Reveal a Locus that Coordinates Expression of Multiple Genes

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    Benedetta Izzi

    2018-04-01

    Full Text Available Chromosomal interactions connect distant enhancers and promoters on the same chromosome, activating or repressing gene expression. PEAR1 encodes the Platelet-Endothelial Aggregation Receptor 1, a contact receptor involved in platelet function and megakaryocyte and endothelial cell proliferation. PEAR1 expression during megakaryocyte differentiation is controlled by DNA methylation at its first CpG island. We identified a PEAR1 cell-specific methylation sensitive region in endothelial cells and megakaryocytes that showed strong chromosomal interactions with ISGL20L2, RRNAD1, MRLP24, HDGF and PRCC, using available promoter capture Hi-C datasets. These genes are involved in ribosome processing, protein synthesis, cell cycle and cell proliferation. We next studied the methylation and expression profile of these five genes in Human Umbilical Vein Endothelial Cells (HUVECs and megakaryocyte precursors. While cell-specific PEAR1 methylation corresponded to variability in expression for four out of five genes, no methylation change was observed in their promoter regions across cell types. Our data suggest that PEAR1 cell-type specific methylation changes may control long distance interactions with other genes. Further studies are needed to show whether such interaction data might be relevant for the genome-wide association data that showed a role for non-coding PEAR1 variants in the same region and platelet function, platelet count and cardiovascular risk.

  3. Cytokeratin 19 promoter directs the expression of Cre recombinase in various epithelia of transgenic mice.

    Science.gov (United States)

    Zhao, Gui-Feng; Zhao, Shuang; Liu, Jia-Jie; Wu, Ji-Cheng; He, Hao-Yu; Ding, Xiao-Qing; Yu, Xue-Wen; Huang, Ke-Qiang; Li, Zhi-Jie; Zheng, Hua-Chuan

    2017-03-14

    Cytokeratin 19 (K19) is expressed in various differentiated cells, including gastric, intestinal and bronchial epithelial cells, and liver duct cells. Here, we generated a transgenic mouse line, K19-Cre, in which the expression of Cre recombinase was controlled by the promoter of K19. To test the tissue distribution and excision activity of Cre recombinase, K19-Cre transgenic mice were bred with Rosa26 reporter strain and a mouse strain that carries PTEN conditional alleles (PTENLoxp/Loxp). At mRNA level, Cre was strongly expressed in the stomach, lung and intestine, while in stomach, lung, and liver at protein level. The immunoreactivity to Cre was strongly observed the cytoplasm of gastric, bronchial and intestinal epithelial cells. Cre activity was detectable in gastric, bronchial and intestinal epithelial cells, according to LacZ staining. In K19-Cre/PTEN Loxp/Loxp mice, PTEN was abrogated in stomach, intestine, lung, liver and breast, the former two of which were verified by in situ PCR. There appeared breast cancer with PTEN loss. These data suggest that K19 promoter may be a useful tool to study the pathophysiological functions of cytokeratin 19-positive cells, especially gastrointestinal epithelial cells. Cell specificity of neoplasia is not completely attributable to the cell-specific expression of oncogenes and cell-specific loss of tumor suppressor genes.

  4. Vitamin K2 promotes mesenchymal stem cell differentiation by inhibiting miR‑133a expression.

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    Zhang, Yuelei; Weng, Shiyang; Yin, Junhui; Ding, Hao; Zhang, Changqing; Gao, Youshui

    2017-05-01

    Vitamin K2 has been demonstrated to promote the osteogenic differentiation of mesenchymal stem cells; however, the mechanisms underlying this effect remain unclear. As microRNA (miR)‑133a has been identified as a negative regulator of osteogenic differentiation, the present study hypothesized that vitamin K2 promoted osteogenesis by inhibiting miR‑133a. Using human bone marrow stromal cells (hBMSCs) overexpressing miR‑133a, or a control, the expression levels of osteogenesis‑associated proteins, including runt‑related transcription factor 2, alkaline phosphatase and osteocalcin, were analyzed. miR‑133a significantly suppressed the osteogenic differentiation of hBMSCs. To determine the effect of vitamin K2 on miR‑133a expression and osteogenesis, hBMSCs were treated with vitamin K2. Vitamin K2 inhibited miR‑133a expression, which was accompanied by enhanced osteogenic differentiation. Furthermore, the expression levels of vitamin K epoxide reductase complex subunit 1, the key protein in γ‑carboxylation, were downregulated by miR‑133a overexpression and upregulated by vitamin K2 treatment, indicating a positive feedback on γ‑carboxylation. The results of the present study suggested that vitamin K2 targets miR‑133a to regulate osteogenesis.

  5. The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona.

    Science.gov (United States)

    Gaji, Rajshekhar Y; Howe, Daniel K

    2009-07-01

    The apicomplexan parasite Sarcocystis neurona undergoes a complex process of intracellular development, during which many genes are temporally regulated. The described study was undertaken to begin identifying the basic promoter elements that control gene expression in S. neurona. Sequence analysis of the 5'-flanking region of five S. neurona genes revealed a conserved heptanucleotide motif GAGACGC that is similar to the WGAGACG motif described upstream of multiple genes in Toxoplasma gondii. The promoter region for the major surface antigen gene SnSAG1, which contains three heptanucleotide motifs within 135 bases of the transcription start site, was dissected by functional analysis using a dual luciferase reporter assay. These analyses revealed that a minimal promoter fragment containing all three motifs was sufficient to drive reporter molecule expression, with the presence and orientation of the 5'-most heptanucleotide motif being absolutely critical for promoter function. Further studies should help to identify additional sequence elements important for promoter function and for controlling gene expression during intracellular development by this apicomplexan pathogen.

  6. HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

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    Chen-Ye Shi

    2013-08-01

    Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

  7. NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

    International Nuclear Information System (INIS)

    Zhao Guohua; Shi Lingfang; Qiu Daoming; Hu Hong; Kao, Peter N.

    2005-01-01

    NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2

  8. Light-promoted rhodopsin expression and starvation survival in the marine dinoflagellate Oxyrrhis marina.

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    Zhiling Guo

    Full Text Available The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR and sensory type (SR rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.

  9. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

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    Zhichun Zhang

    Full Text Available Mutation of distal-less homeobox 3 (DLX3 is responsible for human tricho-dento-osseous syndrome (TDO with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  10. p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

    Science.gov (United States)

    Di Vinci, Angela; Perdelli, Luisa; Banelli, Barbara; Salvi, Sandra; Casciano, Ida; Gelvi, Ilaria; Allemanni, Giorgio; Margallo, Edoardo; Gatteschi, Beatrice; Romani, Massimo

    2005-04-10

    The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma. (c) 2004 Wiley-Liss, Inc.

  11. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

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    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  12. Expression of the central growth regulator BIG BROTHER is regulated by multiple cis-elements

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    Breuninger Holger

    2012-03-01

    Full Text Available Abstract Background Much of the organismal variation we observe in nature is due to differences in organ size. The observation that even closely related species can show large, stably inherited differences in organ size indicates a strong genetic component to the control of organ size. Despite recent progress in identifying factors controlling organ growth in plants, our overall understanding of this process remains limited, partly because the individual factors have not yet been connected into larger regulatory pathways or networks. To begin addressing this aim, we have studied the upstream regulation of expression of BIG BROTHER (BB, a central growth-control gene in Arabidopsis thaliana that prevents overgrowth of organs. Final organ size and BB expression levels are tightly correlated, implying the need for precise control of its expression. BB expression mirrors proliferative activity, yet the gene functions to limit proliferation, suggesting that it acts in an incoherent feedforward loop downstream of growth activators to prevent over-proliferation. Results To investigate the upstream regulation of BB we combined a promoter deletion analysis with a phylogenetic footprinting approach. We were able to narrow down important, highly conserved, cis-regulatory elements within the BB promoter. Promoter sequences of other Brassicaceae species were able to partially complement the A. thaliana bb-1 mutant, suggesting that at least within the Brassicaceae family the regulatory pathways are conserved. Conclusions This work underlines the complexity involved in precise quantitative control of gene expression and lays the foundation for identifying important upstream regulators that determine BB expression levels and thus final organ size.

  13. Andrographolide protects mouse astrocytes against hypoxia injury by promoting autophagy and S100B expression

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    Juan Du

    2018-04-01

    Full Text Available Andrographolide (ANDRO has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains were subjected to 3 and 21% of O2 for various times (0–12 h to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.

  14. Fine-mapping identifies multiple prostate cancer risk loci at 5p15, one of which associates with TERT expression

    Science.gov (United States)

    Kote-Jarai, Zsofia; Saunders, Edward J.; Leongamornlert, Daniel A.; Tymrakiewicz, Malgorzata; Dadaev, Tokhir; Jugurnauth-Little, Sarah; Ross-Adams, Helen; Al Olama, Ali Amin; Benlloch, Sara; Halim, Silvia; Russel, Roslin; Dunning, Alison M.; Luccarini, Craig; Dennis, Joe; Neal, David E.; Hamdy, Freddie C.; Donovan, Jenny L.; Muir, Ken; Giles, Graham G.; Severi, Gianluca; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A.; Schumacher, Fredrick; Henderson, Brian E.; Le Marchand, Loic; Lindstrom, Sara; Kraft, Peter; Hunter, David J.; Gapstur, Susan; Chanock, Stephen; Berndt, Sonja I.; Albanes, Demetrius; Andriole, Gerald; Schleutker, Johanna; Weischer, Maren; Canzian, Federico; Riboli, Elio; Key, Tim J.; Travis, Ruth C.; Campa, Daniele; Ingles, Sue A.; John, Esther M.; Hayes, Richard B.; Pharoah, Paul; Khaw, Kay-Tee; Stanford, Janet L.; Ostrander, Elaine A.; Signorello, Lisa B.; Thibodeau, Stephen N.; Schaid, Dan; Maier, Christiane; Vogel, Walther; Kibel, Adam S.; Cybulski, Cezary; Lubinski, Jan; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y.; Kaneva, Radka; Batra, Jyotsna; Spurdle, Amanda; Clements, Judith A.; Teixeira, Manuel R.; Govindasami, Koveela; Guy, Michelle; Wilkinson, Rosemary A.; Sawyer, Emma J.; Morgan, Angela; Dicks, Ed; Baynes, Caroline; Conroy, Don; Bojesen, Stig E.; Kaaks, Rudolf; Vincent, Daniel; Bacot, François; Tessier, Daniel C.; Easton, Douglas F.; Eeles, Rosalind A.

    2013-01-01

    Associations between single nucleotide polymorphisms (SNPs) at 5p15 and multiple cancer types have been reported. We have previously shown evidence for a strong association between prostate cancer (PrCa) risk and rs2242652 at 5p15, intronic in the telomerase reverse transcriptase (TERT) gene that encodes TERT. To comprehensively evaluate the association between genetic variation across this region and PrCa, we performed a fine-mapping analysis by genotyping 134 SNPs using a custom Illumina iSelect array or Sequenom MassArray iPlex, followed by imputation of 1094 SNPs in 22 301 PrCa cases and 22 320 controls in The PRACTICAL consortium. Multiple stepwise logistic regression analysis identified four signals in the promoter or intronic regions of TERT that independently associated with PrCa risk. Gene expression analysis of normal prostate tissue showed evidence that SNPs within one of these regions also associated with TERT expression, providing a potential mechanism for predisposition to disease. PMID:23535824

  15. Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic.

    Science.gov (United States)

    Pugach, Emily K; Richmond, Phillip A; Azofeifa, Joseph G; Dowell, Robin D; Leinwand, Leslie A

    2015-09-01

    Studying the importance of genetic factors in a desired cell type or tissue necessitates the use of precise genetic tools. With the introduction of bacteriophage Cre recombinase/loxP mediated DNA editing and promoter-specific Cre expression, it is feasible to generate conditional knockout mice in which particular genes are disrupted in a cell type-specific manner in vivo. In cardiac myocytes, this is often achieved through α-myosin heavy chain promoter (αMyHC)-driven Cre expression in conjunction with a loxP-site flanked gene of interest. Recent studies in other cell types demonstrate toxicity of Cre expression through induction of DNA damage. However, it is unclear to what extent the traditionally used αMyHC-Cre line [1] may exhibit cardiotoxicity. Further, the genotype of αMyHC-Cre(+/-) is not often included as a control group in cardiac myocyte-specific knockout studies. Here we present evidence that these αMyHC-Cre(+/-) mice show molecular signs of cardiac toxicity by 3months of age and exhibit decreased cardiac function by 6months of age compared to wild-type littermates. Hearts from αMyHC-Cre(+/-) mice also display evidence of fibrosis, inflammation, and DNA damage. Interestingly, some of the early functional changes observed in αMyHC-Cre(+/-) mice are sexually dimorphic. Given the high level of Cre recombinase expression resulting from expression from the αMyHC promoter, we asked if degenerate loxP-like sites naturally exist in the mouse genome and if so, whether they are affected by Cre in the absence of canonical loxP-sites. Using a novel bioinformatics search tool, we identified 619 loxP-like sites with 4 or less mismatches to the canonical loxP-site. 227 sites overlapped with annotated genes and 55 of these genes were expressed in cardiac muscle. Expression of ~26% of the 27 genes tested was disrupted in αMyHC-Cre(+/-) mice indicating potential targeting by Cre. Taken together, these results highlight both the importance of using αMyHC-Cre mice

  16. Heme-Oxygenase-1 Expression Contributes to the Immunoregulation Induced by Fasciola hepatica and Promotes Infection

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    Paula Carasi

    2017-07-01

    Full Text Available Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1, the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand, cells expressing intermediate levels of HO-1 expressed high levels of F4/80, CD68, Ly6C, and FIZZ-1, indicating that they might correspond to alternatively activated macrophages. Furthermore, the pharmacological induction of HO-1 with the synthetic metalloporphyrin CoPP promoted F. hepatica infection increasing the clinical signs associated with the disease. In contrast, treatment with the HO-1 inhibitor SnPP protected mice from parasite infection, indicating that HO-1 plays an essential role during F. hepatica infection. Finally, HO-1 expression during F. hepatica infection was associated with TGFβ and IL-10 levels in liver and peritoneum, suggesting that HO-1 controls the expression of these immunoregulatory cytokines during infection favoring parasite survival in the host. These results contribute to the elucidation of the immunoregulatory mechanisms induced by F. hepatica in the host and provide alternative checkpoints to control fasciolosis.

  17. Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

    International Nuclear Information System (INIS)

    Lague, Eric; Tremblay, Jacques J.

    2009-01-01

    There are increasing evidence in the literature reporting the detrimental effects of endocrine disruptors on the development and function of the male reproductive system. One example is cryptorchidism, or undescended testis, caused by exposure to excessive estrogens. Estrogens, acting through the estrogen receptor α (ERα), have been shown to repress expression of the gene encoding insulin-like 3 (INSL3), a small peptide produced by testicular Leydig cells that is essential for normal testis descent. The molecular mechanism of estrogen/ER action on Insl3 expression, however, remains poorly understood. Here we report estradiol (E 2 ) represses Insl3 mRNA levels in MA-10 cells, a Leydig cell line model. We also found that E 2 represses the activity of the human and mouse Insl3 promoter in these cells. The E 2 -responsive region of the human INSL3 promoter was located to the proximal INSL3 promoter. This region does not contain a consensus estrogen response element indicating an indirect mechanism of action. In agreement with this, we found that E 2 -responsiveness was lost when two previously characterized binding sites for the nuclear receptors NUR77 and SF1 were mutated. Finally we show that the E 2 repressive effect could be overcome by cotreatment with testosterone, a positive regulator of Insl3 transcription. Collectively our data provide important new insights into the molecular mechanism of estrogen action in Insl3 transcription in Leydig cells

  18. The food processing contaminant glyoxal promotes tumour growth in the multiple intestinal neoplasia (Min) mouse model.

    Science.gov (United States)

    Svendsen, Camilla; Høie, Anja Hortemo; Alexander, Jan; Murkovic, Michael; Husøy, Trine

    2016-08-01

    Glyoxal is formed endogenously and at a higher rate in the case of hyperglycemia. Glyoxal is also a food processing contaminant and has been shown to be mutagenic and genotoxic in vitro. The tumourigenic potential of glyoxal was investigated using the multiple intestinal neoplasia (Min) mouse model, which spontaneously develops intestinal tumours and is susceptible to intestinal carcinogens. C57BL/6J females were mated with Min males. Four days after mating and throughout gestation and lactation, the pregnant dams were exposed to glyoxal through drinking water (0.0125%, 0.025%, 0.05%, 0.1%) or regular tap water. Female and male offspring were housed separately from PND21 and continued with the same treatment. One group were only exposed to 0.1% glyoxal from postnatal day (PND) 21. There was no difference in the number of intestinal tumours between control and treatment groups. However, exposure to 0.1% glyoxal starting in utero and at PND21 caused a significant increase in tumour size in the small intestine for male and female mice in comparison with respective control groups. This study suggests that glyoxal has tumour growth promoting properties in the small intestine in Min mice. Copyright © 2016 Norwegian Institute of Public Health. Published by Elsevier Ltd.. All rights reserved.

  19. A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

    Science.gov (United States)

    Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G

    2004-04-29

    Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

  20. A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC system

    Directory of Open Access Journals (Sweden)

    Golemis Erica A

    2004-04-01

    Full Text Available Abstract Background Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. Results In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. Conclusion This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

  1. Regulation of the CD56 promoter and its association with proliferation, anti-apoptosis and clinical factors in multiple myeloma

    DEFF Research Database (Denmark)

    Damgaard, Tina; Knudsen, Lene M; Dahl, Inger Marie S

    2009-01-01

    the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively...... associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved...

  2. Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

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    Ma Xin-Ming

    2011-02-01

    Full Text Available Abstract Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of

  3. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cleasby, Mark E. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Millard, Susan; Leong, Gary M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cooney, Gregory J. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Muscat, George E.O., E-mail: g.muscat@imb.uq.edu.au [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia)

    2009-10-30

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  4. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

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    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  5. Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

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    Richard W Daniels

    Full Text Available Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A and Thosea asigna virus (T2A worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

  6. Lynch syndrome associated with two MLH1 promoter variants and allelic imbalance of MLH1 expression.

    Science.gov (United States)

    Hesson, Luke B; Packham, Deborah; Kwok, Chau-To; Nunez, Andrea C; Ng, Benedict; Schmidt, Christa; Fields, Michael; Wong, Jason W H; Sloane, Mathew A; Ward, Robyn L

    2015-06-01

    Lynch syndrome is a hereditary cancer syndrome caused by a constitutional mutation in one of the mismatch repair genes. The implementation of predictive testing and targeted preventative surveillance is hindered by the frequent finding of sequence variants of uncertain significance in these genes. We aimed to determine the pathogenicity of previously reported variants (c.-28A>G and c.-7C>T) within the MLH1 5'untranslated region (UTR) in two individuals from unrelated suspected Lynch syndrome families. We investigated whether these variants were associated with other pathogenic alterations using targeted high-throughput sequencing of the MLH1 locus. We also determined their relationship to gene expression and epigenetic alterations at the promoter. Sequencing revealed that the c.-28A>G and c.-7C>T variants were the only potentially pathogenic alterations within the MLH1 gene. In both individuals, the levels of transcription from the variant allele were reduced to 50% compared with the wild-type allele. Partial loss of expression occurred in the absence of constitutional epigenetic alterations within the MLH1 promoter. We propose that these variants may be pathogenic due to constitutional partial loss of MLH1 expression, and that this may be associated with intermediate penetrance of a Lynch syndrome phenotype. Our findings provide further evidence of the potential importance of noncoding variants in the MLH1 5'UTR in the pathogenesis of Lynch syndrome. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  7. Autocrine CSF-1 and CSF-1 Receptor Co-expression Promotes Renal Cell Carcinoma Growth

    Science.gov (United States)

    Menke, Julia; Kriegsmann, Jörg; Schimanski, Carl Christoph; Schwartz, Melvin M.; Schwarting, Andreas; Kelley, Vicki R.

    2011-01-01

    Renal cell carcinoma is increasing in incidence but the molecular mechanisms regulating its growth remain elusive. Co-expression of the monocytic growth factor CSF-1 and its receptor CSF-1R on renal tubular epithelial cells (TEC) will promote proliferation and anti-apoptosis during regeneration of renal tubules. Here we show that a CSF-1-dependent autocrine pathway is also responsible for the growth of renal cell carcinoma (RCC). CSF-1 and CSF-1R were co-expressed in RCC and TEC proximally adjacent to RCC. CSF-1 engagement of CSF-1R promoted RCC survival and proliferation and reduced apoptosis, in support of the likelihood that CSF-1R effector signals mediate RCC growth. In vivo CSF-1R blockade using a CSF-1R tyrosine kinase inhibitor decreased RCC proliferation and macrophage infiltration in a manner associated with a dramatic reduction in tumor mass. Further mechanistic investigations linked CSF-1 and EGF signaling in RCC. Taken together, our results suggest that budding RCC stimulates the proximal adjacent microenvironment in the kidney to release mediators of CSF-1, CSF-1R and EGF expression in RCC. Further, our findings imply that targeting CSF-1/CSF-1R signaling may be therapeutically effective in RCC. PMID:22052465

  8. CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

    DEFF Research Database (Denmark)

    Caldeira, José Roberto F; Prando, Erika C; Quevedo, Francisco C

    2006-01-01

    prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. METHODS: The aim of our study was to assess...... not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association......BACKGROUND: The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor...

  9. Pseudogene PHBP1 promotes esophageal squamous cell carcinoma proliferation by increasing its cognate gene PHB expression.

    Science.gov (United States)

    Feng, Feiyue; Qiu, Bin; Zang, Ruochuan; Song, Peng; Gao, Shugeng

    2017-04-25

    Natural antisense transcripts (NATs) as one of the most diverse classes of long noncoding RNAs (lncRNAs), have been demonstrated involved in fundamental biological processes in human. Here, we reported that human prohibitin gene pseudogene 1 (PHBP1) was upregulated in ESCC, and increased PHBP1 expression in ESCC was associated with clinical advanced stage. Functional experiments showed that PHBP1 knockdown inhibited ESCC cells proliferation, colony formation and xenograft tumor growth in vitro and in vivo by causing cell-cycle arrest at the G1-G0 phase. Mechanisms analysis revealed that PHBP1 transcript as an antisense transcript of PHB is partially complementary to PHB mRNA and formed an RNA-RNA hybrid with PHB, consequently inducing an increase of PHB expression at both the mRNA and protein levels. Furthermore, PHBP1 expression is strongly correlated with PHB expression in ESCC tissues. Collectively, this study elucidates an important role of PHBP1 in promoting ESCC partly via increasing PHB expression.

  10. TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors

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    Peh Bee

    2009-01-01

    Full Text Available Abstract Background Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2. Methods Oncogenic potential of TRIP-Br2 was demonstrated by (1 inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2 comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs. Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target. Results Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro. Conclusion This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.

  11. Expression and promoter DNA methylation of MLH1 in colorectal cancer and lung cancer.

    Science.gov (United States)

    Ma, Yunxia; Chen, Yuan; Petersen, Iver

    2017-04-01

    Aberrant DNA methylation is a common molecular feature in human cancer. The aims of this study were to analyze the methylation status of MLH1, one of the DNA mismatch repair (MMR) genes, in human colorectal and lung cancer and to evaluate its clinical relevance. The expression of MLH1 was analyzed in 8 colorectal cancer (CRC) and 8 lung cancer cell lines by real-time RT-PCR and western blotting. The MLH1 protein expression was evaluated by immunohistochemistry on tissue microarrays including 121 primary CRC and 90 lung cancer patient samples. In cancer cell lines, the methylation status of MLH1 promoter and exon 2 was investigated by bisulfite sequencing (BS). Methylation-specific-PCR (MSP) was used to evaluate methylation status of MLH1. The expression of MLH1 mRNA was detected in 8 CRC cell lines as well as normal colonic fibroblast cells CCD-33Co. At protein levels, MLH1 was lost in one CRC cell line HCT-116 and normal cells CCD-33Co. No methylation was found in the promoter and exon 2 of MLH1 in CRC cell lines. MLH1 was expressed in 8 lung cancer cell lines at both mRNA and protein levels. Compared to cancer cells, normal bronchial epithelial cells (HBEC) had lower expression of MLH1 protein. In primary CRC, 54.5% of cases exhibited positive staining, while 47.8% of lung tumors were positive for MLH1 protein. MSP analysis showed that 58 out of 92 (63.0%) CRC and 41 out of 73 (56.2%) lung cancer exhibited MLH1 methylation. In CRC, the MLH1 methylation was significantly associated with tumor invasion in veins (P=0.012). However, no significant links were found between MLH1 expression and promoter methylation in both tumor entities. MLH1 methylation is a frequent molecular event in CRC and lung cancer patients. In CRC, methylation of MLH1 could be linked to vascular invasiveness. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin

    Science.gov (United States)

    Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

    2018-01-01

    To investigate the roles of B-cell lymphoma-2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC-a and in SW1353 chondrosarcoma cells by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony-formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β-catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt

  13. BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin.

    Science.gov (United States)

    Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

    2018-04-01

    To investigate the roles of B‑cell lymphoma‑2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC‑a and in SW1353 chondrosarcoma cells by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony‑formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β‑catenin expression at the mRNA and protein levels. In addition, BAG3 induced the

  14. ATF3 expression improves motor function in the ALS mouse model by promoting motor neuron survival and retaining muscle innervation.

    Science.gov (United States)

    Seijffers, Rhona; Zhang, Jiangwen; Matthews, Jonathan C; Chen, Adam; Tamrazian, Eric; Babaniyi, Olusegun; Selig, Martin; Hynynen, Meri; Woolf, Clifford J; Brown, Robert H

    2014-01-28

    ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and atrophy of distal axon terminals in muscle, resulting in loss of motor function. Motor end plates denervated by axonal retraction of dying motor neurons are partially reinnervated by remaining viable motor neurons; however, this axonal sprouting is insufficient to compensate for motor neuron loss. Activating transcription factor 3 (ATF3) promotes neuronal survival and axonal growth. Here, we reveal that forced expression of ATF3 in motor neurons of transgenic SOD1(G93A) ALS mice delays neuromuscular junction denervation by inducing axonal sprouting and enhancing motor neuron viability. Maintenance of neuromuscular junction innervation during the course of the disease in ATF3/SOD1(G93A) mice is associated with a substantial delay in muscle atrophy and improved motor performance. Although disease onset and mortality are delayed, disease duration is not affected. This study shows that adaptive axonal growth-promoting mechanisms can substantially improve motor function in ALS and importantly, that augmenting viability of the motor neuron soma and maintaining functional neuromuscular junction connections are both essential elements in therapy for motor neuron disease in the SOD1(G93A) mice. Accordingly, effective protection of optimal motor neuron function requires restitution of multiple dysregulated cellular pathways.

  15. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    Directory of Open Access Journals (Sweden)

    Jack W Hickmott

    2016-01-01

    Full Text Available Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6 gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

  16. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

    Science.gov (United States)

    Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

  17. Resveratrol Promotes Remyelination in Cuprizone Model of Multiple Sclerosis: Biochemical and Histological Study.

    Science.gov (United States)

    Ghaiad, Heba R; Nooh, Mohammed M; El-Sawalhi, Maha M; Shaheen, Amira A

    2017-07-01

    Multiple sclerosis (MS) is a demyelinating neurodegenerative disease, representing a major cause of neurological disability in young adults. Resveratrol is a stilbenoid polyphenol, known to pass blood brain barrier and exhibit antioxidant, anti-inflammatory, and neuroprotective effects in several brain injuries. Cuprizone model of MS is particularly beneficial in studying demyelination/remyelination. Our study examined the potential neuroprotective and pro-remyelination effects of resveratrol in cuprizone-intoxicated C57Bl/6 mice. Mice were fed with chow containing 0.7 % cuprizone for 7 days, followed by 3 weeks on 0.2 % cuprizone diet. Resveratrol (250 mg/kg/day, p.o.) was given for 3 weeks starting from the second week. At the end of the experiment, animals were tested on rotarod to evaluate changes in balance and motor coordination. Mice were then sacrificed to measure the brain content of glutathione, lipid peroxidation products, adenosine triphosphate, and phospho-inhibitory subunit of nuclear factor κB-α. The activities of cytochrome oxidase and superoxide dismutase were also assessed. The gene expression of myelin basic protein, 2',3'-cyclic nucleotide 3' phosphodiesterase, oligodendrocyte transcription factor-1 (Olig1), NF-κB p65 subunit, and tumor necrosis factor-α was also estimated. Luxol fast blue/periodic acid-Schiff stained brain sections were blindly scored to assess the myelin status. Resveratrol effectively enhanced motor coordination and balance, reversed cuprizone-induced demyelination, improved mitochondrial function, alleviated oxidative stress, and inhibited NF-κB signaling. Interestingly, resveratrol increased Olig1 expression that is positively correlated to active remyelination. The present study may be the first to indicate a pro-remyelinative effect for resveratrol which might represent a potential additive benefit in treating MS.

  18. Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans.

    Science.gov (United States)

    Fischer, Alexandra; Niklowitz, Petra; Menke, Thomas; Döring, Frank

    2014-10-03

    Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Histone Deacetylase 7 Promotes Toll-like Receptor 4-dependent Proinflammatory Gene Expression in Macrophages*

    Science.gov (United States)

    Shakespear, Melanie R.; Hohenhaus, Daniel M.; Kelly, Greg M.; Kamal, Nabilah A.; Gupta, Praveer; Labzin, Larisa I.; Schroder, Kate; Garceau, Valerie; Barbero, Sheila; Iyer, Abishek; Hume, David A.; Reid, Robert C.; Irvine, Katharine M.; Fairlie, David P.; Sweet, Matthew J.

    2013-01-01

    Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target. PMID:23853092

  20. Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages.

    Science.gov (United States)

    Shakespear, Melanie R; Hohenhaus, Daniel M; Kelly, Greg M; Kamal, Nabilah A; Gupta, Praveer; Labzin, Larisa I; Schroder, Kate; Garceau, Valerie; Barbero, Sheila; Iyer, Abishek; Hume, David A; Reid, Robert C; Irvine, Katharine M; Fairlie, David P; Sweet, Matthew J

    2013-08-30

    Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.

  1. Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

    Science.gov (United States)

    Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

    2015-01-01

    In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

  2. TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

    International Nuclear Information System (INIS)

    Sun, Jing; Liu, Su-zhi; Lin, Yan; Cao, Xiao-pan; Liu, Jia-ming

    2014-01-01

    Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy

  3. CNPY2 promoted the proliferation of renal cell carcinoma cells and increased the expression of TP53

    International Nuclear Information System (INIS)

    Taniguchi, Hidefumi; Ito, Saya; Ueda, Takashi; Morioka, Yukako; Kayukawa, Naruhiro; Ueno, Akihisa; Nakagawa, Hideo; Fujihara, Atsuko; Ushijima, So; Kanazawa, Motohiro; Hongo, Fumiya; Ukimura, Osamu

    2017-01-01

    Renal cell carcinoma (RCC) is the most common type of kidney cancer. However, the mechanisms underlying the progression of the disease are not well understood. The data in this report suggest that canopy FGF signaling regulator 2 (CNPY2) is a promoter of RCC progression. We found that CNPY2 significantly promoted growth of RCC cells and upregulated TP53 gene expression. Although TP53 is widely known as a tumor suppressor, in RCC TP53 promoted tumor cell growth. A typical p53 target gene, CDKN1A, was upregulated by both p53 and CNPY2 in RCC cells, suggesting that CNPY2 increased the expression level of TP53. Consistent with these results, CNPY2 and TP53 expression levels were positively correlated in RCC patients. These findings suggested that CNPY2 promoted cancer cell growth in RCC through regulating TP53 gene expression. - Highlights: • CNPY2 promoted growth of renal cell carcinoma (RCC) cells. • TP53 expression levels were increased by CNPY2 in RCC cells. • Growth of RCC cells was promoted by TP53. • CNPY2 expression positively correlated with TP53 expression in RCC patients.

  4. Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells.

    Science.gov (United States)

    Dong, Hongli; Carlton, Michael E; Lerner, Adam; Epstein, Paul M

    2015-01-01

    Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad.

  5. Multiple memory systems, multiple time points: how science can inform treatment to control the expression of unwanted emotional memories.

    Science.gov (United States)

    Visser, Renée M; Lau-Zhu, Alex; Henson, Richard N; Holmes, Emily A

    2018-03-19

    Memories that have strong emotions associated with them are particularly resilient to forgetting. This is not necessarily problematic, however some aspects of memory can be. In particular, the involuntary expression of those memories, e.g. intrusive memories after trauma, are core to certain psychological disorders. Since the beginning of this century, research using animal models shows that it is possible to change the underlying memory, for example by interfering with its consolidation or reconsolidation. While the idea of targeting maladaptive memories is promising for the treatment of stress and anxiety disorders, a direct application of the procedures used in non-human animals to humans in clinical settings is not straightforward. In translational research, more attention needs to be paid to specifying what aspect of memory (i) can be modified and (ii) should be modified. This requires a clear conceptualization of what aspect of memory is being targeted, and how different memory expressions may map onto clinical symptoms. Furthermore, memory processes are dynamic, so procedural details concerning timing are crucial when implementing a treatment and when assessing its effectiveness. To target emotional memory in its full complexity, including its malleability, science cannot rely on a single method, species or paradigm. Rather, a constructive dialogue is needed between multiple levels of research, all the way 'from mice to mental health'.This article is part of a discussion meeting issue 'Of mice and mental health: facilitating dialogue between basic and clinical neuroscientists'. © 2018 The Authors.

  6. Expression of Nanog gene promotes NIH3T3 cell proliferation

    International Nuclear Information System (INIS)

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu

    2005-01-01

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion

  7. Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression.

    Science.gov (United States)

    Lu, Wanlu; Lu, Libing; Feng, Yun; Chen, Jiao; Li, Yan; Kong, Xiangli; Chen, Sixiu; Li, Xiaoyu; Chen, Qianming; Zhang, Ping

    2013-05-01

    The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8 + T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment.

  8. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Science.gov (United States)

    Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

    2006-12-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  9. Over-expression of Gene FaASR Promotes Strawberry Fruit Coloring

    Directory of Open Access Journals (Sweden)

    Liu Zhongjie

    2015-11-01

    Full Text Available Fruit development and ripening is a complicate process. Although much progress has been made on the ripenig process, the molecular mechamism of fruit development is not yet clear. In this study, we used ‘Sweet Charlie’ strawberry as test materials, based on cloning the strawberries ASR homologous gene, we carried out the bioinformatics and temporal expression analysis of FaASR, by manipulating ASR gene expression level in strawberry fruit, we tested the changes of physiological indicators, including sugar, ABA, pigments content, and fruit firmness, as well as phenotypic changes. In addition, we measured the expression changes of some anthocyanin-related gene, such as CHS and UFGT, by which we revealed the regulation mechanisms of ASR gene over strawberry fruit ripening. Strawberry ASR contained a typical domain of ABA/WDS that was related to fruit ripening and stress-resistance, and ASR gene over-expression could promote strawberry fruit coloring, endogenous ABA and sucrose accumulation, fruit softening, and induced the transcription levels of anthocyanin-related genes CHS and UFGT. The present study will further reveal the molecular mechanisms of information transmission in fruit development, and will also play an important foundation for future molecular improvement of strawberries breeding.

  10. Combined therapy of interferon plus ribavirin promotes multiple adaptive solutions in hepatitis C virus.

    Science.gov (United States)

    Cuevas, José M; Torres-Puente, Manuela; Jiménez-Hernández, Nuria; Bracho, María A; García-Robles, Inmaculada; Carnicer, Fernando; Olmo, Juan Del; Ortega, Enrique; González-Candelas, Fernando; Moya, Andrés

    2009-04-01

    Hepatitis C virus (HCV) presents several regions involved potentially in evading antiviral treatment and host immune system. Two regions, known as PKR-BD and V3 domains, have been proposed to be involved in resistance to interferon. Additionally, hypervariable regions in the envelope E2 glycoprotein are also good candidates to participate in evasion from the immune system. In this study, we have used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples obtained just before initiation of therapy and after 6 or/and 12 months of treatment were available. A range of 25-100 clones per patient, genome region and time sample were obtained. The predominant amino acid sequences for each time sample and patient were determined. Next, the sequences of the PKR-BD and V3 domains and the hypervariable regions from different time samples were compared for each patient. The highest levels of variability were detected at the three hypervariable regions of the E2 protein and, to a lower extent, at the V3 domain of the NS5A protein. However, no clear patterns of adaptation to the host immune system or to antiviral treatment were detected. In summary, although high levels of variability are correlated to viral adaptive response, antiviral treatment does not seem to promote convergent adaptive changes. Consequently, other regions must be involved in evasion strategies likely based on a combination of multiple mechanisms, in which pools of changes along the HCV genome could confer viruses the ability to overcome strong selective pressures. (c) 2009 Wiley-Liss, Inc.

  11. Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

    Science.gov (United States)

    Ge, Haitao; Mu, Luyan; Jin, Linchun; Yang, Changlin; Chang, Yifan Emily; Long, Yu; DeLeon, Gabriel; Deleyrolle, Loic; Mitchell, Duane A; Kubilis, Paul S; Lu, Dunyue; Qi, Jiping; Gu, Yunhe; Lin, Zhiguo; Huang, Jianping

    2017-10-01

    Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM. © 2017 UICC.

  12. Chronic intermittent hypoxia promotes expression of 3-mercaptopyruvate sulfurtransferase in adult rat medulla oblongata.

    Science.gov (United States)

    Li, Mingqiang; Nie, Lihong; Hu, Yajie; Yan, Xiang; Xue, Lian; Chen, Li; Zhou, Hua; Zheng, Yu

    2013-12-01

    The present experiments were carried out to investigate the expression of 3-mercaptopyruvate sulfurtransferase (3MST) in medulla oblongata of rats and effects of chronic intermittent hypoxia (CIH) on its expression. Sprague Dawley adult rats were randomly divided into two groups, including control (Con) group and CIH group. The endogenous production of hydrogen sulfide (H2S) in medulla oblongata tissue homogenates was measured using the methylene blue assay method, 3MST mRNA and protein expression were analyzed by RT-PCR and Western blotting, respectively, and the expression of 3MST in the neurons of respiratory-related nuclei in medulla oblongata of rats was investigated with immunohistochemical technique. CIH elevated the endogenous H2S production in rat medulla oblongata (Pmedulla oblongata of rats and CIH promoted their expression (P<0.01). Immunohistochemical staining indicated that 3MST existed in the neurons of pre-Bötzinger complex (pre-BötC), hypoglossal nucleus (12N), ambiguous nucleus (Amb), facial nucleus (FN) and nucleus tractus solitarius (NTS) in the animals and the mean optical densities of 3MST-positive neurons in the pre-BötC, 12N and Amb, but not in FN and NTS, were significantly increased in CIH group (P<0.05). In conclusion, 3MST exists in the neurons of medullary respiratory nuclei and its expression can be up-regulated by CIH in adult rat, suggesting that 3MST-H2S pathway may be involved in regulation of respiration and protection on medullary respiratory centers from injury induced by CIH. © 2013.

  13. Differentially expressed microRNA in multiple sclerosis: A window into pathogenesis?

    DEFF Research Database (Denmark)

    Martin, Nellie Anne; Illés, Zsolt

    2014-01-01

    MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery, and sile......MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery......RNA related to multiple sclerosis has increased significantly in recent years. Differentially expressed microRNA have been identified in the whole blood, serum, plasma, cerebrospinal fluid, peripheral blood mononuclear cells, blood-derived cell subsets and brain lesions of patients with multiple sclerosis....... Most studies applied a non-candidate approach of screening by microarray and validation by quantitative polymerase chain reaction or next generation sequencing; others used a candidate-driven approach. Despite a relatively high number of multiple sclerosis-associated microRNA, just a few could...

  14. Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

    Directory of Open Access Journals (Sweden)

    Wen-Qin Bai

    Full Text Available Bioactive gibberellins (GAs comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

  15. MeCP2 Expression and Promoter Methylation of Cyclin D1 Gene Are Associated with Cyclin D1 Expression in Developing Rat Epididymal Duct

    International Nuclear Information System (INIS)

    Darwanto, Agus; Kitazawa, Riko; Mori, Kiyoshi; Kondo, Takeshi; Kitazawa, Sohei

    2008-01-01

    Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation

  16. Transcriptional coactivator NT-PGC-1α promotes gluconeogenic gene expression and enhances hepatic gluconeogenesis.

    Science.gov (United States)

    Chang, Ji Suk; Jun, Hee-Jin; Park, Minsung

    2016-10-01

    The transcriptional coactivator PGC-1α plays a central role in hepatic gluconeogenesis. We previously reported that alternative splicing of the PGC-1α gene produces an additional transcript encoding the truncated protein NT-PGC-1α NT-PGC-1α is co-expressed with PGC-1α and highly induced by fasting in the liver. NT-PGC-1α regulates tissue-specific metabolism, but its role in the liver has not been investigated. Thus, the objective of this study was to determine the role of hepatic NT-PGC-1α in the regulation of gluconeogenesis. Adenovirus-mediated expression of NT-PGC-1α in primary hepatocytes strongly stimulated the expression of key gluconeogenic enzyme genes (PEPCK and G6Pase), leading to increased glucose production. To further understand NT-PGC-1α function in hepatic gluconeogenesis in vivo, we took advantage of a previously reported FL-PGC-1α -/- mouse line that lacks full-length PGC-1α (FL-PGC-1α) but retains a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α 254 ). In FL-PGC-1α -/- mice, NT-PGC-1α 254 was induced by fasting in the liver and recruited to the promoters of PEPCK and G6Pase genes. The enrichment of NT-PGC-1α 254 at the promoters was closely associated with fasting-induced increase in PEPCK and G6Pase gene expression and efficient production of glucose from pyruvate during a pyruvate tolerance test in FL-PGC-1α -/- mice. Moreover, FL-PGC-1α -/- primary hepatocytes showed a significant increase in gluconeogenic gene expression and glucose production after treatment with dexamethasone and forskolin, suggesting that NT-PGC-1α 254 is sufficient to stimulate the gluconeogenic program in the absence of FL-PGC-1α Collectively, our findings highlight the role of hepatic NT-PGC-1α in stimulating gluconeogenic gene expression and glucose production. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  17. Screening of Toll-like receptors expression in multiple system atrophy brains

    DEFF Research Database (Denmark)

    Brudek, Tomasz; Winge, Kristian; Agander, Tina Klitmøller

    2013-01-01

    The family of Toll-like receptors (TLRs) plays a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules. It was recently suggested that TLRs have an important role in the crosstalk between neurons and glial cells in the central nervous system, thus...... inclusions in oligodendrocytes. α-Synuclein can act as a danger-associated molecular pattern and alter TLR expression thereby activating inflammatory responses in the brain. In this study, using real-time PCR, we assessed the expression of TLRs (TLR1-10) in selected areas of MSA brains (substantia nigra......TLR-1 mRNA were elevated in substantia nigra and striatum whereas levels of hTLR-8 and hTLR-9 mRNAs were significantly higher in cerebella from MSA patients. The concerted alteration of expression of multiple TLRs in MSA brains can be of relevance for understanding the pathogenesis of the disease....

  18. Six1 promotes proliferation of pancreatic cancer cells via upregulation of cyclin D1 expression.

    Directory of Open Access Journals (Sweden)

    Zhaoming Li

    Full Text Available Six1 is one of the transcription factors that act as master regulators of development and are frequently dysregulated in cancers. However, the role of Six1 in pancreatic cancer is not clear. Here we show that the relative expression of Six1 mRNA is increased in pancreatic cancer and correlated with advanced tumor stage. In vitro functional assays demonstrate that forced overexpression of Six1 significantly enhances the growth rate and proliferation ability of pancreatic cancer cells. Knockdown of endogenous Six1 decreases the proliferation of these cells dramatically. Furthermore, Six1 promotes the growth of pancreatic cancer cells in a xenograft assay. We also show that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Overexpression of Six1 upregulates cyclin D1 mRNA and protein, and significantly enhances the activity of the cyclin D1 promoter in PANC-1 cells. We demonstrate that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. These data suggest that Six1 is overexpressed in pancreatic cancer and may contribute to the increased cell proliferation through upregulation of cyclin D1.

  19. Upregulation of miR-3607 promotes lung adenocarcinoma proliferation by suppressing APC expression.

    Science.gov (United States)

    Lin, Yong; Gu, Qiangye; Sun, Zongwen; Sheng, Baowei; Qi, Congcong; Liu, Bing; Fu, Tian; Liu, Cun; Zhang, Yan

    2017-11-01

    Lung cancer is the leading cause of worldwide cancer-related deaths, although many drugs and new therapeutic approaches have been used, the 5-years survival rate is still low for lung cancer patients. microRNAs have been shown to regulate lung cancer initiation and development, here we studied the role of miR-3607 in lung cancer cell proliferation. We found miR-3607 was upregulated in lung cancer tissues and cells, miR-3607 overexpression promoted lung cancer cell A549 proliferation determined by MTT assay, colony formation assay, anchorage-independent growth ability assay and bromodeoxyuridine incorporation assay, while the opposite phenotypes were shown when miR-3607 was knocked down. Predicted analysis suggested a Wnt signaling pathway regulator adenomatous polyposis coli (APC) was the target of miR-3607, miR-3607 could directly bind to the 3'UTR of APC, and promoted Cyclin D1 and c-Myc expression which can be suppressed by APC. Double knockdown of miR-3607 and APC copied the phenotypes of miR-3607 overexpression, suggesting miR-3607 promoted lung cancer cell A549 proliferation by targeting APC. In conclusion, our study suggested miR-3607 contributes to lung cancer cell proliferation by inhibiting APC. Copyright © 2017. Published by Elsevier Masson SAS.

  20. Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

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    Green Carla B

    2001-05-01

    Full Text Available Abstract Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

  1. Promotion of Metastasis-associated Gene Expression in Survived PANC-1 Cells Following Trichostatin A Treatment.

    Science.gov (United States)

    Chen, Zongjing; Yang, Yunxiu; Liu, Biao; Wang, Benquan; Sun, Meng; Zhang, Ling; Chen, Bicheng; You, Heyi; Zhou, Mengtao

    2015-01-01

    Histone deacetylase inhibitors represent a promising class of potential anticancer agents for the treatment of human malignancies. In this study, the effects of trichostatin A (TSA) on apoptosis, metastasis-associated gene expression, and activation of the Notch pathway in human pancreatic cancer cell lines were investigated. After treatment with TSA, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide] assay, Hoechst 33258 staining, and flow cytometry. Moreover, RT-PCR and western blot analyses were performed to measure the expression levels of apoptosis-associated genes (Bcl-2, Bax, and caspase-3), metastasis-associated genes (E-cadherin, vimentin, and matrix metalloproteinases), and Notch pathway activation (Notch intracellular domain, NICD). The levels of matrix metalloproteinase 2 and NICD were also semi-quantified by immunoassay. Following treatment with TSA for 24 h, PANC-1, SW1990, and MIATACA-2 cells exhibited cell death. The MTT assay revealed that TSA significantly decreased cell viability in a dose-dependent manner in PANC-1 cells. The Hoechst 33258 staining and flow cytometry results evidenced a significant increase in PANC-1 cell apoptosis following TSA treatment. The expression levels of Bax and caspase-3 were increased significantly, whereas Bcl-2 was down-regulated after TSA treatment. In the PANC-1 cells that survived after TSA treatment, the expression levels of vimentin, E-cadherin, and MMP genes were altered by the promotion of potential metastasis and increased expression of NICD. TSA can induce apoptosis of pancreatic cancer cells. In addition, the up-regulation of metastasis-related genes and the activation of the Notch pathway in the survived PANC-1 cells may be associated with a too-low level of TSA or resistance to TSA.

  2. MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival

    Directory of Open Access Journals (Sweden)

    Pezzella Francesco

    2011-05-01

    Full Text Available Abstract Background MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0 of purified tumor (CD138+ cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS and 9 controls. Results Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129 were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40% suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59% of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14 and t(11;14 and del(13q. Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS in MM, ten of which were significant by univariate (logrank survival analysis. Conclusions In summary, this work has identified aberrantly expressed microRNAs associated with the

  3. Efficient construction of an inverted minimal H1 promoter driven siRNA expression cassette: facilitation of promoter and siRNA sequence exchange.

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    Hoorig Nassanian

    2007-08-01

    Full Text Available RNA interference (RNAi, mediated by small interfering RNA (siRNA, is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC. The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each. Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette.

  4. Isolation, Expression, and Promoter Analysis of GbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba

    Directory of Open Access Journals (Sweden)

    Yong-Ling Liao

    2015-01-01

    Full Text Available WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28 WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.

  5. Ectopic expression of PTTG1/securin promotes tumorigenesis in human embryonic kidney cells

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    Malik Mohammed T

    2005-01-01

    Full Text Available Abstract Background Pituitary tumor transforming gene1 (PTTG1 is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3 cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293 cells. Results We report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8. Conclusions Our results

  6. Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Guitao Zhong

    2017-06-01

    Full Text Available Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.

  7. Promoter Methylation and BDNF and DAT1 Gene Expression Profiles in Patients with Drug Addiction.

    Science.gov (United States)

    Kordi-Tamandani, Dor Mohammad; Tajoddini, Shahrad; Salimi, Farzaneh

    2015-01-01

    Drug addiction is a brain disorder that has negative consequences for individuals and society. Addictions are chronic relapsing diseases of the brain that are caused by direct drug-induced effects and persevering neuroadaptations at the epigenetic, neuropeptide and neurotransmitter levels. Because the dopaminergic system has a significant role in drug abuse, the purpose of this study was to analyze the methylation and expression profile of brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in individuals with drug addiction. BDNF and DAT1 promoter methylation were investigated with a methylation-specific polymerase chain reaction (PCR) technique in blood samples from 75 individuals with drug addiction and 65 healthy controls. The expression levels of BDNF and DAT1 were assessed in 12 mRNA samples from the blood of patients and compared to the samples of healthy controls (n = 12) with real-time quantitative reverse transcription PCR. No significant differences were found in the methylation of BDNF and DAT1 between patients and controls, but the relative levels of expression of BDNF and DAT1 mRNA differed significantly in the patients compared to controls (p drug addiction.

  8. Health-Promoting Changes with Children as Agents: Findings from a Multiple Case Study Research

    Science.gov (United States)

    Simovska, Venka; Carlsson, Monica

    2012-01-01

    Purpose: With the aim of contributing to the evidence base on school-based health promotion, the authors discuss the outcomes and processes of a European intervention project aiming to prevent obesity among children (4-16 years) and promote their health and well-being, titled "Shape Up: a school-community approach to influencing determinants…

  9. Meeting multiple demands: Water transaction opportunities for environmental benefits promoting adaptation to climate change

    Science.gov (United States)

    McCoy, Amy

    2015-04-01

    In arid regions, the challenge of balancing water use among a diversity of sectors expands in lock step with conditions of water stress that are exacerbated by climate variability, prolonged drought, and growing water-use demands. The elusiveness of achieving a sustainable balance under conditions of environmental change in the southwestern United States is evidenced by reductions in both overall water availability and freshwater ecosystem health, as well as by recent projections of shortages on the Colorado River within the next five years. The water sustainability challenge in this region, as well as drylands throughout the world, can therefore be viewed through the lens of water stress, a condition wherein demands on land and water -- including the needs of freshwater ecosystems -- exceed reliable supplies, and the full range of water needs cannot be met without tradeoffs across multiple uses. Water stress influences not only ecosystems, but a region's economy, land management, quality of life, and cultural heritage -- each of which requires water to thrive. With respect to promoting successful adaptation to climate change, achieving full water sustainability would allow for water to be successfully divided among water users -- including municipalities, agriculture, and freshwater ecosystems -- at a level that meets the goals of water users and the governing body. Over the last ten to fifteen years, the use of transactional approaches in the western U.S., Mexico, and Australia has proven to be a viable management tool for achieving stream flow and shallow aquifer restoration. By broad definition, environmental water transactions are an equitable and adaptable tool that brings diverse stakeholders to the table to facilitate a fair-market exchange of rights to use water in a manner that benefits both water users and the environment. This talk will present a basic framework of necessary stakeholder engagement, hydrologic conditions, enabling laws and policies

  10. Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

    2013-01-01

    auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...... the production strain with the proper phenotype and product yield. However, the sequential number of metabolic engineering is time-consuming. Furthermore, the number of available selectable markers is also limiting the number of genetic modifications. To overcome these limitations, we have developed a new set...... of shuttle vectors for convenience of use for high-throughput cloning and selectable marker recycling. The new USER-based cloning vectors consist of a unique USER site and a CRE-loxP-mediated marker recycling system. The USER site allows insertion of genes of interest along with a bidirectional promoter...

  11. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    International Nuclear Information System (INIS)

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng

    2005-01-01

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10 9 PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases

  12. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  13. Capacity limitations to extract the mean emotion from multiple facial expressions depend on emotion variance.

    Science.gov (United States)

    Ji, Luyan; Pourtois, Gilles

    2018-04-20

    We examined the processing capacity and the role of emotion variance in ensemble representation for multiple facial expressions shown concurrently. A standard set size manipulation was used, whereby the sets consisted of 4, 8, or 16 morphed faces each uniquely varying along a happy-angry continuum (Experiment 1) or a neutral-happy/angry continuum (Experiments 2 & 3). Across the three experiments, we reduced the amount of emotion variance in the sets to explore the boundaries of this process. Participants judged the perceived average emotion from each set on a continuous scale. We computed and compared objective and subjective difference scores, using the morph units and post-experiment ratings, respectively. Results of the subjective scores were more consistent than the objective ones across the first two experiments where the variance was relatively large, and revealed each time that increasing set size led to a poorer averaging ability, suggesting capacity limitations in establishing ensemble representations for multiple facial expressions. However, when the emotion variance in the sets was reduced in Experiment 3, both subjective and objective scores remained unaffected by set size, suggesting that the emotion averaging process was unlimited in these conditions. Collectively, these results suggest that extracting mean emotion from a set composed of multiple faces depends on both structural (attentional) and stimulus-related effects. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage.

    Science.gov (United States)

    Taguchi, Y-h

    2015-01-01

    Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study.

  15. Understanding Selective Downregulation of c-Myc Expression through Inhibition of General Transcription Regulators in Multiple Myeloma

    Science.gov (United States)

    2015-06-01

    We next tested whether BET bromodomain inhibition mitigated the acti- vation of proadhesion pathways in aortic endothelium, which oc- curs during the...tinuum of activity as Myc flickers on and off of weakly bound, weakly expressed promoters, but stays longer or more frequently at high output promoters

  16. The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

    Directory of Open Access Journals (Sweden)

    Sarah L Noton

    Full Text Available Respiratory syncytial virus (RSV is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

  17. PAI-1 expression and its regulation by promoter 4G/5G polymorphism in clear cell renal cell carcinoma.

    Science.gov (United States)

    Choi, Jung-Woo; Lee, Ju-Han; Park, Hong Seok; Kim, Young-Sik

    2011-10-01

    To characterise patients with high plasminogen activator inhibitor-1 (PAI-1) expression as oral PAI-1 antagonists are currently in preclinical trials, and to determine whether the PAI-1 promoter 4G/5G polymorphism regulates PAI-1 expression in clear cell renal cell carcinoma (CCRCC). PAI-1 expression was examined by immunohistochemistry in 69 CCRCC specimens. In addition, the promoter 4G/5G polymorphism was investigated by both allele-specific PCR and direct DNA sequencing. PAI-1 was overexpressed in 25/69 (36.2%) patients with CCRCC. PAI-1 staining was intense in tumour cells with a high Fuhrman nuclear grade and in spindle-shaped tumour cells. PAI-1 expression was significantly associated with older age at diagnosis (p=0.027), high nuclear grade (p5G and 31.9% (22/69) 5G/5G. The homozygous 4G/4G or 5G/5G group showed a tendency for a high nuclear grade (p=0.05) but the 4G/5G polymorphism was not related to other prognostic parameters. PAI-1 expression was poorly correlated with its promoter 4G/5G polymorphism (Spearman ρ=0.088). CCRCC with high PAI-1 expression is characterised by older age, high nuclear grade, advanced stage, distant metastasis and/or shortened disease-free survival. PAI-1 expression is not affected by the promoter 4G/5G polymorphism.

  18. Wheat chloroplast targeted sHSP26 promoter confers heat and abiotic stress inducible expression in transgenic Arabidopsis Plants.

    Directory of Open Access Journals (Sweden)

    Neetika Khurana

    Full Text Available The small heat shock proteins (sHSPs have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs in the promoter of sHSP26 was performed. Moreover, the importance of 5' untranslated region (UTR has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress.

  19. HBx induced AFP receptor expressed to activate PI3K/AKT signal to promote expression of Src in liver cells and hepatoma cells

    International Nuclear Information System (INIS)

    Zhu, Mingyue; Guo, Junli; Li, Wei; Xia, Hua; Lu, Yan; Dong, Xu; Chen, Yi; Xie, Xieju; Fu, Shigan; Li, Mengsen

    2015-01-01

    Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. A total of 71 clinical patients’ liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC. The online version of this article (doi:10.1186/s12885-015-1384-9) contains supplementary material, which is available to authorized users

  20. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    International Nuclear Information System (INIS)

    Rieken, Stefan; Simon, Florian; Habermehl, Daniel; Dittmar, Jan Oliver; Combs, Stephanie E.; Weber, Klaus; Debus, Juergen; Lindel, Katja

    2014-01-01

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.) [de

  1. Structure of the gene for human β2-adrenergic receptor: expression and promoter characterization

    International Nuclear Information System (INIS)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.

    1987-01-01

    The genomic gene coding for the human β 2 -adrenergic receptor (β 2 AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with β 2 AR properties. Southern blot analyses with β 2 AR-specific probes show that a single β 2 AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the β 2 AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins

  2. Multiple growth hormone-binding proteins are expressed on insulin-producing cells

    DEFF Research Database (Denmark)

    Møldrup, A; Billestrup, N; Thorn, N A

    1989-01-01

    The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as d....... It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production....

  3. Using multiple schedules during functional communication training to promote rapid transfer of treatment effects.

    Science.gov (United States)

    Fisher, Wayne W; Greer, Brian D; Fuhrman, Ashley M; Querim, Angie C

    2015-12-01

    Multiple schedules with signaled periods of reinforcement and extinction have been used to thin reinforcement schedules during functional communication training (FCT) to make the intervention more practical for parents and teachers. We evaluated whether these signals would also facilitate rapid transfer of treatment effects across settings and therapists. With 2 children, we conducted FCT in the context of mixed (baseline) and multiple (treatment) schedules introduced across settings or therapists using a multiple baseline design. Results indicated that when the multiple schedules were introduced, the functional communication response came under rapid discriminative control, and problem behavior remained at near-zero rates. We extended these findings with another individual by using a more traditional baseline in which problem behavior produced reinforcement. Results replicated those of the previous participants and showed rapid reductions in problem behavior when multiple schedules were implemented across settings. © Society for the Experimental Analysis of Behavior.

  4. Isolation and characterization of the Jatropha curcas APETALA1 (JcAP1) promoter conferring preferential expression in inflorescence buds.

    Science.gov (United States)

    Tao, Yan-Bin; He, Liang-Liang; Niu, Longjian; Xu, Zeng-Fu

    2016-08-01

    The 1.5 kb JcAP1 promoter from the biofuel plant Jatropha curcas is predominantly active in the inflorescence buds of transgenic plants, in which the -1313/-1057 region is essential for maintaining the activity. Arabidopsis thaliana APETALA1 (AP1) is a MADS-domain transcription factor gene that functions primarily in flower development. We isolated a homolog of AP1 from Jatropha curcas (designated JcAP1), which was shown to exhibit flower-specific expression in Jatropha. JcAP1 is first expressed in inflorescence buds and continues to be primarily expressed in the sepals. We isolated a 1.5 kb JcAP1 promoter and evaluated its activity in transgenic Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis and Jatropha, the inflorescence buds exhibited notable GUS activity, whereas the sepals did not. Against expectations, the JcAP1 promoter was active in the anthers of Arabidopsis and Jatropha and was highly expressed in Jatropha seeds. An analysis of promoter deletions in transgenic Arabidopsis revealed that deletion of the -1313/-1057 region resulted in loss of JcAP1 promoter activity in the inflorescence buds and increased activity in the anthers. These results suggested that some regulatory sequences in the -1313/-1057 region are essential for maintaining promoter activity in inflorescence buds and can partly suppress activity in the anthers. Based on these findings, we hypothesized that other elements located upstream of the 1.5 kb JcAP1 promoter may be required for flower-specific activation. The JcAP1 promoter characterized in this study can be used to drive transgene expression in both the inflorescence buds and seeds of Jatropha.

  5. The effects of multiple UV exposures on HIV-LTR (long terminal repeat) expression

    International Nuclear Information System (INIS)

    Schreck, S.; Milton, J.; Panozzo, J.; Libertin, C.R.; Woloschak, G.E.; Loyola Univ., Maywood, IL

    1995-01-01

    Previous studies have shown that cellular stress agents such as UV radiation induce transcription from the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Using HeLa cells stably transfected with the HIV-LTR sequence, which transcriptionally drives the chloramphenicol acetyl transferase (CAT) reporter gene, we examined the effects of multiple exposures to UVC (254 nm) on HIV-LTR-CAT expression. Low doses (≤ 5 J m -2 ) had no effect on CAT expression, but up to 29-fold induction was observed with 10 J m -2 when cells were harvested 48 h after completion of the exposure. Little difference was noted in induction levels when cells were exposed to one 25 J m -2 dose, viable cells were harvested at 24 h, 48 h or 72 h, and cell lysates were assayed for CAT expression. Two sequential 12.5 J m -2 exposures, given 24 h apart, resulted in an additive effect on CAT expression; these two exposures produced CAT activity equivalent to that induced following a single 25 J m -2 dose. Our data suggest that HIV-LTR requires a specific threshold UV dose in order to elicit induction; a maximal induction dose is also evident; exposures higher than this maximal dose contribute no more to HIV-LTR induction in viable cells. (author)

  6. Cerebrospinal Fluid Cytokine Expression Profile in Multiple Sclerosis and Chronic Inflammatory Demyelinating Polyneuropathy.

    Science.gov (United States)

    Bonin, Serena; Zanotta, Nunzia; Sartori, Arianna; Bratina, Alessio; Manganotti, Paolo; Trevisan, Giusto; Comar, Manola

    2018-02-01

    Cerebrospinal fluid (CSF) analysis in patients with particular neurologic disorders is a powerful tool to evaluate specific central nervous system inflammatory markers for diagnostic needs, because CSF represents the specific immune micro-environment to the central nervous system. CSF samples from 49 patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), and non-inflammatory neurologic disorders (NIND) as controls were submitted to protein expression profiles of 47 inflammatory biomarkers by multiplex Luminex bead assay to investigate possible differences in the inflammatory process for MS and CIDP. Our results showed differences in CSF cytokine levels in MS and CIDP; in particular, IL12 (p40) was significantly highly expressed in MS in comparison with CIDP and NIND, while SDF-1α and SCGF-β were significantly highly expressed in CIDP cohort when compared to MS and NIND. IL-9, IL-13, and IL-17 had higher expression levels in NIND if compared with the other groups. Our study showed that, despite some common pathogenic mechanisms, central and peripheral nervous system demyelinating diseases, such as MS and CIDP, differ in some specific inflammatory soluble proteins in CSF, underlining differences in the immune response involved in those autoimmune diseases.

  7. Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea.

    Science.gov (United States)

    Zhu, Changfu; Yang, Qingjie; Ni, Xiuzhen; Bai, Chao; Sheng, Yanmin; Shi, Lianxuan; Capell, Teresa; Sandmann, Gerhard; Christou, Paul

    2014-04-01

    Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea. © 2013 Scandinavian Plant Physiology Society.

  8. Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

    Directory of Open Access Journals (Sweden)

    Norazlina Ahmad

    2012-01-01

    Full Text Available Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

  9. Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.

    Directory of Open Access Journals (Sweden)

    Francescopaolo Di Cello

    Full Text Available Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA. Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+ breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.

  10. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  11. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  12. Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

    Directory of Open Access Journals (Sweden)

    Shane D. Sykes

    2015-01-01

    Full Text Available The intrauterine renin angiotensin system (RAS is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AGTR1, and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

  13. Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression.

    Science.gov (United States)

    O'Riordan, N; O'Callaghan, J; Buttò, L F; Kilcoyne, M; Joshi, L; Hickey, R M

    2018-05-23

    Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Hair growth is promoted by BeauTop via expression of EGF and FGF‑7.

    Science.gov (United States)

    Lee, Chien-Ying; Yang, Chi-Yu; Lin, Ching-Che; Yu, Min-Chien; Sheu, Shuenn-Jyi; Kuan, Yu-Hsiang

    2018-06-01

    Minoxidil and finasteride have been approved to treat hair loss by the Food and Drug Administration. However, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. BeauTop (BT) is a health food supplement which contains Ginseng radix, Astragali radix, Radix Angelicae sinensis, Ligustri fructus, Rehmannia glutinosa and Eclipta prostrata (Linn). Susbsequent to oral administration of BT at 0.6 g/kg/day to wax/rosin‑induced alopecia in C57BL/6 mice, BT significantly induced hair growth at day 8 compared with control treatment (P<0.05). The expression levels of epidermal growth factor (EGF), and fibroblast growth factor (FGF)‑7 were increased compared with control animals on day 8. In contrast, levels of FGF‑5 of the BT group were reduced compared with the control on day 12. There were no effects on the expression of insulin‑like growth factor 1. The results demonstrated that the mechanism of BT improving alopecia is potentially associated with modulation of EGF and FGF‑7 levels. Taken together, it is suggested that BT may have a potential effect of the promotion of hair growth.

  15. Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Manuel Hiss

    2017-10-01

    Full Text Available The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

  16. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  17. Multiple mutualist effects on genomewide expression in the tripartite association between Medicago truncatula, nitrogen-fixing bacteria and mycorrhizal fungi.

    Science.gov (United States)

    Afkhami, Michelle E; Stinchcombe, John R

    2016-10-01

    While all species interact with multiple mutualists, the fitness consequences and molecular mechanisms underlying these interactions remain largely unknown. We combined factorial ecological experiments with genomewide expression analyses to examine the phenotypic and transcriptomic responses of model legume Medicago truncatula to rhizobia and mycorrhizal fungi. We found synergistic effects of these mutualists on plant performance and examined unique features of plant gene expression responses to multiple mutualists. There were genomewide signatures of mutualists and multiple mutualists on expression, with partners often affecting unique sets of genes. Mycorrhizal fungi had stronger effects on plant expression than rhizobia, with 70% of differentially expressed genes affected by fungi. Fungal and bacterial mutualists had joint effects on 10% of differentially expressed genes, including unexpected, nonadditive effects on some genes with important functions such as nutrient metabolism. For a subset of genes, interacting with multiple mutualists even led to reversals in the direction of expression (shifts from up to downregulation) compared to interacting with single mutualists. Rhizobia also affected the expression of several mycorrhizal genes, including those involved in nutrient transfer to host plants, indicating that partner species can also impact each other's molecular phenotypes. Collectively, these data illustrate the diverse molecular mechanisms and transcriptional responses associated with the synergistic benefits of multiple mutualists. © 2016 John Wiley & Sons Ltd.

  18. Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy.

    Directory of Open Access Journals (Sweden)

    Deepak Kumar

    Full Text Available BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s. Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27 and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31. Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in

  19. A search engine to identify pathway genes from expression data on multiple organisms

    Directory of Open Access Journals (Sweden)

    Zambon Alexander C

    2007-05-01

    Full Text Available Abstract Background The completion of several genome projects showed that most genes have not yet been characterized, especially in multicellular organisms. Although most genes have unknown functions, a large collection of data is available describing their transcriptional activities under many different experimental conditions. In many cases, the coregulatation of a set of genes across a set of conditions can be used to infer roles for genes of unknown function. Results We developed a search engine, the Multiple-Species Gene Recommender (MSGR, which scans gene expression datasets from multiple organisms to identify genes that participate in a genetic pathway. The MSGR takes a query consisting of a list of genes that function together in a genetic pathway from one of six organisms: Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana, and Helicobacter pylori. Using a probabilistic method to merge searches, the MSGR identifies genes that are significantly coregulated with the query genes in one or more of those organisms. The MSGR achieves its highest accuracy for many human pathways when searches are combined across species. We describe specific examples in which new genes were identified to be involved in a neuromuscular signaling pathway and a cell-adhesion pathway. Conclusion The search engine can scan large collections of gene expression data for new genes that are significantly coregulated with a pathway of interest. By integrating searches across organisms, the MSGR can identify pathway members whose coregulation is either ancient or newly evolved.

  20. Global map of physical interactions among differentially expressed genes in multiple sclerosis relapses and remissions.

    Science.gov (United States)

    Tuller, Tamir; Atar, Shimshi; Ruppin, Eytan; Gurevich, Michael; Achiron, Anat

    2011-09-15

    Multiple sclerosis (MS) is a central nervous system autoimmune inflammatory T-cell-mediated disease with a relapsing-remitting course in the majority of patients. In this study, we performed a high-resolution systems biology analysis of gene expression and physical interactions in MS relapse and remission. To this end, we integrated 164 large-scale measurements of gene expression in peripheral blood mononuclear cells of MS patients in relapse or remission and healthy subjects, with large-scale information about the physical interactions between these genes obtained from public databases. These data were analyzed with a variety of computational methods. We find that there is a clear and significant global network-level signal that is related to the changes in gene expression of MS patients in comparison to healthy subjects. However, despite the clear differences in the clinical symptoms of MS patients in relapse versus remission, the network level signal is weaker when comparing patients in these two stages of the disease. This result suggests that most of the genes have relatively similar expression levels in the two stages of the disease. In accordance with previous studies, we found that the pathways related to regulation of cell death, chemotaxis and inflammatory response are differentially expressed in the disease in comparison to healthy subjects, while pathways related to cell adhesion, cell migration and cell-cell signaling are activated in relapse in comparison to remission. However, the current study includes a detailed report of the exact set of genes involved in these pathways and the interactions between them. For example, we found that the genes TP53 and IL1 are 'network-hub' that interacts with many of the differentially expressed genes in MS patients versus healthy subjects, and the epidermal growth factor receptor is a 'network-hub' in the case of MS patients with relapse versus remission. The statistical approaches employed in this study enabled us

  1. G-Protein-Coupled Receptor Gpr17 Expression in Two Multiple Sclerosis Remyelination Models.

    Science.gov (United States)

    Nyamoya, Stella; Leopold, Patrizia; Becker, Birte; Beyer, Cordian; Hustadt, Fabian; Schmitz, Christoph; Michel, Anne; Kipp, Markus

    2018-06-05

    In multiple sclerosis patients, demyelination is prominent in both the white and gray matter. Chronic clinical deficits are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. The underlying molecular mechanisms of remyelination and its failure remain currently unclear. Recent studies have recognized G protein-coupled receptor 17 (GPR17) as an important regulator of oligodendrocyte development and remyelination. So far, the relevance of GPR17 for myelin repair was mainly tested in remyelinating white matter lesions. The relevance of GPR17 for gray matter remyelination as well as remyelination of chronic white matter lesions was not addressed so far. Here, we provide a detailed characterization of GPR17 expression during experimental de- and remyelination. Experimental lesions with robust and limited endogenous remyelination capacity were established by either acute or chronic cuprizone-induced demyelination. Furthermore, remyelinating lesions were induced by the focal injection of lysophosphatidylcholine (LPC) into the corpus callosum. GPR17 expression was analyzed by complementary techniques including immunohistochemistry, in situ hybridization, and real-time PCR. In control animals, GPR17 + cells were evenly distributed in the corpus callosum and cortex and displayed a highly ramified morphology. Virtually all GPR17 + cells also expressed the oligodendrocyte-specific transcription factor OLIG2. After acute cuprizone-induced demyelination, robust endogenous remyelination was evident in the white matter corpus callosum but not in the gray matter cortex. Endogenous callosal remyelination was paralleled by a robust induction of GPR17 expression which was absent in the gray matter cortex. Higher numbers of GPR17 + cells were as well observed after LPC-induced focal white matter demyelination. In contrast, densities of GPR17 + cells were comparable to control animals after chronic cuprizone-induced demyelination indicating

  2. EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing's Sarcoma.

    Science.gov (United States)

    Hossain, Mohammad Motarab; Ray, Swapan Kumar

    2014-10-01

    Ewing's sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing's sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing's sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing's sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing's sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing's sarcoma in cell culture and animal models.

  3. Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression.

    Science.gov (United States)

    Lim, Eun Jin; Heo, Jeonghoon; Kim, Young-Ho

    2015-08-01

    Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies.

  4. Phase I metabolic genes and risk of lung cancer: multiple polymorphisms and mRNA expression.

    Directory of Open Access Journals (Sweden)

    Melissa Rotunno

    2009-05-01

    Full Text Available Polymorphisms in genes coding for enzymes that activate tobacco lung carcinogens may generate inter-individual differences in lung cancer risk. Previous studies had limited sample sizes, poor exposure characterization, and a few single nucleotide polymorphisms (SNPs tested in candidate genes. We analyzed 25 SNPs (some previously untested in 2101 primary lung cancer cases and 2120 population controls from the Environment And Genetics in Lung cancer Etiology (EAGLE study from six phase I metabolic genes, including cytochrome P450s, microsomal epoxide hydrolase, and myeloperoxidase. We evaluated the main genotype effects and genotype-smoking interactions in lung cancer risk overall and in the major histology subtypes. We tested the combined effect of multiple SNPs on lung cancer risk and on gene expression. Findings were prioritized based on significance thresholds and consistency across different analyses, and accounted for multiple testing and prior knowledge. Two haplotypes in EPHX1 were significantly associated with lung cancer risk in the overall population. In addition, CYP1B1 and CYP2A6 polymorphisms were inversely associated with adenocarcinoma and squamous cell carcinoma risk, respectively. Moreover, the association between CYP1A1 rs2606345 genotype and lung cancer was significantly modified by intensity of cigarette smoking, suggesting an underlying dose-response mechanism. Finally, increasing number of variants at CYP1A1/A2 genes revealed significant protection in never smokers and risk in ever smokers. Results were supported by differential gene expression in non-tumor lung tissue samples with down-regulation of CYP1A1 in never smokers and up-regulation in smokers from CYP1A1/A2 SNPs. The significant haplotype associations emphasize that the effect of multiple SNPs may be important despite null single SNP-associations, and warrants consideration in genome-wide association studies (GWAS. Our findings emphasize the necessity of post

  5. Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

    Science.gov (United States)

    Kathiria, Palak; Sidler, Corinne; Woycicki, Rafal; Yao, Youli; Kovalchuk, Igor

    2013-07-01

    The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.

  6. IL-6 Promotes FSH-Induced VEGF Expression Through JAK/STAT3 Signaling Pathway in Bovine Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Meng Yang

    2017-11-01

    Full Text Available Background/Aims: Vascular endothelial growth factor (VEGF has been demonstrated to play a pivotal role in the regulation of angiogenesis in ovarian follicular development, particularly during the preovulatory period. Although numerous studies have shown that interleukin-6 (IL-6 is one of the major inducing factors that regulate the expression of VEGF in non-ovarian cells, whether it involved in regulating the expression of VEGF in normal ovarian granulosa cells is still unknown. The aim of this study was to elucidate the mechanisms underlying the effect of IL-6 on FSH-induced VEGF expression in bovine granulosa cells derived from large follicles. Methods: VEGF mRNA expression in granulosa cells after IL-6 with/without inhibitors treatment was analyzed by RT-qPCR. Phosphorylation levels of ERK1/2 and STAT3 proteins induced by IL-6 were analyzed by western blotting. The protein levels produced by granulosa cells were detected by ELISA. Results: High concentration of IL-6 (10ng/ml can significantly up-regulate FSH-induced VEGF gene and protein expression levels in granulosa cells, and also promote the VEGF upstream regulators HIF-1α and COX2 mRNA expression. VEGF expression levels were significantly decreased after specifically blocking HIF-1α and COX2 by using inhibitors. The up-regulation effect of IL-6 on FSH-induced VEGF expression in granulosa cells mainly through activating the JAK/STAT3 signaling pathway, which can be impaired by JAK inhibitors. Conclusion: IL-6 can promote FSH-induced VEGF expression in granulosa cells, which is mainly achieved by increasing the expression of HIF-1α and COX2.This promoting effect is mediated by activating the JAK/STAT3 pathway. Moreover, there may be a synergistic relationship between FSH and IL-6 in the regulation of VEGF expression.

  7. Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Makoto Seo

    2008-01-01

    Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

  8. Buying in multiple stores : Shopping strategies beyond price promotions and their effects on Store Competition

    NARCIS (Netherlands)

    Gijsbrechts, E.; Campo, K.; Nisol, P.

    2013-01-01

    Grocery-store switching has typically been viewed as evidence of cherry-picking behavior, with consumers switching stores to benefit from temporary promotional offers. However, research reveals that it may also result from a longer-term planning process based on stable store characteristics. Even in

  9. Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

    Science.gov (United States)

    Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

    2010-01-01

    Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (ptrend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

  10. Reduced MeCP2 expression is frequent in autism frontal cortex and correlates with aberrant MECP2 promoter methylation.

    Science.gov (United States)

    Nagarajan, Raman P; Hogart, Amber R; Gwye, Ynnez; Martin, Michelle R; LaSalle, Janine M

    2006-01-01

    Mutations in MECP2, encoding methyl CpG binding protein 2 (MeCP2), cause most cases of Rett syndrome (RTT), an X-linked neurodevelopmental disorder. Both RTT and autism are "pervasive developmental disorders" and share a loss of social, cognitive and language skills and a gain in repetitive stereotyped behavior, following apparently normal perinatal development. Although MECP2 coding mutations are a rare cause of autism, MeCP2 expression defects were previously found in autism brain. To further study the role of MeCP2 in autism spectrum disorders (ASDs), we determined the frequency of MeCP2 expression defects in brain samples from autism and other ASDs. We also tested the hypotheses that MECP2 promoter mutations or aberrant promoter methylation correlate with reduced expression in cases of idiopathic autism. MeCP2 immunofluorescence in autism and other neurodevelopmental disorders was quantified by laser scanning cytometry and compared with control postmortem cerebral cortex samples on a large tissue microarray. A significant reduction in MeCP2 expression compared to age-matched controls was found in 11/14 autism (79%), 9/9 RTT (100%), 4/4 Angelman syndrome (100%), 3/4 Prader-Willi syndrome (75%), 3/5 Down syndrome (60%), and 2/2 attention deficit hyperactivity disorder (100%) frontal cortex samples. One autism female was heterozygous for a rare MECP2 promoter variant that correlated with reduced MeCP2 expression. A more frequent occurrence was significantly increased MECP2 promoter methylation in autism male frontal cortex compared to controls. Furthermore, percent promoter methylation of MECP2 significantly correlated with reduced MeCP2 protein expression. These results suggest that both genetic and epigenetic defects lead to reduced MeCP2 expression and may be important in the complex etiology of autism.

  11. CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

    Science.gov (United States)

    Zinzow-Kramer, W M; Long, A B; Youngblood, B A; Rosenthal, K M; Butler, R; Mohammed, A-U-R; Skountzou, I; Ahmed, R; Evavold, B D; Boss, J M

    2012-06-01

    Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

  12. Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer

    OpenAIRE

    Metelli, Alessandra; Wu, Bill X; Fugle, Caroline W; Rachidi, Saleh; Sun, Shaoli; Zhang, Yongliang; Wu, Jennifer; Tomlinson, Stephen; Howe, Philip; Yang, Yi; Garrett-Mayer, Elizabeth; Liu, Bei; Li, Zihai

    2016-01-01

    GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGF-β which is expressed naturally by platelets and regulatory T cells. Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGF-β in the tumor microenvironment. We found that human breast, lung and colon cancers expres...

  13. 5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.

    Science.gov (United States)

    Zheng, Xiaomei; Zheng, Ping; Zhang, Kun; Cairns, Timothy C; Meyer, Vera; Sun, Jibin; Ma, Yanhe

    2018-04-30

    The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.

  14. The expression of p53 protein in patients with multiple myeloma

    Directory of Open Access Journals (Sweden)

    Marković Olivera

    2007-01-01

    Full Text Available Introduction: Although mutations of p53 are one of the most often acquired genetic changes in malignant tumors, these mutations are rare events in patients with newly diagnosed multiple myeloma (MM. Moreover, there are a few literature data about clinical significance of p53 overexpression in multiple myeloma. Objective The aim of our study was to evaluate the clinical significance of p53 immunoexpression in multiple myeloma. Method A total of 58 patients with newly diagnosed MM (26 females and 32 males, mean age 62 years were enrolled in the study. The diagnosis of MM was made according to criteria of Chronic Leukemia-Myeloma Task Force. Clinical staging was done according to Durie and Salmon classification (4 patients had disease stage I, 15 patients stage II and 39 patients stage III. The histological grade and histological stage were determined according to predominant plasma cell morphology and volume of myeloma infiltration, respectively. Standard immunohistochemical analysis with p53 antibody in B5-fixed and paraffin- embedded bone marrow specimens was used to evaluate the expression of p53 in myeloma cells. The specimens were considered positive when ≥5% of plasma cells exhibited clear nuclear positivity. Results Out of 58 patients, p53 expression was detected in 9 (15.52%. No significant correlation was found between p53 expression and clinical stage (I+II vs. III, Я2-microglobulin level (≤6 mg/L vs. >6mg/L, histological grade (I vs. II+III, histological stage (<20% vs. 21-50% vs. >50% and the extent of osteolytic lesions (≤3 vs. >3 lesions. Median survival of patients with p53 immunoreactivity in =>5% of plasma cells was 10 months, whilst median survival of patients with p53 immunoreactivity in <5% of plasma cells was 36 months. However, such difference was not significant (p=0.2. Conclusion The frequency of p53 immunoexpression in our group of newly diagnosed MM was relatively low. Although p53 immunoexpression was not

  15. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

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    Oliveira Jorge

    2007-07-01

    Full Text Available Abstract Background Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. Methods A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC, 13 papillary (pRCC, 10 chromophobe (chRCC, and 10 oncocytomas and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Results Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007, PTGS2 (p = 0.002, and RASSF1A (p = 0.0001. CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively, whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004. RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035. In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031 and nuclear grade (p = 0.022, respectively. Conclusion The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

  16. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    International Nuclear Information System (INIS)

    Costa, Vera L; Henrique, Rui; Ribeiro, Franclim R; Pinto, Mafalda; Oliveira, Jorge; Lobo, Francisco; Teixeira, Manuel R; Jerónimo, Carmen

    2007-01-01

    Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses

  17. Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

    International Nuclear Information System (INIS)

    Noetzel, Erik; Veeck, Jürgen; Niederacher, Dieter; Galm, Oliver; Horn, Felicitas; Hartmann, Arndt; Knüchel, Ruth; Dahl, Edgar

    2008-01-01

    Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level

  18. IN-MACA-MCC: Integrated Multiple Attractor Cellular Automata with Modified Clonal Classifier for Human Protein Coding and Promoter Prediction

    Directory of Open Access Journals (Sweden)

    Kiran Sree Pokkuluri

    2014-01-01

    Full Text Available Protein coding and promoter region predictions are very important challenges of bioinformatics (Attwood and Teresa, 2000. The identification of these regions plays a crucial role in understanding the genes. Many novel computational and mathematical methods are introduced as well as existing methods that are getting refined for predicting both of the regions separately; still there is a scope for improvement. We propose a classifier that is built with MACA (multiple attractor cellular automata and MCC (modified clonal classifier to predict both regions with a single classifier. The proposed classifier is trained and tested with Fickett and Tung (1992 datasets for protein coding region prediction for DNA sequences of lengths 54, 108, and 162. This classifier is trained and tested with MMCRI datasets for protein coding region prediction for DNA sequences of lengths 252 and 354. The proposed classifier is trained and tested with promoter sequences from DBTSS (Yamashita et al., 2006 dataset and nonpromoters from EID (Saxonov et al., 2000 and UTRdb (Pesole et al., 2002 datasets. The proposed model can predict both regions with an average accuracy of 90.5% for promoter and 89.6% for protein coding region predictions. The specificity and sensitivity values of promoter and protein coding region predictions are 0.89 and 0.92, respectively.

  19. Using Multiple Schedules during Functional Communication Training to Promote Rapid Transfer of Treatment Effects

    Science.gov (United States)

    Fisher, Wayne W.; Greer, Brian D.; Fuhrman, Ashley M.; Querim, Angie C.

    2015-01-01

    Multiple schedules with signaled periods of reinforcement and extinction have been used to thin reinforcement schedules during functional communication training (FCT) to make the intervention more practical for parents and teachers. We evaluated whether these signals would also facilitate rapid transfer of treatment effects across settings and…

  20. Health promotion for young people with profound and multiple learning disabilities.

    Science.gov (United States)

    Davis, Kathy; Carter, Simone; Myers, Elizabeth; Rocca, Nicola

    2018-02-07

    Research confirms that children and young people with severe learning disabilities do not have the same level of access to high-quality care, health education and health promotion activities as children and young people without disabilities. This article discusses a quality improvement, action research project to investigate alternative approaches to health promotion that enhance the health and well-being of children and young people with complex neurodisabilities. The project involved assessment of school records and completion by staff of an eight-question survey. It found that the proactive approach of school nurses in raising awareness and understanding through questioning was positively received, and reinforced how meaningful and relevant information could be delivered to these young people. The project also had unexpected benefits, including more integrated team working, increased knowledge, greater awareness and understanding of the importance of health promotion participation, and student satisfaction. ©2018 RCN Publishing Company Ltd. All rights reserved. Not to be copied, transmitted or recorded in any way, in whole or part, without prior permission of the publishers.

  1. Fine tuning of RFX/DAF-19-regulated target gene expression through binding to multiple sites in Caenorhabditis elegans

    OpenAIRE

    Chu, Jeffery S. C.; Tarailo-Graovac, Maja; Zhang, Di; Wang, Jun; Uyar, Bora; Tu, Domena; Trinh, Joanne; Baillie, David L.; Chen, Nansheng

    2011-01-01

    In humans, mutations of a growing list of regulatory factor X (RFX) target genes have been associated with devastating genetics disease conditions including ciliopathies. However, mechanisms underlying RFX transcription factors (TFs)-mediated gene expression regulation, especially differential gene expression regulation, are largely unknown. In this study, we explore the functional significance of the co-existence of multiple X-box motifs in regulating differential gene expression in Caenorha...

  2. MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

    2016-11-15

    Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

  3. LHCSR Expression under HSP70/RBCS2 Promoter as a Strategy to Increase Productivity in Microalgae

    Directory of Open Access Journals (Sweden)

    Federico Perozeni

    2018-01-01

    Full Text Available Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, limited biomass productivity is commonly experienced in their cultivating system despite their high potential. One of the reasons for this limitation is the high thermal dissipation of the light absorbed by the outer layers of the cultures exposed to high light caused by the activation of a photoprotective mechanism called non-photochemical quenching (NPQ. In the model organism for green algae Chlamydomonas reinhardtii, NPQ is triggered by pigment binding proteins called light-harvesting-complexes-stress-related (LHCSRs, which are over-accumulated in high light. It was recently reported that biomass productivity can be increased both in microalgae and higher plants by properly tuning NPQ induction. In this work increased light use efficiency is reported by introducing in C. reinhardtii a LHCSR3 gene under the control of Heat Shock Protein 70/RUBISCO small chain 2 promoter in a npq4 lhcsr1 background, a mutant strain knockout for all LHCSR genes. This complementation strategy leads to a low expression of LHCSR3, causing a strong reduction of NPQ induction but is still capable of protecting from photodamage at high irradiance, resulting in an improved photosynthetic efficiency and higher biomass accumulation.

  4. Bringing numerous methods for expression and promoter analysis to a public cloud computing service.

    Science.gov (United States)

    Polanski, Krzysztof; Gao, Bo; Mason, Sam A; Brown, Paul; Ott, Sascha; Denby, Katherine J; Wild, David L

    2018-03-01

    Every year, a large number of novel algorithms are introduced to the scientific community for a myriad of applications, but using these across different research groups is often troublesome, due to suboptimal implementations and specific dependency requirements. This does not have to be the case, as public cloud computing services can easily house tractable implementations within self-contained dependency environments, making the methods easily accessible to a wider public. We have taken 14 popular methods, the majority related to expression data or promoter analysis, developed these up to a good implementation standard and housed the tools in isolated Docker containers which we integrated into the CyVerse Discovery Environment, making these easily usable for a wide community as part of the CyVerse UK project. The integrated apps can be found at http://www.cyverse.org/discovery-environment, while the raw code is available at https://github.com/cyversewarwick and the corresponding Docker images are housed at https://hub.docker.com/r/cyversewarwick/. info@cyverse.warwick.ac.uk or D.L.Wild@warwick.ac.uk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  5. LHCSR Expression under HSP70/RBCS2 Promoter as a Strategy to Increase Productivity in Microalgae.

    Science.gov (United States)

    Perozeni, Federico; Stella, Giulio Rocco; Ballottari, Matteo

    2018-01-05

    Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, limited biomass productivity is commonly experienced in their cultivating system despite their high potential. One of the reasons for this limitation is the high thermal dissipation of the light absorbed by the outer layers of the cultures exposed to high light caused by the activation of a photoprotective mechanism called non-photochemical quenching (NPQ). In the model organism for green algae Chlamydomonas reinhardtii , NPQ is triggered by pigment binding proteins called light-harvesting-complexes-stress-related (LHCSRs), which are over-accumulated in high light. It was recently reported that biomass productivity can be increased both in microalgae and higher plants by properly tuning NPQ induction. In this work increased light use efficiency is reported by introducing in C. reinhardtii a LHCSR3 gene under the control of Heat Shock Protein 70 / RUBISCO small chain 2 promoter in a npq4 lhcsr1 background, a mutant strain knockout for all LHCSR genes. This complementation strategy leads to a low expression of LHCSR3 , causing a strong reduction of NPQ induction but is still capable of protecting from photodamage at high irradiance, resulting in an improved photosynthetic efficiency and higher biomass accumulation.

  6. CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yongyan [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Ai, Zhiying [Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); College of Life Sciences, Northwest A and F University, Yangling 712100, Shaanxi (China); Yao, Kezhen [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Cao, Lixia; Du, Juan; Shi, Xiaoyan [Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); College of Life Sciences, Northwest A and F University, Yangling 712100, Shaanxi (China); Guo, Zekun, E-mail: gzk@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhylab@hotmail.com [College of Veterinary Medicine, Northwest A and F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi (China)

    2013-10-15

    Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR.

  7. CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

    International Nuclear Information System (INIS)

    Wu, Yongyan; Ai, Zhiying; Yao, Kezhen; Cao, Lixia; Du, Juan; Shi, Xiaoyan; Guo, Zekun; Zhang, Yong

    2013-01-01

    Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR

  8. Insulin stimulates the expression of the SHARP-1 gene via multiple signaling pathways.

    Science.gov (United States)

    Takagi, K; Asano, K; Haneishi, A; Ono, M; Komatsu, Y; Yamamoto, T; Tanaka, T; Ueno, H; Ogawa, W; Tomita, K; Noguchi, T; Yamada, K

    2014-06-01

    The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKCλ) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKCλ/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression. © Georg Thieme Verlag KG Stuttgart · New York.

  9. A randomized controlled trial of single versus multiple health behavior change: promoting physical activity and nutrition among adolescents.

    Science.gov (United States)

    Prochaska, Judith J; Sallis, James F

    2004-05-01

    Targeting multiple behaviors for change may provide significant health benefits. This study compared interventions targeting physical activity and nutrition (PAN) concurrently versus physical activity (PA) alone. Adolescents (N=138) were randomized to the PAN or PA intervention or control condition (n=46 per group). Primary outcomes were change in PA accelerometer and 3-day dietary recording from baseline to 3-month follow-up. The PAN and PA interventions were efficacious in supporting boys' (pdecrement to PA promotion when a nutrition intervention was added, neither do they reveal any additional benefit. More studies comparing single versus multibehavioral interventions are needed. ((c) 2004 APA, all rights reserved)

  10. Expression of osteoblast and osteoclast regulatory genes in the bone marrow microenvironment in multiple myeloma

    DEFF Research Database (Denmark)

    Kristensen, Ida B; Christensen, Jacob Haaber; Lyng, Maria Bibi

    2014-01-01

    Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression...... of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies...... radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our...

  11. Automatic feedback to promote safe walking and speech loudness control in persons with multiple disabilities: two single-case studies.

    Science.gov (United States)

    Lancioni, Giulio E; Singh, Nirbhay N; O'Reilly, Mark F; Green, Vanessa A; Alberti, Gloria; Boccasini, Adele; Smaldone, Angela; Oliva, Doretta; Bosco, Andrea

    2014-08-01

    Assessing automatic feedback technologies to promote safe travel and speech loudness control in two men with multiple disabilities, respectively. The men were involved in two single-case studies. In Study I, the technology involved a microprocessor, two photocells, and a verbal feedback device. The man received verbal alerting/feedback when the photocells spotted an obstacle in front of him. In Study II, the technology involved a sound-detecting unit connected to a throat and an airborne microphone, and to a vibration device. Vibration occurred when the man's speech loudness exceeded a preset level. The man included in Study I succeeded in using the automatic feedback in substitution of caregivers' alerting/feedback for safe travel. The man of Study II used the automatic feedback to successfully reduce his speech loudness. Automatic feedback can be highly effective in helping persons with multiple disabilities improve their travel and speech performance.

  12. Co-regulated expression of HAND2 and DEIN by a bidirectional promoter with asymmetrical activity in neuroblastoma

    Directory of Open Access Journals (Sweden)

    Berthold Frank

    2009-04-01

    Full Text Available Abstract Background HAND2, a key regulator for the development of the sympathetic nervous system, is located on chromosome 4q33 in a head-to-head orientation with DEIN, a recently identified novel gene with stage specific expression in primary neuroblastoma (NB. Both genes are expressed in primary NB as well as most NB cell lines and are separated by a genomic sequence of 228 bp. The similar expression profile of both genes suggests a common transcriptional regulation mediated by a bidirectional promoter. Results Northern Blot analysis of DEIN and HAND2 in 20 primary NBs indicated concurrent expression levels of the two genes, which was confirmed by microarray analysis of 236 primary NBs (Pearson's correlation coefficient r = 0.65. While DEIN expression in the latter cohort was associated with stage 4S (p = 0.02, HAND2 expression was not associated with tumor stage. In contrast, both HAND2 and DEIN transcript levels were highly associated with age at diagnosis DEIN orientation, an average 3.4 fold increase in activity was observed as compared to the promoterless vector, whereas an average 15.4 fold activation was detected in HAND2 orientation. The presence of two highly conserved putative regulatory elements, one of which was shown to enhance HAND2 expression in branchial arches previously, displayed weak repressor activity for both genes. Conclusion HAND2 and DEIN represent a gene pair that is tightly linked by a bidirectional promoter in an evolutionary highly conserved manner. Expression of both genes in NB is co-regulated by asymmetrical activity of this promoter and modulated by the activity of two cis-regulatory elements acting as weak repressors. The concurrent quantitative and tissue specific expression of HAND2 and DEIN suggests a functional link between both genes.

  13. Brn3a regulates neuronal subtype specification in the trigeminal ganglion by promoting Runx expression during sensory differentiation

    Directory of Open Access Journals (Sweden)

    Raisa Eng S

    2010-01-01

    Full Text Available Abstract The transcription factor Brn3a, product of the pou4f1 gene, is expressed in most sensory neurons throughout embryogenesis. Prior work has demonstrated a role for Brn3a in the repression of early neurogenic genes; here we describe a second major role for Brn3a in the specification of sensory subtypes in the trigeminal ganglion (TG. Sensory neurons initially co-express multiple Trk-family neurotrophin receptors, but are later marked by the unique expression of TrkA, TrkB or TrkC. Maturation of these sensory subtypes is known to depend on the expression of Runx transcription factors. Newborn Brn3a knockout mice fail to express TrkC, which is associated in the TG with mechanoreceptors, plus a set of functional genes associated with nociceptor subtypes. In embryonic Brn3a-/- ganglia, the normal expression of Runx3 is never initiated in TrkC+ neurons, and Runx1 expression is greatly attenuated in TrkA+ nociceptors. These changes are accompanied by expanded expression of TrkB in neurons that abnormally express multiple Trks, followed by the loss of TrkC and TrkA expression. In transgenic embryos expressing a Brn3a-VP16 dominant transactivator, Runx3 mRNA expression is increased, suggesting that it is a direct regulatory target of Brn3a. Chromatin immunoprecipitation confirms that Brn3a binds in vivo to a conserved upstream enhancer element within histone H3-acetylated chromatin in the Runx3 locus. Together these data show that Brn3a acts upstream of the Runx factors, which then repress TrkB expression to allow establishment of the non-overlapping Trk receptor profiles and correct terminally differentiated phenotypes.

  14. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella

    NARCIS (Netherlands)

    Mengesha, Asferd; Dubois, Ludwig; Lambin, Philippe; Landuyt, Willy; Chiu, Roland K; Wouters, Bradly G; Theys, Jan

    To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells

  15. Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Rasmussen, Anders Aamann; Byriel, Lene

    2013-01-01

    In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim...... of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management....

  16. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

    Directory of Open Access Journals (Sweden)

    Peter J Romanienko

    Full Text Available The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6. However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

  17. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

    Science.gov (United States)

    Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

    2017-02-20

    We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, P amyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the P amyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter P HpaII -P amyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter P HpaII -P amyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter P HpaII -P amyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter P HpaII -P amyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

  18. Association of diminished expression of RASSF1A with promoter methylation in primary gastric cancer from patients of central China

    Directory of Open Access Journals (Sweden)

    Zhou Feng

    2007-07-01

    Full Text Available Abstract Background Although methylation-mediated inactivation of expression of RASSF1A, a candidate tumor suppressor gene, has been observed in several human cancers, the data concerning alteration of RASSF1A expression and methylation in Chinese primary gastric cancer are scarce. Moreover, direct evidence showing the association between protein expression of RASSF1A and primary human cancers is lacking. The aim of this study was to investigate RASSF1A expression in tissue of primary gastric cancer (GC at mRNA and protein levels, and to establish the possible relationship between DNA methylation status and protein expression of RASSF1A in Chinese. Methods Fifty-four patients with primary gastric cancers were included in the study of RASSF1A mRNA expression and methylation status between the cancer tissue and the corresponding adjacent normal tissue. 20 out of 54 patients were included for study of RASSF1A protein expression. The expression of RASSF1A at mRNA and protein levels was determined by RT-PCR and Western-blotting, respectively. The RASSF1A promoter methylation was detected by methylation-specific PCR. Results RASSF1A mRNA and protein expressions in GC were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2589 ± 0.2407 vs 0.5448 ± 0.2971, P P P P Conclusion Expression of RASSF1A was reduced in tissue of GC at mRNA and protein levels. Diminished expression of RASSF1A was associated with the promoter methylation.

  19. Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.).

    Science.gov (United States)

    Lipka, A; Paukszto, L; Majewska, M; Jastrzebski, J P; Myszczynski, K; Panasiewicz, G; Szafranska, B

    2017-09-01

    The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α 7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α 1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber

  20. The silkworm (Bombyx mori microRNAs and their expressions in multiple developmental stages.

    Directory of Open Access Journals (Sweden)

    Xiaomin Yu

    multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.

  1. Novel system uses probasin-based promoter, transcriptional silencers and amplification loop to induce high-level prostate expression

    Directory of Open Access Journals (Sweden)

    Yu Hong

    2007-02-01

    Full Text Available Abstract Background Despite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity. Results To address this issue, we have developed a regulation strategy that includes feedback amplification of gene expression along with a differentially suppressible tetracycline regulated expression system (DiSTRES. By differentially suppressing expression of the tetracycline-regulated transcriptional activator (tTA and silencer (tTS genes based on the cell origin, this leads to the activation and silencing of the TRE promoter, respectively. In vitro transduction of LNCaP cells with Ad/GFPDiSTRES lead to GFP expression levels that were over 30-fold higher than Ad/CMV-GFP. Furthermore, Ad/FasL-GFPDiSTRES demonstrated cytotoxic effects in prostate cancer cells known to be resistant to Fas-mediated apoptosis. Conclusion Prostate-specific regulation from the DiSTRES system, therefore, serves as a promising new regulation strategy for future applications in the field of cancer gene therapy and gene therapy as a whole.

  2. DNMT 1 maintains hypermethylation of CAG promoter specific region and prevents expression of exogenous gene in fat-1 transgenic sheep.

    Science.gov (United States)

    Yang, Chunrong; Shang, Xueying; Cheng, Lei; Yang, Lei; Liu, Xuefei; Bai, Chunling; Wei, Zhuying; Hua, Jinlian; Li, Guangpeng

    2017-01-01

    Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721-1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals.

  3. Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3

    International Nuclear Information System (INIS)

    Uranishi, Kousuke; Akagi, Tadayuki; Koide, Hiroshi; Yokota, Takashi

    2016-01-01

    Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1–Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb. Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 promoter is activated by Esrrb in association with Ncoa3, and Dax1 inhibited activities of Esrrb and Ncoa3, resulting maintenance of the undifferentiated status of ES cells. - Highlights: • Esrrb induced Gata6 expression in ES cells. • Gata6 promoter activity was enhanced by Esrrb, which was repressed by Dax1. • Dax1 associated with the Gata6 promoter via Esrrb. • Dax1 associated with Ncoa3 and repressed its transcriptional activity.

  4. A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

    Directory of Open Access Journals (Sweden)

    Lu Ying

    2004-04-01

    Full Text Available Abstract Background TGM1(transglutaminase 1 is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. Methods In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. Results In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the

  5. Expression and Genetic Analysis of MicroRNAs Involved in Multiple Sclerosis

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    Daniela Galimberti

    2013-02-01

    Full Text Available Evidence underlines the importance of microRNAs (miRNAs in the pathogenesis of multiple sclerosis (MS. Based on the fact that miRNAs are present in human biological fluids, we previously showed that miR-223, miR-23a and miR-15b levels were downregulated in the sera of MS patients versus controls. Here, the expression levels of these candidate miRNAs were determined in peripheral blood mononuclear cells (PBMCs and the serum of MS patients, in addition to three genotyped single nucleotide polymorphisms (SNPs. Mapping in the genomic regions of miR-223, miR-23a and miR-15b genes, 399 cases and 420 controls were tested. Expression levels of miR-223 and miR-23a were altered in PBMCs from MS patients versus controls. Conversely, there were no differences in the expression levels of miR-15b. A significantly decreased genotypic frequency of miR-223 rs1044165 T/T genotype was observed in MS patients. Moreover, the allelic frequency of miR-23a rs3745453 C allele was significantly increased in patients versus controls. In contrast, there were no differences in the distribution of miR-15b SNP. In conclusion, our results suggest that miR-223 and miR-23a could play a role in the pathogenesis of MS. Moreover, miR-223 rs1044165 polymorphism likely acts as a protective factor, while miR-23a rs3745453 variant seems to act as a risk factor for MS.

  6. Expression and Genetic Analysis of MicroRNAs Involved in Multiple Sclerosis.

    Science.gov (United States)

    Ridolfi, Elisa; Fenoglio, Chiara; Cantoni, Claudia; Calvi, Alberto; De Riz, Milena; Pietroboni, Anna; Villa, Chiara; Serpente, Maria; Bonsi, Rossana; Vercellino, Marco; Cavalla, Paola; Galimberti, Daniela; Scarpini, Elio

    2013-02-25

    Evidence underlines the importance of microRNAs (miRNAs) in the pathogenesis of multiple sclerosis (MS). Based on the fact that miRNAs are present in human biological fluids, we previously showed that miR-223, miR-23a and miR-15b levels were downregulated in the sera of MS patients versus controls. Here, the expression levels of these candidate miRNAs were determined in peripheral blood mononuclear cells (PBMCs) and the serum of MS patients, in addition to three genotyped single nucleotide polymorphisms (SNPs). Mapping in the genomic regions of miR-223, miR-23a and miR-15b genes, 399 cases and 420 controls were tested. Expression levels of miR-223 and miR-23a were altered in PBMCs from MS patients versus controls. Conversely, there were no differences in the expression levels of miR-15b. A significantly decreased genotypic frequency of miR-223 rs1044165 T/T genotype was observed in MS patients. Moreover, the allelic frequency of miR-23a rs3745453 C allele was significantly increased in patients versus controls. In contrast, there were no differences in the distribution of miR-15b SNP. In conclusion, our results suggest that miR-223 and miR-23a could play a role in the pathogenesis of MS. Moreover, miR-223 rs1044165 polymorphism likely acts as a protective factor, while miR-23a rs3745453 variant seems to act as a risk factor for MS.

  7. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

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    Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F

    2010-02-04

    Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  8. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I

    2010-02-01

    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  9. Promoter motifs required for c-mpl gene expression induced by thrombopoietin in CMK cells.

    Science.gov (United States)

    Sunohara, Masataka; Sato, Iwao; Morikawa, Shigeru

    2017-11-30

    Thrombopoietin (TPO) and its receptor, c-Mpl, are the central regulators of megakaryocyte development and platelet production and are also crucial to regulate megakaryocytopoiesis. TPO remarkably elevated c-mpl promoter activity, while the protein kinase C (PKC) inhibitors, GF109203, H7 and Calphostin C, clearly reduced the steady level of its promoter activity.  In the present study, motifs crucial for c-mpl promoter activity induced by TPO treatment have been analyzed using a human megakaryoblastic cell line, CMK. Destruction of the -107Sp1 and the -57Sp1 sites in the c-mpl promoter enhancer region resulted in decrease of the promoter activity by 53.1% and 64.4%, respectively, and destruction of -69Ets and -28Ets elements dramatically decreased the promoter activity by 96.4% and 87.8%, respectively, while mutation of -77GATA moderately reduced the activity by 31.4%. The result was in agreement with our previous report that showed the crucial motifs in the c-mpl promoter for the promoter activity induced by PMA-treatment. This indicates that TPO-induced activation of the c-mpl promoter activity is fully modulated by transcription through a PKC-dependent pathway and the two Sp1 and two Ets motifs are crucial for the activation of the c-mpl promoter activity rather than a GATA motif in the c-mpl promoter of CMK cells.

  10. Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

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    Kawe Martin

    2009-01-01

    Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

  11. Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis.

    Science.gov (United States)

    Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua

    2011-11-04

    The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

    2011-01-01

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

  13. Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host.

    Science.gov (United States)

    Song, Wan; Nie, Yao; Mu, Xiao Qing; Xu, Yan

    2016-08-01

    Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Progressive practice promotes motor learning and repeated transient increases in corticospinal excitability across multiple days

    DEFF Research Database (Denmark)

    Christiansen, Lasse; Madsen, Mads Alexander Just; Bojsen-Møller, Emil

    2018-01-01

    Background: A session of motor skill learning is accompanied by transient increases in corticospinal excitability (CSE), which are thought to reflect acute changes in neuronal connectivity associated with improvements in sensorimotor performance. Factors influencing changes in excitability...... and motor skill with continued practice remain however to be elucidated. Objective/Hypothesis: Here we investigate the hypothesis that progressive motor practice during consecutive days can induce repeated transient increases in corticospinal excitability and promote motor skill learning. Methods: Changes...... in motor performance and CSE were assessed during 4 consecutive days of skill learning and 8 days after the last practice session. CSE was assessed as area under recruitment curves (RC) using transcranial magnetic stimulation (TMS). Two groups of participants (n = 12) practiced a visuomotor tracking...

  15. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

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    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  16. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    Energy Technology Data Exchange (ETDEWEB)

    Rieken, Stefan; Simon, Florian; Habermehl, Daniel; Dittmar, Jan Oliver; Combs, Stephanie E.; Weber, Klaus; Debus, Juergen; Lindel, Katja [University Hospital of Heidelberg, Department of Radiation Therapy and Radiation Oncology, Heidelberg (Germany)

    2014-10-15

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.) [German] Persistierende Infektionen mit humanen Papillomaviren 16 (HPV16) sind ein Hauptausloeser des Zervixkarzinoms. Die Integration der viralen DNS in das Wirtszellgenom fuehrt zum Integritaetsverlust des E2-Gens, wodurch in der Wirtszelle Apoptose verhindert und Motilitaet gesteigert werden. In

  17. Process evaluation of a community-based intervention promoting multiple maternal and neonatal care practices in rural Nepal

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    Silwal Ram C

    2010-06-01

    Full Text Available Abstract Background The challenge of delivering multiple, complex messages to promote maternal and newborn health in the terai region of Nepal was addressed through training Female Community Health Volunteers (FCHVs to counsel pregnant women and their families using a flipchart and a pictorial booklet that was distributed to clients. The booklet consists of illustrated messages presented on postcard-sized laminated cards that are joined by a ring. Pregnant women were encouraged to discuss booklet content with their families. Methods We examined use of the booklet and factors affecting adoption of practices through semi-structured interviews with district and community-level government health personnel, staff from the Nepal Family Health Program, FCHVs, recently delivered women and their husbands and mothers-in-law. Results The booklet is shared among household members, promotes discussion, and is referred to when questions arise or during emergencies. Booklet cards on danger signs and nutritious foods are particularly well-received. Cards on family planning and certain aspects of birth preparedness generate less interest. Husbands and mothers-in-law control decision-making for maternal and newborn care-seeking and related household-level behaviors. Conclusions Interpersonal peer communication through trusted community-level volunteers is an acceptable primary strategy in Nepal for promotion of household-level behaviors. The content and number of messages should be simplified or streamlined before being scaled-up to minimize intervention complexity and redundant communication.

  18. A novel Meloidogyne graminicola effector, MgMO237, interacts with multiple host defence-related proteins to manipulate plant basal immunity and promote parasitism.

    Science.gov (United States)

    Chen, Jiansong; Hu, Lili; Sun, Longhua; Lin, Borong; Huang, Kun; Zhuo, Kan; Liao, Jinling

    2018-02-27

    Plant-parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up-regulated in parasitic third-/fourth-stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two-hybrid and co-immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3-β-glucan synthase component (OsGSC), cysteine-rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis-related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence-related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence-related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes. © 2018 BSPP AND JOHN WILEY & SONS LTD.

  19. A Computerized Lifestyle Application to Promote Multiple Health Behaviors at the Workplace: Testing Its Behavioral and Psychological Effects.

    Science.gov (United States)

    Lippke, Sonia; Fleig, Lena; Wiedemann, Amelie U; Schwarzer, Ralf

    2015-10-01

    Preventive health behaviors, such as regular physical activity and healthy nutrition, are recommended to maintain employability and to facilitate the health of employees. Theory-based workplace health promotion needs to include psychological constructs and consider the motivational readiness (so-called stages of change) of employees. According to the stages, people can be grouped as nonintenders (not motivated to change and not performing the goal behavior), intenders (decided to adopt the goal behavior but not started yet), or actors (performing the goal behavior already). The tailoring to these stages can be done computer based and should make workplace health promotion more effective. It was tested whether a parsimonious computer-based health promotion program implemented at the workplace was effective in terms of lifestyle changes and psychological outcomes as well as body weight. We hypothesized that the stage-matched intervention would outperform the one-size-fits-all active control condition (standard care intervention). In a randomized controlled trial, a total of 1269 employees were recruited by a trained research assistant at their workplace during a routine medical examination. After excluding noneligible employees, 560 completed Time 1 (T1), and 384 also completed Time 2 (T2), achieving a retention rate of 68.6%. Two fully automated computer-based treatments were adopted: (1) an active control condition with information about benefits of exercise and healthy nutrition (n=52), or (2) a stage-matched multiple-behavior intervention that provided different psychological treatments to 9 subgroups, addressing stages of change (nonintenders, intenders, and actors per behavior; n=332). Baseline assessments (T1) on behavior, psychological constructs, and body weight were repeated after 4 weeks (T2). The stage-matched intervention outperformed the active control condition for lifestyle changes containing physical activity and nutrition (χ(2) 1=3.5; P=.04, for N

  20. A Baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori.

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    Mika Masumoto

    Full Text Available Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively; however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.

  1. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

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    Guo, Kai, E-mail: gk161@163.com [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Department of Respiration, 161th Hospital, PLA, Wuhan 430015 (China); Jin, Faguang, E-mail: jinfag@fmmu.edu.cn [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  2. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    International Nuclear Information System (INIS)

    Guo, Kai; Jin, Faguang

    2015-01-01

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells

  3. Expression of multiple sexual signals by fathers and sons in the East-Mediterranean barn swallow: are advertising strategies heritable?

    Science.gov (United States)

    Vortman, Yoni; Safran, Rebecca J; Reiner Brodetzki, Tali; Dor, Roi; Lotem, Arnon

    2015-01-01

    The level of expression of sexually selected traits is generally determined by genes, environment and their interaction. In species that use multiple sexual signals which may be costly to produce, investing in the expression of one sexual signal may limit the expression of the other, favoring the evolution of a strategy for resource allocation among signals. As a result, even when the expression of sexual signals is condition dependent, the relative level of expression of each signal may be heritable. We tested this hypothesis in the East-Mediterranean barn swallow (Hirundo rustica transitiva), in which males have been shown to express two uncorrelated sexual signals: red-brown ventral coloration, and long tail streamers. We show that variation in both signals may partially be explained by age, as well as by paternal origin (genetic father-son regressions), but that the strongest similarity between fathers and sons is the relative allocation towards one trait or the other (relative expression index), rather than the expression of the traits themselves. These results suggest that the expression of one signal is not independent of the other, and that genetic strategies for resource allocation among sexual signals may be selected for during the evolution of multiple sexual signals.

  4. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    International Nuclear Information System (INIS)

    Zuo, Luning; Li, Qiang; Sun, Bei; Xu, Zhiying; Ge, Zhiming

    2013-01-01

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  5. AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

    Science.gov (United States)

    Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

    2018-04-01

    The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

  6. Multiple independent regulatory pathways control UBI4 expression after heat shock in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, J R; Treger, J M; McEntee, K

    1999-02-01

    Transcription of the polyubiquitin gene UBI4 of Saccharomyces cerevisiae is strongly induced by a variety of environmental stresses, such as heat shock, nutrient depletion and exposure to DNA-damaging agents. This transcriptional response of UBI4 is likely to be the primary mechanism for increasing the pool of ubiquitin for degradation of stress-damaged proteins. Deletion and promoter fusion studies of the 5' regulatory sequences indicated that two different elements, heat shock elements (HSEs) and stress response element (STREs), contributed independently to heat shock regulation of the UBI4 gene. In the absence of HSEs, STRE sequences localized to the intervals -264 to -238 and -215 to -183 were needed for stress control of transcription after heat shock. Site-directed mutagenesis of the STRE (AG4) at -252 to -248 abolished heat shock induction of UBI4 transcription. Northern analysis demonstrated that cells containing either a temperature-sensitive HSF or non-functional Msn2p/Msn4p transcription factors induced high levels of UBI4 transcripts after heat shock. In cells deficient in both heat stress pathways, heat-induced UBI4 transcript levels were considerably lower but not abolished, suggesting a role for another factor(s) in stress control of its expression.

  7. Distinct profiles of TERT promoter mutations and telomerase expression in head and neck cancer and cervical carcinoma.

    Science.gov (United States)

    Annunziata, Clorinda; Pezzuto, Francesca; Greggi, Stefano; Ionna, Franco; Losito, Simona; Botti, Gerardo; Buonaguro, Luigi; Buonaguro, Franco M; Tornesello, Maria Lina

    2018-03-31

    Two recurrent mutations (-124 G > A and -146 G > A) in the core promoter region of the human telomerase reverse transcriptase (TERT) gene create consensus binding sites for ETS transcription factors and cause increased TERT expression in several tumour types. We analyzed TERT promoter mutations and TERT mRNA levels in head and neck cancer, cervical carcinoma and cervical intraepithelial neoplasia (CIN) as well as in C-4I, CaSki, HeLa and SiHa cervical cell lines. Nucleotide sequence analysis of TERT promoter region showed that 33.3% of oral squamous cell carcinoma (SCC) and 16.8% of cervical SCC harboured mutually exclusive G to A transitions at nucleotide position -124 or -146. TERT promoter was mutated at nucleotide -146 (G > A) in SiHa cell line. Other nucleotide changes creating in some cases putative ETS binding sites were more frequent in oral SCC (26.7%) than in cervical carcinoma (4.8%). The frequency of mutations was independent of human papillomavirus (HPV) tumour status in both cervical and oral cancer. Expression of TERT gene was significantly higher in TERT promoter mutated (-124G > A or -146G > A) cervical SCC compared to not mutated SCC irrespective of HPV16 E6 and E7 levels. Such hot spot changes were not detected in oropharyngeal SCC, cervical adenocarcinoma and CIN lesions. Our results suggest that TERT promoter mutations play a relevant role in oral SCC as well as in cervical SCC, besides the already known effect of HPV16 E6 protein on TERT expression. © 2018 UICC.

  8. Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.

    Science.gov (United States)

    Blount, Benjamin A; Weenink, Tim; Vasylechko, Serge; Ellis, Tom

    2012-01-01

    Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex "multi-wire" logic functions.

  9. Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

    Science.gov (United States)

    Blount, Benjamin A.; Weenink, Tim; Vasylechko, Serge; Ellis, Tom

    2012-01-01

    Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions. PMID:22442681

  10. Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways.

    Science.gov (United States)

    Jung, Hyun-Joo; Jeon, Yong-Heui; Bokara, Kiran Kumar; Koo, Bon-Nyeo; Lee, Won Taek; Park, Kyung Ah; Lee, Jong-Eun

    2013-01-17

    The combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. The effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. Agmatine treatment (50, 100 and 200 μM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. Taken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer

    Science.gov (United States)

    Metelli, Alessandra; Wu, Bill X; Fugle, Caroline W; Rachidi, Saleh; Sun, Shaoli; Zhang, Yongliang; Wu, Jennifer; Tomlinson, Stephen; Howe, Philip; Yang, Yi; Garrett-Mayer, Elizabeth; Liu, Bei; Li, Zihai

    2016-01-01

    GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGF-β which is expressed naturally by platelets and regulatory T cells. Although Lrrc32 is amplified frequently in breast cancer, the expression and relevant functions of GARP in cancer have not been explored. Here we report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGF-β in the tumor microenvironment. We found that human breast, lung and colon cancers expressed GARP aberrantly. In genetic studies in normal mammary gland epithelial and carcinoma cells, GARP expression increased TGF-β bioactivity and promoted malignant transformation in immune deficient mice. In breast carcinoma-bearing mice that were immune competent, GARP overexpression promoted Foxp3+ regulatory T cell activity, which in turn contributed to enhancing cancer progression and metastasis. Notably, administration of a panel of GARP-specific monoclonal antibodies limited metastasis in an orthotopic model of human breast cancer. Overall, these results define the oncogenic effects of the GARP-TGF-β axis in the tumor microenvironment and suggest mechanisms that might be exploited for diagnostic and therapeutic purposes. PMID:27913437

  12. Meta-analysis methods for combining multiple expression profiles: comparisons, statistical characterization and an application guideline.

    Science.gov (United States)

    Chang, Lun-Ching; Lin, Hui-Min; Sibille, Etienne; Tseng, George C

    2013-12-21

    As high-throughput genomic technologies become accurate and affordable, an increasing number of data sets have been accumulated in the public domain and genomic information integration and meta-analysis have become routine in biomedical research. In this paper, we focus on microarray meta-analysis, where multiple microarray studies with relevant biological hypotheses are combined in order to improve candidate marker detection. Many methods have been developed and applied in the literature, but their performance and properties have only been minimally investigated. There is currently no clear conclusion or guideline as to the proper choice of a meta-analysis method given an application; the decision essentially requires both statistical and biological considerations. We performed 12 microarray meta-analysis methods for combining multiple simulated expression profiles, and such methods can be categorized for different hypothesis setting purposes: (1) HS(A): DE genes with non-zero effect sizes in all studies, (2) HS(B): DE genes with non-zero effect sizes in one or more studies and (3) HS(r): DE gene with non-zero effect in "majority" of studies. We then performed a comprehensive comparative analysis through six large-scale real applications using four quantitative statistical evaluation criteria: detection capability, biological association, stability and robustness. We elucidated hypothesis settings behind the methods and further apply multi-dimensional scaling (MDS) and an entropy measure to characterize the meta-analysis methods and data structure, respectively. The aggregated results from the simulation study categorized the 12 methods into three hypothesis settings (HS(A), HS(B), and HS(r)). Evaluation in real data and results from MDS and entropy analyses provided an insightful and practical guideline to the choice of the most suitable method in a given application. All source files for simulation and real data are available on the author's publication website.

  13. Increased expression of hepatocyte nuclear factor 4 alpha transcribed by promoter 2 indicates a poor prognosis in hepatocellular carcinoma

    OpenAIRE

    Cai, Shao-hang; Lu, Shi-xun; Liu, Li-li; Zhang, Chris Zhiyi; Yun, Jing-ping

    2017-01-01

    Background: Hepatocyte nuclear factor 4 alpha (HNF4α) plays an important role in tumourigenesis. There is growing evidence indicating that HNF4α transcribed by promoter 1 (P1-HNF4α) is expressed at relatively low levels in HCC and its presence predicts a favourable outcome for hepatocellular carcinoma (HCC) patients. However, the role of HNF4α transcribed by promoter 2 (P2-HNF4α) in HCC remains unclear. Methods: A total of 615 HCC specimens were obtained to construct tissue microarrays and pe...

  14. Impact of genetic risk loci for multiple sclerosis on expression of proximal genes in patients

    KAUST Repository

    James, Tojo

    2018-01-06

    Despite advancements in genetic studies, it is difficult to understand and characterize the functional relevance of disease-associated genetic variants, especially in the context of a complex multifactorial disease such as Multiple Sclerosis (MS). Since a large proportion of expression quantitative trait loci (eQTLs) are context-specific, we performed RNA-Seq in peripheral blood mononuclear cells (PBMCs) from MS patients (n=145) to identify eQTLs in regions centered on 109 MS risk SNPs and seven associated HLA variants. We identified 77 statistically significant eQTL associations, including pseudogenes and non-coding RNAs. Thirty-eight out of 40 testable eQTL effects were colocalised with the disease association signal. Since many eQTLs are tissue specific, we aimed to detail their significance in different cell types. Approximately 70% of the eQTLs were replicated and characterized in at least one major PBMC derived cell type. Furthermore, 40% of eQTLs were found to be more pronounced in MS patients compared to noninflammatory neurological diseases patients. In addition, we found two SNPs to be significantly associated with the proportions of three different cell types. Mapping to enhancer histone marks and predicted transcription factor binding sites added additional functional evidence for eight eQTL regions. As an example, we found that rs71624119, shared with three other autoimmune diseases and located in a primed enhancer (H3K4me1) with potential binding for STAT transcription factors, significantly associates with ANKRD55 expression. This study provides many novel and validated targets for future functional characterization of MS and other diseases.

  15. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  16. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

    2011-01-01

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  17. Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

    Science.gov (United States)

    Schausi, Diane; Tiffoche, Christophe; Thieulant, Marie-Lise

    2003-07-01

    We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

  18. A Hybrid One-Way ANOVA Approach for the Robust and Efficient Estimation of Differential Gene Expression with Multiple Patterns.

    Directory of Open Access Journals (Sweden)

    Mohammad Manir Hossain Mollah

    Full Text Available Identifying genes that are differentially expressed (DE between two or more conditions with multiple patterns of expression is one of the primary objectives of gene expression data analysis. Several statistical approaches, including one-way analysis of variance (ANOVA, are used to identify DE genes. However, most of these methods provide misleading results for two or more conditions with multiple patterns of expression in the presence of outlying genes. In this paper, an attempt is made to develop a hybrid one-way ANOVA approach that unifies the robustness and efficiency of estimation using the minimum β-divergence method to overcome some problems that arise in the existing robust methods for both small- and large-sample cases with multiple patterns of expression.The proposed method relies on a β-weight function, which produces values between 0 and 1. The β-weight function with β = 0.2 is used as a measure of outlier detection. It assigns smaller weights (≥ 0 to outlying expressions and larger weights (≤ 1 to typical expressions. The distribution of the β-weights is used to calculate the cut-off point, which is compared to the observed β-weight of an expression to determine whether that gene expression is an outlier. This weight function plays a key role in unifying the robustness and efficiency of estimation in one-way ANOVA.Analyses of simulated gene expression profiles revealed that all eight methods (ANOVA, SAM, LIMMA, EBarrays, eLNN, KW, robust BetaEB and proposed perform almost identically for m = 2 conditions in the absence of outliers. However, the robust BetaEB method and the proposed method exhibited considerably better performance than the other six methods in the presence of outliers. In this case, the BetaEB method exhibited slightly better performance than the proposed method for the small-sample cases, but the the proposed method exhibited much better performance than the BetaEB method for both the small- and large

  19. Human Neural Precursor Cells Promote Neurologic Recovery in a Viral Model of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Lu Chen

    2014-06-01

    Full Text Available Using a viral model of the demyelinating disease multiple sclerosis (MS, we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4+CD25+FOXP3+ regulatory T cells (Tregs within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments.

  20. A Computerized Lifestyle Application to Promote Multiple Health Behaviors at the Workplace: Testing Its Behavioral and Psychological Effects

    Science.gov (United States)

    Fleig, Lena; Wiedemann, Amelie U; Schwarzer, Ralf

    2015-01-01

    Background Preventive health behaviors, such as regular physical activity and healthy nutrition, are recommended to maintain employability and to facilitate the health of employees. Theory-based workplace health promotion needs to include psychological constructs and consider the motivational readiness (so-called stages of change) of employees. According to the stages, people can be grouped as nonintenders (not motivated to change and not performing the goal behavior), intenders (decided to adopt the goal behavior but not started yet), or actors (performing the goal behavior already). The tailoring to these stages can be done computer based and should make workplace health promotion more effective. Objective It was tested whether a parsimonious computer-based health promotion program implemented at the workplace was effective in terms of lifestyle changes and psychological outcomes as well as body weight. We hypothesized that the stage-matched intervention would outperform the one-size-fits-all active control condition (standard care intervention). Methods In a randomized controlled trial, a total of 1269 employees were recruited by a trained research assistant at their workplace during a routine medical examination. After excluding noneligible employees, 560 completed Time 1 (T1), and 384 also completed Time 2 (T2), achieving a retention rate of 68.6%. Two fully automated computer-based treatments were adopted: (1) an active control condition with information about benefits of exercise and healthy nutrition (n=52), or (2) a stage-matched multiple-behavior intervention that provided different psychological treatments to 9 subgroups, addressing stages of change (nonintenders, intenders, and actors per behavior; n=332). Baseline assessments (T1) on behavior, psychological constructs, and body weight were repeated after 4 weeks (T2). Results The stage-matched intervention outperformed the active control condition for lifestyle changes containing physical activity and

  1. Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer

    International Nuclear Information System (INIS)

    Thangapazham, Rajesh; Saenz, Francisco; Katta, Shilpa; Mohamed, Ahmed A; Tan, Shyh-Han; Petrovics, Gyorgy; Srivastava, Shiv; Dobi, Albert

    2014-01-01

    In normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context. Comparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay. Comparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion. These observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis

  2. Etanercept Promotes Bone Formation via Suppression of Dickkopf-1 Expression in Rats with Collagen-Induced Arthritis

    Science.gov (United States)

    Tanida, Atsushi; Kishimoto, Yuji; Okano, Toru; Hagino, Hiroshi

    2013-01-01

    Background Various clinical reports suggest etanercept (ETN) has some efficacy in bone formation in rheumatoid arthritis (RA). To examine this effect, we investigated the gene expression of cytokines relevant to osteoblast/osteoclast differentiation, and evaluated histomorphometric findings in mature rats with collagen-induced arthritis (CIA). Methods Total RNA was extracted from knee joints with CIA after ETN or placebo administration. Subsequently, realtime-PCR was carried out to quantify the mRNAs encoding Wnt-1, Dickkopf-1 (DKK-1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegelin (OPG) and TNF (tumor necrosis factor)-alpha. In histomorphometric analysis, the infiltrating pannus volume and pannus surface, and the following items in contact with pannus surface were measured: osteoclast number, osteoid surface, osteoid volume and labeling surface. These were evaluated in the distal femur with CIA with or without ETN administration. Results TNF-alpha, RANKL and OPG mRNA expressions, linked to osteoclastogenesis, were not significantly different with or without ETN administration. ETN administration significantly increased Wnt-1 mRNA expression, the osteoblast promoter, and decreased DKK-1 mRNA expression, the Wnt signal inhibitor. In histomorphometric analysis, pannus volume, pannus surface and osteoclast number, parameters of bone destruction, were not significantly different among groups. Osteoid volume, osteoid surface and labeling surface, parameters of bone formation, increased significantly with ETN administration. Conclusion Our results suggest that ETN suppresses DDK-1 expression, and, as a result, Wnt expression is promoted and osteoblastogenesis becomes more active, independent of the regulation of osteoclast activity. Marked bone formation is attributed to the fact that ETN directly promotes osteoblastogenesis, not as a result of suppressing osteoclastogenesis. PMID:24031147

  3. Identification of gene expression patterns crucially involved in experimental autoimmune encephalomyelitis and multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Martin M. Herrmann

    2016-10-01

    Full Text Available After encounter with a central nervous system (CNS-derived autoantigen, lymphocytes leave the lymph nodes and enter the CNS. This event leads only rarely to subsequent tissue damage. Genes relevant to CNS pathology after cell infiltration are largely undefined. Myelin-oligodendrocyte-glycoprotein (MOG-induced experimental autoimmune encephalomyelitis (EAE is an animal model of multiple sclerosis (MS, a chronic autoimmune disease of the CNS that results in disability. To assess genes that are involved in encephalitogenicity and subsequent tissue damage mediated by CNS-infiltrating cells, we performed a DNA microarray analysis from cells derived from lymph nodes and eluted from CNS in LEW.1AV1 (RT1av1 rats immunized with MOG 91-108. The data was compared to immunizations with adjuvant alone or naive rats and to immunizations with the immunogenic but not encephalitogenic MOG 73-90 peptide. Here, we show involvement of Cd38, Cxcr4 and Akt and confirm these findings by the use of Cd38-knockout (B6.129P2-Cd38tm1Lnd/J mice, S1P-receptor modulation during EAE and quantitative expression analysis in individuals with MS. The hereby-defined underlying pathways indicate cellular activation and migration pathways mediated by G-protein-coupled receptors as crucial events in CNS tissue damage. These pathways can be further explored for novel therapeutic interventions.

  4. Multiple embryonic origins of nitric oxide synthase-expressing GABAergic neurons of the neocortex

    Directory of Open Access Journals (Sweden)

    Lorenza eMagno

    2012-09-01

    Full Text Available Cortical GABAergic interneurons in rodents originate in three subcortical regions: the medial ganglionic eminence (MGE, the lateral/caudal ganglionic eminence (LGE/CGE and the preoptic area (POA. Each of these neuroepithelial precursor domains contributes different interneuron subtypes to the cortex. nNOS-expressing neurons represent a heterogenous population of cortical interneurons. We examined the development of these cells in the mouse embryonic cortex and their abundance and distribution in adult animals. Using genetic lineage tracing in transgenic mice we find that nNOS type I cells originate only in the MGE whereas type II cells have a triple origin in the MGE, LGE/CGE and POA. The two populations are born at different times during development, occupy different layers in the adult cortex and have distinct neurochemical profiles. nNOS neurons are more numerous in the adult cortex than previously reported and constitute a significant proportion of the cortical interneuron population. Our data suggest that the heterogeneity of nNOS neurons in the cortex can be attributed to their multiple embryonic origins which likely impose distinct genetic specification programs.

  5. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  6. The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

    Science.gov (United States)

    2012-01-01

    Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available

  7. Promoting Physical Activity Through a Manual Wheelchair Propulsion Intervention in Persons With Multiple Sclerosis.

    Science.gov (United States)

    Rice, Ian M; Rice, Laura A; Motl, Robert W

    2015-10-01

    To examine the efficacy and feasibility of a multifactorial intervention to increase lifestyle physical activity in nonambulatory persons with multiple sclerosis (MS) based on wheelchair optimization, propulsion skill/technique training, and behavioral strategies based on social cognitive theory. Randomized controlled trial, 3-month postintervention follow-up. Home and general community, and university research laboratory. Nonambulatory individuals with MS (N=14; mean age ± SD, 53.6±8.7y) were randomly assigned to an intervention group (IG) or a control group (CG). After baseline testing, the IG participants received custom-fit, ultralightweight manual wheelchairs with propulsion/skills training, followed by 3 months of at-home use with the custom ultralightweight wheelchair and weekly phone calls to deliver support through a multifactorial intervention. The CG participants received no training and used their own wheelchairs at home during this time. All subjects were assessed at baseline and 3 months later for fatigue (Fatigue Severity Scale), upper extremity strength (digital handheld dynamometer), and propulsion technique (on a treadmill [0.5m/s] with instrumented wheels). Two 1-week bouts of physical activity were measured in both groups from home with wrist-worn accelerometry at the beginning (IG and CG in own wheelchairs) and end (IG in study wheelchair, CG in own) of the 3-month period of home use. The intervention was well tolerated, and no adverse events were reported. The IG demonstrated increased strength (P=.008) and a trend toward less fatigue (P=.068), both with large effect sizes (d>0.8), as well as reduced application of braking torque during propulsion (P=.003) with a moderate/large effect size (d=.73), compared with the CG. Findings suggest a 3-month physical activity intervention based on manual wheelchair propulsion and training is safe and feasible for some wheelchair users living with MS and may produce secondary benefits in strength

  8. Expression of the 90K immunostimulator gene is controlled by a promoter with unique features

    DEFF Research Database (Denmark)

    Brakebusch, C; Jallal, B; Fusco, O

    1997-01-01

    T3 cells; however, it contained neither a TATA-box nor a SP1 site and was not GC-rich. No known initiator motif was found around the transcription start site. 5'- and 3'-deletions defined a minimal promoter of 51 base pairs (-66 --> -16), not including the start site, essential and sufficient...... for promoter activity. This minimal promoter showed increased activity after stimulation with interferon-gamma or poly(I.C), a substance mimicking viral infection. Essential for both inductions was the integrity of an interferon regulatory factor element within this sequence, a potential binding site...

  9. LncRNA CCAT2 promotes tumorigenesis by over-expressed Pokemon in non-small cell lung cancer.

    Science.gov (United States)

    Zhao, Zhihong; Wang, Ju; Wang, Shengfa; Chang, Hao; Zhang, Tiewa; Qu, Junfeng

    2017-03-01

    Non-small cell lung cancer (NSCLC) remains one of the most important death-related diseases, with poor effective diagnosis and less therapeutic biomarkers. LncRNA colon cancer-associated transcript 2 (CCAT2) was identified as an oncogenic lncRNA and over-expressed in many tumor cells. The aims of this study were to detect the correlation between CCAT2 and its regulatory genes and then explore the potential mechanism between them in NSCLC. In this study, qRT-PCR was used to detect CCAT2, Pokemon and p21 expression. Western-blot was used to detect protein levels of Pokemon and p21. CCK-8 assay and Transwell chambers were used to assess cell viability and invasion. CCAT2 and Pokemon were over-expressed in NSCLC tissue and cells. In NSCLC cells, CCAT2 knockdown significantly decreased cell viability and invasion as well as Pokemon expression, but increased the expression of p21; then CCAT2 overexpression revealed an opposite result. In addition, over-expressed Pokemon reversed the results that induced by si-CCAT2, while down-regulation of Pokemon significantly reversed the results that induced by CCAT2 overexpression. The results indicated that CCAT2 promotes tumorigenesis by over-expression of Pokemon, and the potential mechanism might relate to the Pokemon related gene p21. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. [Adipose-derived stem cell transplantation promotes the expression of netrin-1 in the rat cortex after focal cerebral ischemia].

    Science.gov (United States)

    Wang, Jiehua; Hong, Zhuquan; Pan, Ying; Li, Guoqian

    2017-01-01

    Objective To observe the effect of adipose-derived stem cells (ADSCs) transplantation on the expression of netrin-1 in rats after focal cerebral ischemia. Methods Male SD rats were randomly divided into control group, model group and ADSC group. ADSCs were harvested and purified. Focal cerebral ischemia models were established in rats by the suture method. ADSCs were injected into the lateral ventricle of ADSC group rats and the same does of PBS was given to model group rats. At day 4, 7 and 14 after reperfusion, six rats were sacrificed to remove the brain tissues at each time point. The expression of netrin-1 was detected by reverse-transcription PCR, Western blotting and immunohistochemistry. Results Compared with the control group, the expression of netrin-1 in the brain tissues of the model group increased after focal cerebral ischemia, reached the peak at 4 days, and the expression of netrin-1 was significantly higher than that of the control group at each time point. Compared with the model group, the expression of netrin-1 in the ADSC group increased further, reached the peak at 7 days, and the expression of netrin-1 in the ADSC group was significantly higher than that of the model group at each time point. Conclusion ADSC transplantation could up-regulate the expression of netrin-1, and promote axon regeneration and the recovery of neurological functions.

  11. Exercise promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body β-hydroxybutyrate.

    Science.gov (United States)

    Sleiman, Sama F; Henry, Jeffrey; Al-Haddad, Rami; El Hayek, Lauretta; Abou Haidar, Edwina; Stringer, Thomas; Ulja, Devyani; Karuppagounder, Saravanan S; Holson, Edward B; Ratan, Rajiv R; Ninan, Ipe; Chao, Moses V

    2016-06-02

    Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.

  12. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

    Directory of Open Access Journals (Sweden)

    Tung Shu-Yun

    2011-04-01

    Full Text Available Abstract Background Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. Results Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR. A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI genes (OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. Conclusions By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

  13. Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

    Science.gov (United States)

    Bando, Hiroki; Hisada, Hiromoto; Ishida, Hiroki; Hata, Yoji; Katakura, Yoshio; Kondo, Akihiko

    2011-11-01

    A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

  14. Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter

    Directory of Open Access Journals (Sweden)

    Hiu Man Grisch-Chan

    2017-06-01

    Full Text Available Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH by delivery of naked DNA/minicircle (MC-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU. Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of 95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

  15. Expression analysis of four flower-specific promoters of Brassica spp ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... power of the promoter. Besides, several enhancer and suppressor regions were also identified in the ... of the target gene and decrement of the burden of plant growth and ..... Meyerowitz's ABC model of flower development.

  16. The impact of self-transcendence on physical health status promotion in multiple sclerosis patients attending peer support groups.

    Science.gov (United States)

    JadidMilani, Maryam; Ashktorab, Tahereh; AbedSaeedi, Zhila; AlaviMajd, Hamid

    2015-12-01

    This study aimed to investigate the effect of self-transcendence on the physical health of multiple sclerosis (MS) patients attending peer support groups. This study was a quasi-experimental before-and-after design including 33 MS patients in three groups: 10 men in the men-only group, 11 women in the women-only group, and 12 men and women in the mixed group. Participants were required to attend eight weekly sessions of 2 h each. Instruments included the physical health section of the Multiple Sclerosis Quality of Life Inventory and Reed's Self-Transcendence Scale. Peer support group attendance was found to have a significant positive effect on the physical health and self-transcendence of MS patients when comparing average scores before and after attendance. Regression analysis showed that improvement in self-transcendence predicted improvement in physical health. Results show the positive effects of peer support groups on self-transcendence and physical health in MS patients, and suggest that improvement in well-being can be gained by promoting self-transcendence and physical health. © 2015 Wiley Publishing Asia Pty Ltd.

  17. All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

    Directory of Open Access Journals (Sweden)

    Fang Bo

    2009-07-01

    Full Text Available Abstract Background All-trans retinoic acid (RA is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs, we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture. Results Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation. Conclusion RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.

  18. Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response

    Directory of Open Access Journals (Sweden)

    Wei Jian-Chao

    2011-07-01

    Full Text Available Abstract Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV. Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865 and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716, were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine than that of mono-epitope peptide(rE2-a or rE2-b. Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

  19. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo

    International Nuclear Information System (INIS)

    Jones, M.H.; Learned, R.M.; Tjian, R.

    1988-01-01

    The authors have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream regions is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of ≥ 600 nucleotides. In addition, they demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them

  1. Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

    Science.gov (United States)

    Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

    2011-01-01

    Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

  2. Hepatitis Bx Antigen Stimulates Expression of a Novel Cellular Gene, URG4, that Promotes Hepatocellular Growth and Survival

    Directory of Open Access Journals (Sweden)

    N. Lale Satiroglu Tufan

    2002-01-01

    Full Text Available Hepatitis B virus encoded X antigen (HBxAg may contribute to the development of hepatocellular carcinoma (HCC by up-or downregulating the expression of cellular genes that promote cell growth and survival. To test this hypothesis, HBxAg-positive and-negative HepG2 cells were constructed, and the patterns of cellular gene expression compared by polymerase chain reaction select cDNA subtraction. The full-length clone of one of these upregulated genes (URG, URG4, encoded a protein of about 104 kDa. URG4 was strongly expressed in hepatitis 13-infected liver and in HCC cells, where it costained with HBxAg, and was weakly expressed in uninfected liver, suggesting URG4 was an effector of HBxAg in vivo. Overexpression of URG4 in HepG2 cells promoted hepatocellular growth and survival in tissue culture and in soft agar, and accelerated tumor development in nude mice. Hence, URG4 may be a natural effector of HBxAg that contributes importantly to multistep hepatocarcinogenesis.

  3. An Expression of Multiple Values: The Relationship Between Community, Landscape and Natural Resource

    Directory of Open Access Journals (Sweden)

    Darrell Smith

    2016-10-01

    Full Text Available To aid political and institutional decision making in the sustainable use of natural resources the value of ecological, socio-cultural and economic assets has increasingly been communicated in terms of monetary units. Despite reliance upon natural resources, the impact of human activities has now reached a stage where cumulative losses are forcing society to re-appraise the evaluation process and how to better incorporate these values in to the decision-making process. This paper examines the attributes of value held by natural resources within ecological, socio-cultural and economic value domains from the perspective of a rural UK community. Here we reflect upon the continued primacy of monetary valuation of natural resource using two approaches, a scaled preference-based value typology and a place-based map measure. We demonstrate that the societal relationships which inform the evaluation of natural resources are both multi-faceted and hierarchical. Moreover, whilst aware of the utilitarian character of society’s relationship with natural resource, the societal value-for-natural-resource relationship is primarily expressed using social-ecological qualities. These results add weight to the call for a new approach towards natural resource evaluation and how these values contribute to the sustainability agenda. New methods of evaluation must adopt multiple values that extend beyond a solely economic-based commodification concern to encompass the human relationship with the resource itself. Wherein, a multi-faceted approach to attributing value to natural resource, set within an experiential framework, can provide a focal point for discussion and the decision-making process.

  4. Over Expression of Long Non-Coding RNA PANDA Promotes Hepatocellular Carcinoma by Inhibiting Senescence Associated Inflammatory Factor IL8.

    Science.gov (United States)

    Peng, Chuanhui; Hu, Wendi; Weng, Xiaoyu; Tong, Rongliang; Cheng, Shaobing; Ding, Chaofeng; Xiao, Heng; Lv, Zhen; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2017-06-23

    It has been reported that long non-coding RNA PANDA was disregulated in varieties types of tumor, but its expression level and biological role in hepatocellular carcinoma (HCC) remains contradictory. We detected PANDA expression in two independent cohorts (48 HCC patients following liver transplantation and 84 HCC patients following liver resection), and found that PANDA was down-regulated in HCC. Thereafter we explored its function in cancer biology by inversing its low expression. Surprisingly, overexpression of PANDA promoted HCC proliferation and carcinogenesis in vitro and in vivo. Mechanistically, PANDA repressed transcriptional activity of senescence associated inflammatory factor IL8, which leaded to inhibition of cellular senescence. Therefore, our research help to better understand the complex role of PANDA in HCC, and suggest more thoughtful strategies should be applied before it can be treated as a potential therapeutic target.

  5. Aberrant expression of CKLF-like MARVEL transmembrane member 5 (CMTM5) by promoter methylation in myeloid leukemia.

    Science.gov (United States)

    Niu, Jihong; Li, Henan; Zhang, Yao; Li, Jinlan; Xie, Min; Li, Lingdi; Qin, Xiaoying; Qin, Yazhen; Guo, Xiaohuan; Jiang, Qian; Liu, Yanrong; Chen, Shanshan; Huang, Xiaojun; Han, Wenling; Ruan, Guorui

    2011-06-01

    CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. The positives of negative emotions: willingness to express negative emotions promotes relationships.

    Science.gov (United States)

    Graham, Steven M; Huang, Julie Y; Clark, Margaret S; Helgeson, Vicki S

    2008-03-01

    Four studies support the hypothesis that expressing negative emotion is associated with positive relationship outcomes, including elicitation of support, building of new close relationships, and heightening of intimacy in the closest of those relationships. In Study 1, participants read vignettes in which another person was experiencing a negative emotion. Participants reported they would provide more help when the person chose to express the negative emotion. In Study 2, participants watched a confederate preparing for a speech. Participants provided more help to her when she expressed nervousness. In Study 3, self-reports of willingness to express negative emotions predicted having more friends, controlling for demographic variables and extraversion. In Study 4, self-reports of willingness to express negative emotion measured prior to arrival at college predicted formation of more relationships, greater intimacy in the closest of those relationships, and greater received support from roommates across participants' first semester of college.

  7. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  8. ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy.

    Science.gov (United States)

    Antonelli, Martina; Strappazzon, Flavie; Arisi, Ivan; Brandi, Rossella; D'Onofrio, Mara; Sambucci, Manolo; Manic, Gwenola; Vitale, Ilio; Barilà, Daniela; Stagni, Venturina

    2017-03-28

    The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one.Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype.

  9. Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

    Science.gov (United States)

    Coffelt, Seth B; Chen, Yung-Yi; Muthana, Munitta; Welford, Abigail F; Tal, Andrea O; Scholz, Alexander; Plate, Karl H; Reiss, Yvonne; Murdoch, Craig; De Palma, Michele; Lewis, Claire E

    2011-04-01

    Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.

  10. Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

    Science.gov (United States)

    Athwal, T; Huang, W; Mukherjee, R; Latawiec, D; Chvanov, M; Clarke, R; Smith, K; Campbell, F; Merriman, C; Criddle, D; Sutton, R; Neoptolemos, J; Vlatković, N

    2014-01-01

    Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore

  11. Suppression of LFA-1 expression by spermine is associated with enhanced methylation of ITGAL, the LFA-1 promoter area.

    Directory of Open Access Journals (Sweden)

    Yoshihiko Kano

    Full Text Available Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1 expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt activity, enhanced demethylation of the LFA-1 gene (ITGAL promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM, and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM-such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase and SAM supplementation-successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL.

  12. Increased expression of hepatocyte nuclear factor 4 alpha transcribed by promoter 2 indicates a poor prognosis in hepatocellular carcinoma.

    Science.gov (United States)

    Cai, Shao-Hang; Lu, Shi-Xun; Liu, Li-Li; Zhang, Chris Zhiyi; Yun, Jing-Ping

    2017-10-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) plays an important role in tumourigenesis. There is growing evidence indicating that HNF4α transcribed by promoter 1 (P1-HNF4α) is expressed at relatively low levels in HCC and its presence predicts a favourable outcome for hepatocellular carcinoma (HCC) patients. However, the role of HNF4α transcribed by promoter 2 (P2-HNF4α) in HCC remains unclear. A total of 615 HCC specimens were obtained to construct tissue microarrays and perform immunohistochemistry. The relationship between P2-HNF4α and clinical features of HCC patients were analysed. Kaplan-Meier analysis was conducted to assess the prognostic value of P2-HNF4α. The results showed that the expression of P2-HNF4α in HCC was noticeably increased in HCC tissues compared with the nontumourous tissues. In addition, P1-HNF4α expression was negatively correlated with P2-HNF4α expression ( p  = 0.023). High P2-HNF4α expression was significantly associated with poor differentiation of HCC ( p = 0.002) and vascular invasion ( p = 0.017). Kaplan-Meier analysis showed that P2-HNF4α expression was closely correlated with overall survival in the training group ( p = 0.01), validation group ( p = 0.034), and overall group of patients with HCC ( p P2-HNF4α, different from P1-HNF4α, is markedly upregulated and serves as an oncogene-associated protein in HCC. Our study therefore provides a promising biomarker for prognostic prediction and a potential therapeutic target for HCC.

  13. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  14. Dynamic T-lymphocyte chemokine receptor expression induced by interferon-beta therapy in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, M; Sorensen, P S; Khademi, M

    2006-01-01

    chemokine receptor (CXCR)3 was unaltered. Conversely, at 9-12 h after the most recent IFN-beta injection, CCR4, CCR5 and CCR7 expressions were unaltered, while CXCR3 expression was reduced. CD4(+) T-cell surface expression of CCR4 was significantly lower in untreated MS patients compared with healthy...

  15. Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants.

    Science.gov (United States)

    Halpin, C; Cooke, S E; Barakate, A; El Amrani, A; Ryan, M D

    1999-02-01

    Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.

  16. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  17. Inducible Costimulator Expressing T Cells Promote Parasitic Growth During Blood Stage Plasmodium berghei ANKA Infection

    Directory of Open Access Journals (Sweden)

    Gajendra M. Jogdand

    2018-05-01

    Full Text Available The lethality of blood stage Plasmodium berghei ANKA (PbA infection is associated with the expression of T-bet and production of cytokine IFN-γ. Expression of inducible costimulator (ICOS and its downstream signaling has been shown to play a critical role in the T-bet expression and IFN-γ production. Although earlier studies have examined the role of ICOS in the control of acute blood-stage infection of Plasmodium chabaudi chabaudi AS (a non-lethal model of malaria infection, its significance in the lethal blood-stage of PbA infection remains unclear. Thus, to address the seminal role of ICOS in lethal blood-stage of PbA infection, we treated PbA-infected mice with anti-ICOS antibody and observed that these mice survived longer than their infected counterparts with significantly lower parasitemia. Anti-ICOS treatment notably depleted ICOS expressing CD4+ and CD8+ T cells with a concurrent reduction in plasma IFN-γ, which strongly indicated that ICOS expressing T cells are major IFN-γ producers. Interestingly, we observed that while ICOS expressing CD4+ and CD8+ T cells produced IFN-γ, ICOS−CD8+ T cells were also found to be producers of IFN-γ. However, we report that ICOS+CD8+ T cells were higher producers of IFN-γ than ICOS−CD8+ T cells. Moreover, correlation of ICOS expression with IFN-γ production in ICOS+IFN-γ+ T cell population (CD4+ and CD8+ T cells suggested that ICOS and IFN-γ could positively regulate each other. Further, master transcription factor T-bet importantly involved in regulating IFN-γ production was also found to be expressed by ICOS expressing CD4+ and CD8+ T cells during PbA infection. As noted above with IFN-γ and ICOS, a positive correlation of expression of ICOS with the transcription factor T-bet suggested that both of them could regulate each other. Taken together, our results depicted the importance of ICOS expressing CD4+ and CD8+ T cells in malaria parasite growth and lethality through IFN

  18. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.; Rizzu, Patrizia; Francescatto, Margherita; Vitezic, Morana; Leday, Gwenaë l G.R.; Sanchez, Javier Simon; Khamis, Abdullah M.; Takahashi, Hazuki; van de Berg, Wilma D.J.; Medvedeva, Yulia A.; van de Wiel, Mark A.; Daub, Carsten O.; Carninci, Piero; Heutink, Peter

    2013-01-01

    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites

  19. Overexpression of HMGA2-LPP fusion transcripts promotes expression of the α 2 type XI collagen gene

    International Nuclear Information System (INIS)

    Kubo, Takahiro; Matsui, Yoshito; Goto, Tomohiro; Yukata, Kiminori; Yasui, Natsuo

    2006-01-01

    In a subset of human lipomas, a specific t (3; 12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. In addition to its role in adipogenesis, several observations suggest that HMGA2-LPP is linked to chondrogenesis. Here, we analyzed whether HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the α 2 type XI collagen gene (Col11a2). Real-time PCR analysis showed that HMGA2-LPP and COL11A2 were co-expressed. Luciferase assay demonstrated that either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the Col11a2 promoter in HeLa cells, while the C-terminal LPP did not. RT-PCR analysis revealed that HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. These results indicate that in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains

  20. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Z. N.; Sharma, V. P.; Beaty, B. T.; Roh-Johnson, M.; Peterson, E. A.; Van Rooijen, N.; Kenny, P. A.; Wiley, H. S.; Condeelis, J. S.; Segall, J. E.

    2014-10-13

    Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

  1. Ubiquitous expression of MAKORIN-2 in normal and malignant hematopoietic cells and its growth promoting activity.

    Directory of Open Access Journals (Sweden)

    King Yiu Lee

    Full Text Available Makorin-2 (MKRN2 is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.

  2. Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

    2009-07-01

    The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 kaccase gene contains a putative site for xenobiotics (XRE). (Author)

  3. Expression analysis of four flower-specific promoters of Brassica spp ...

    African Journals Online (AJOL)

    The 5'-flanking region of ca. 1200 bp upstream of the translation start site (TSS) of a putative cell wall protein gene was cloned from Brassica campestris, B. chinensis, B. napus and B. oleracea, and transferred to tobacco via Agrobacterium-mediation after fused to promoter-less beta-glucuronidase (GUS) reporter gene.

  4. Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

    International Nuclear Information System (INIS)

    Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

    2009-01-01

    The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 laccase gene contains a putative site for xenobiotics (XRE). (Author)

  5. The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish

    NARCIS (Netherlands)

    Ward, AC; McPhee, DO; Condron, MM; Varma, S; Cody, SH; Onnebo, SMN; Paw, BH; Zon, LI; Lieschke, GJ

    2003-01-01

    The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3

  6. BRCA1-regulated RRM2 expression protects glioblastoma cells from endogenous replication stress and promotes tumorigenicity

    DEFF Research Database (Denmark)

    Rasmussen, Rikke D.; Gajjar, Madhavsai K.; Tuckova, Lucie

    2016-01-01

    Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Hi...

  7. Acute activation of GLP-1-expressing neurons promotes glucose homeostasis and insulin sensitivity

    Directory of Open Access Journals (Sweden)

    Xuemei Shi

    2017-11-01

    Conclusions: We conclude that acute activation of PPG neurons in the brainstem reduces basal glucose production, enhances intraperitoneal glucose tolerance, and augments hepatic insulin sensitivity, suggesting an important physiological role of PPG neurons-mediated circuitry in promoting glycemic control and insulin sensitivity.

  8. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  9. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    International Nuclear Information System (INIS)

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua

    2011-01-01

    Highlights: → A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. → The promoter was characterized with radiation-inducibility and tumor-specificity. → Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. → Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  10. Promoting Expressive Language in Young Children with or At-Risk for Autism Spectrum Disorder in a Preschool Classroom.

    Science.gov (United States)

    Lane, Justin D; Shepley, Collin; Lieberman-Betz, Rebecca

    2016-10-01

    Young children with autism spectrum disorder (ASD) often demonstrate delays in expressive communication, impacting their ability to independently function in typical environments. Individuals with ASD who develop expressive language during early childhood experience better outcomes later in life; therefore, examination of naturalistic language interventions (NLIs) remain an important area of investigation. The current study used a multiple probe design across participants to examine the effects of a classroom-based NLI on various expressive language targets in three preschool-aged children demonstrating characteristics of ASD. Findings suggest the intervention had positive and maintained effects on trial-based use of language targets, as well as concomitant changes in commenting, requesting, and phrase complexity. Implications regarding implementation of NLIs within typical classroom play activities are discussed.

  11. Expression of scavenger receptor‐AI promotes alternative activation of murine macrophages to limit hepatic inflammation and fibrosis

    Science.gov (United States)

    Labonte, Adam C.; Sung, Sun‐Sang J.; Jennelle, Lucas T.; Dandekar, Aditya P.

    2016-01-01

    The liver maintains an immunologically tolerant environment as a result of continuous exposure to food and bacterial constituents from the digestive tract. Hepatotropic pathogens can take advantage of this niche and establish lifelong chronic infections causing hepatic fibrosis and hepatocellular carcinoma. Macrophages (Mϕ) play a critical role in regulation of immune responses to hepatic infection and regeneration of tissue. However, the factors crucial for Mϕ in limiting hepatic inflammation or resolving liver damage have not been fully understood. In this report, we demonstrate that expression of C‐type lectin receptor scavenger receptor‐AI (SR‐AI) is crucial for promoting M2‐like Mϕ activation and polarization during hepatic inflammation. Liver Mϕ uniquely up‐regulated SR‐AI during hepatotropic viral infection and displayed increased expression of alternative Mϕ activation markers, such as YM‐1, arginase‐1, and interleukin‐10 by activation of mer receptor tyrosine kinase associated with inhibition of mammalian target of rapamycin. Expression of these molecules was reduced on Mϕ obtained from livers of infected mice deficient for the gene encoding SR‐AI (msr1). Furthermore, in vitro studies using an SR‐AI‐deficient Mϕ cell line revealed impeded M2 polarization and decreased phagocytic capacity. Direct stimulation with virus was sufficient to activate M2 gene expression in the wild‐type (WT) cell line, but not in the knockdown cell line. Importantly, tissue damage and fibrosis were exacerbated in SR‐AI–/– mice following hepatic infection and adoptive transfer of WT bone‐marrow–derived Mϕ conferred protection against fibrosis in these mice. Conclusion: SR‐AI expression on liver Mϕ promotes recovery from infection‐induced tissue damage by mediating a switch to a proresolving Mϕ polarization state. (Hepatology 2017;65:32‐43). PMID:27770558

  12. Penta- and octa-bromodiphenyl ethers promote proinflammatory protein expression in human bronchial epithelial cells in vitro.

    Science.gov (United States)

    Koike, Eiko; Yanagisawa, Rie; Takigami, Hidetaka; Takano, Hirohisa

    2014-03-01

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer products. Humans can be exposed to PBDEs mainly through the inhalation of air or dust. Thus, PBDEs can affect respiratory and immune systems. In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells. We examined commercial penta-BDE (DE-71), octa-BDE (DE-79), and deca-BDE (DE-83R). Human bronchial epithelial cells (BEAS-2B) were exposed to each PBDE for 24h. Subsequently, the expression of intercellular adhesion molecule-1 (ICAM-1) and proinflammatory cytokines were investigated. DE-71 and DE-79, but not DE-83R, significantly increased the expression of ICAM-1, interleukin-6 (IL-6), and IL-8 in BEAS-2B. Because these remarkable effects were observed with DE-71, we further investigated the underlying intracellular mechanisms. DE-71 promoted epidermal growth factor receptor (EGFR) phosphorylation. Inhibitors of EGFR-selective tyrosine kinase and p38 mitogen-activated protein kinase effectively blocked the increase of IL-6 and IL-8. Furthermore, antagonists of thyroid hormone receptor and aryl hydrocarbon receptor significantly suppressed the increase in IL-6 and/or IL-8 production. In conclusion, penta- and octa-BDE, but not deca-BDE, might promote the expression of proinflammatory proteins in bronchial epithelial cells possibly by activating protein kinases and/or stimulating nuclear receptors related to subsequent activation of transcriptional factors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

    Directory of Open Access Journals (Sweden)

    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  14. Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

    Science.gov (United States)

    Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

    2017-01-01

    The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

  15. Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression.

    Science.gov (United States)

    Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

    2017-08-01

    Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Clinical research on the expression of Lgr5 in the colon cancer tissues and its transfer promoting role

    Directory of Open Access Journals (Sweden)

    Xin-Jun Shu

    2016-05-01

    Full Text Available Objective: To detect the expression of Lgr5 in the colon cancer tissue, and to explore its correlation with the clinical and pathological characteristics and its role in promoting the tumor invasion and metastasis. Methods: A total of 102 specimens from the patients with colon cancer who were admitted in the General Surgery Department of our hospital for resection from April, 2013 to October, 2015 were included in the study; meanwhile, 35 colorectal adenoma specimens, and 137 normal colon tissue specimens were collected. The immunohistochemical method was used to detect the expression of Lgr5. Results: The positive rate of Lgr5 in the colon cancer tissues (62.75% was significantly higher than that in the adenoma tissues (28.57% and normal tissues (16.06%. The positive expression of Lgr5 in the colon cancer tissues was associated with the adenocarcinoma differentiation degree, Dukes staging, lymphatic metastasis, and distant metastasis. Meanwhile, it was found by the Logistic regression that the adenocarcinoma differentiation degree, lymphatic metastasis, and distant metastasis were the independent predictive factors for the positive expression of Lgr5. Conclusions: The increasement of Lgr5 expression is probably involved in the formation, differentiation, invasion, and metastasis of colon cancer; therefore, Lgr5 is expected to be a new therapeutic target aiming at colon cancer stem cells.

  17. Multiple Taf subunits of TFIID interact with Ino2 activation domains and contribute to expression of genes required for yeast phospholipid biosynthesis.

    Science.gov (United States)

    Hintze, Stefan; Engelhardt, Maike; van Diepen, Laura; Witt, Eric; Schüller, Hans-Joachim

    2017-12-01

    Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2. © 2017 John Wiley & Sons Ltd.

  18. High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease

    KAUST Repository

    Eid, Ayman

    2016-05-28

    Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.

  19. Molecular effects of autoimmune-risk promoter polymorphisms on expression, exon choice, and translational efficiency of interferon regulatory factor 5.

    Science.gov (United States)

    Clark, Daniel N; Lambert, Jared P; Till, Rodney E; Argueta, Lissenya B; Greenhalgh, Kathryn E; Henrie, Brandon; Bills, Trieste; Hawkley, Tyson F; Roznik, Marinya G; Sloan, Jason M; Mayhew, Vera; Woodland, Loc; Nelson, Eric P; Tsai, Meng-Hsuan; Poole, Brian D

    2014-05-01

    The rs2004640 single nucleotide polymorphism and the CGGGG copy-number variant (rs77571059) are promoter polymorphisms within interferon regulatory factor 5 (IRF5). They have been implicated as susceptibility factors for several autoimmune diseases. IRF5 uses alternative promoter splicing, where any of 4 first exons begin the mRNA. The CGGGG indel is in exon 1A's promoter; the rs2004640 allele creates a splicing recognition site, enabling usage of exon 1B. This study aimed at characterizing alterations in IRF5 mRNA due to these polymorphisms. Cells with risk polymorphisms exhibited ~2-fold higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR demonstrated decreased usage of exons 1C and 1D in cell lines with the risk polymorphisms. RNA folding analysis revealed a hairpin in exon 1B; mutational analysis showed that the hairpin shape decreased translation 5-fold. Although translation of mRNA that uses exon 1B is low due to a hairpin, increased IRF5 mRNA levels in individuals with the rs2004640 risk allele lead to higher overall protein expression. In addition, several new splice variants of IRF5 were sequenced. IRF5's promoter polymorphisms alter first exon usage and increase transcription levels. High levels of IRF5 may bias the immune system toward autoimmunity.

  20. The Mysterious Noh Mask: Contribution of Multiple Facial Parts to the Recognition of Emotional Expressions

    Science.gov (United States)

    Miyata, Hiromitsu; Nishimura, Ritsuko; Okanoya, Kazuo; Kawai, Nobuyuki

    2012-01-01

    Background A Noh mask worn by expert actors when performing on a Japanese traditional Noh drama is suggested to convey countless different facial expressions according to different angles of head/body orientation. The present study addressed the question of how different facial parts of a Noh mask, including the eyebrows, the eyes, and the mouth, may contribute to different emotional expressions. Both experimental situations of active creation and passive recognition of emotional facial expressions were introduced. Methodology/Principal Findings In Experiment 1, participants either created happy or sad facial expressions, or imitated a face that looked up or down, by actively changing each facial part of a Noh mask image presented on a computer screen. For an upward tilted mask, the eyebrows and the mouth shared common features with sad expressions, whereas the eyes with happy expressions. This contingency tended to be reversed for a downward tilted mask. Experiment 2 further examined which facial parts of a Noh mask are crucial in determining emotional expressions. Participants were exposed to the synthesized Noh mask images with different facial parts expressing different emotions. Results clearly revealed that participants primarily used the shape of the mouth in judging emotions. The facial images having the mouth of an upward/downward tilted Noh mask strongly tended to be evaluated as sad/happy, respectively. Conclusions/Significance The results suggest that Noh masks express chimeric emotional patterns, with different facial parts conveying different emotions This appears consistent with the principles of Noh which highly appreciate subtle and composite emotional expressions, as well as with the mysterious facial expressions observed in Western art. It was further demonstrated that the mouth serves as a diagnostic feature in characterizing the emotional expressions. This indicates the superiority of biologically-driven factors over the traditionally

  1. Promoter characteristics of two cyp19 genes differentially expressed in the brain and ovary of teleost fish.

    Science.gov (United States)

    Tchoudakova, A; Kishida, M; Wood, E; Callard, G V

    2001-11-01

    Teleost fish are characterized by exceptionally high levels of neural estrogen biosynthesis when compared with the brains of other vertebrates or to the ovaries of the same fish. Two P450arom mRNAs which derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (b>a) and ovary (a>b) and have a different developmental program (b>a) and estrogen upregulation (b only). A polymerase chain reaction (PCR)-based genomic walking strategy was used to isolate the 5'-flanking regions of the goldfish (Carassius auratus) cyp19 genes. Sequence analysis of the cyp19b gene approximately 1.8 kb upstream of the transcription start site revealed a TATA box at nucleotide (nt) -30, two estrogen responsive elements (EREs; nt -351 and -211) and a consensus binding site (NBRE) for nerve growth factor inducible-B protein (NGFI-B/Nur77) at -286, which includes another ERE half-site. Also present were a sequence at nt -399 (CCCTCCT) required for neural specificity of the zebrafish GATA-2 gene, and 16 copies of an SRY/SOX binding motif. The 5'-flanking region ( approximately 1.0 kb) of the cyp19a gene had TATA (nt -48) and CAAT (nt -71) boxes, a steroidogenic factor-1 (SF-1) binding site (nt -265), eight copies of the SRY/SOX motif, and two copies of a recognition site for binding the arylhydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) heterodimer. Both genes had elements previously identified in the brain specific exon I promoter of the mouse aromatase gene. Cyp19a- and -b/luciferase constructs showed basal promoter activity in aromatase-expressing rodent pituitary (GH3) cells, but differences (a>b) did not reflect expression in fish pituitary in vivo (b>a), implying a lack of appropriate cell factors. Consistent with the onset of cyp19b expression in zebrafish embryos, microinjection of a green fluorescent protein (GFP) reporter plasmid into fertilized eggs revealed labeling in neural tissues at 30-48 h post-fertilization (hpf), most

  2. Progranulin is expressed within motor neurons and promotes neuronal cell survival

    Directory of Open Access Journals (Sweden)

    Kay Denis G

    2009-10-01

    Full Text Available Abstract Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. Results In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months. This is mediated at least in part through

  3. Decrease in PSCA expression caused by Helicobacter pylori infection may promote progression to severe gastritis

    Science.gov (United States)

    Toyoshima, Osamu; Tanikawa, Chizu; Yamamoto, Ryuta; Watanabe, Hidenobu; Yamashita, Hiroharu; Sakitani, Kosuke; Yoshida, Shuntaro; Kubo, Michiaki; Matsuo, Keitaro; Ito, Hidemi; Koike, Kazuhiko; Seto, Yasuyuki; Matsuda, Koichi

    2018-01-01

    SNP rs2294008 in Prostate Stem Cell Antigen (PSCA) and decreased PSCA expression are associated with gastric cancer. The objective of this study is to investigate the role of rs2294008 and PSCA expression in the gastritis-gastric cancer carcinogenic pathway. We conducted a case-control association study of H. pylori-infected gastritis and gastric cancer. rs2294008 was associated with the progression to chronic active gastritis (P = 9.4 × 10–5; odds ratio = 3.88, TT + TC vs CC genotype), but not with H. pylori infection per se nor with the progression from active gastritis to gastric cancer. We also assessed the association of rs2294008 with PSCA mRNA expression in the gastric mucosa at various disease stages and found that rs2294008 was associated with PSCA expression (P = 1.3 × 10–12). H. pylori infection (P = 5.1 × 10–8) and eradication therapy (P gastritis compared with mild gastritis only among T allele carriers. Our findings revealed the regulation of PSCA expression by host genetic variation and bacterial infection might contribute to gastritis progression after H. pylori infection. PMID:29423095

  4. Soluble serum VCAM-1, whole blood mRNA expression and treatment response in natalizumab-treated multiple sclerosis

    DEFF Research Database (Denmark)

    Petersen, E R; Søndergaard, H B; Oturai, A B

    2016-01-01

    Background Natalizumab reduces disease activity in multiple sclerosis (MS). Natalizumab binds to the very late antigen-4 and inhibits vascular cell adhesion molecule-1 (VCAM-1)-mediated transmigration of immune cells across the blood-brain-barrier. This is associated with decreased serum...... levels of a panel of immunomarkers, associated with natalizumab-treatment, are not sensitive markers of MS disease activity. However, decreased expression of pro-inflammatory HLX1 and IL1B and increased expression of immunoregulatory EBI3 may indicate a less pathogenic immune activation status...

  5. [Knock-down of BCL11A expression in breast cancer cells promotes MDA-MB-231 cell apoptosis].

    Science.gov (United States)

    Li, Hongli; Gui, Chen; Yan, Lijun

    2016-11-01

    Objective To detect the expression and pathological significance of B-cell CLL/lymphoma 11A (BCL11A) in breast cancer and investigate the effect of its silencing on the apoptosis of human MDA-MB-231 breast cancer cells. MethodsImmunohistochemistry was used to detect the expression of BCL11A in 62 cases of human breast cancer tissues and 8 cases of normal tissues. We synthesized siRNA targeting BCL11A, and then siRNA was transfected into MDA-MB-231 cells. Forty-eight hours later, the suppression effect of siRNA on BCL11A was determined by quantitative real-time PCR and Western blotting. The apoptosis of MDA-MB-231 cells was detected by flow cytometry. Results The BCL11A protein was mainly expressed in cytoplasm. The expression level of BCL11A in breast cancer tissues was higher than that in paracancerous tissues. The expression had correlations with tumor grade, tumor stage, while it had no correlations with the patients' age and tumor size. BCL11A-siRNA significantly suppressed the expression of BCL11A mRNA and protein as compared with the control group. MDA-MB-231 cells transfected with BCL11A-siRNA had higher apoptosis rate compared with the control group. Conclusion The BCL11A protein is highly expressed in breast cancer and knock-down of BCL11A promotes the apoptosis of MDA-MB-231 cells.

  6. IL-15 expression on RA synovial fibroblasts promotes B cell survival.

    Directory of Open Access Journals (Sweden)

    Marta Benito-Miguel

    Full Text Available INTRODUCTION: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib IL-15 expression on B cell survival. METHODS: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib. RESULTS: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001. IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05. Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures. CONCLUSION: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

  7. Promoter Hypermethylation and Decreased Expression of Syncytin-1 in Pancreatic Adenocarcinomas.

    Directory of Open Access Journals (Sweden)

    Qinsheng Lu

    Full Text Available Syncytin-1 is a member of human endogenous retroviral W gene family (HERVW1. Known to be expressed in human placental trophoblast, syncytin-1 protein mediates the fusion of cytotrophoblasts for the formation of syncytiotrophoblasts, the terminally differentiated form of trophoblast lineage. In addition, in vitro studies indicate that syncytin-1 possessed nonfusogenic functions such as those for immune suppression, cell cycle regulation and anti-apoptotic activities. Overexpression of syncytin-1 has been observed in various malignant tissues including breast, endometrial and ovarian cancers. It was reported that syncytin-1 gene expression is associated with dynamic changes of DNA hypomethylation in the 5' LTR. In this study, applying the real-time PCR, Western blot analysis and immunohistochemistry methods, we demonstrate a constitutive expression of syncytin-1 in normal pancreas tissues as well as normal tissues adjacent to cancer lesions. Moreover, a reduced expression is found in the pancreatic adenocarcinoma tissues. The expression levels of syncytin-1 are not correlated with the stage, historical grade and gender, but inversely correlated with patients' age. Furthermore, COBRA and bisulfite sequencing results indicated that the lower expression of syncytin-1 is correlated with the hypermethylation of two CpG dinucleotides in the 5' LTR of syncytin-1 gene. The nonfusogenic function of syncytin-1 in normal pancreas as well as its role(s in the pathogenesis and progression of pancreatic cancers remains to be investigated. Identification of the two CpG dinucleotides around transcription start site as key epigenetic elements has provided valuable information for further studies on the epigenetic regulation of syncytin-1 in pancreatic cancer cells.

  8. Acute activation of GLP-1-expressing neurons promotes glucose homeostasis and insulin sensitivity

    OpenAIRE

    Xuemei Shi; Shaji Chacko; Feng Li; Depei Li; Douglas Burrin; Lawrence Chan; Xinfu Guan

    2017-01-01

    Objective: Glucagon-like peptides are co-released from enteroendocrine L cells in the gut and preproglucagon (PPG) neurons in the brainstem. PPG-derived GLP-1/2 are probably key neuroendocrine signals for the control of energy balance and glucose homeostasis. The objective of this study was to determine whether activation of PPG neurons per se modulates glucose homeostasis and insulin sensitivity in vivo. Methods: We generated glucagon (Gcg) promoter-driven Cre transgenic mice and injected...

  9. The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

    Science.gov (United States)

    Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C

    2014-04-14

    Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. TUG1 promotes osteosarcoma tumorigenesis by upregulating EZH2 expression via miR-144-3p

    OpenAIRE

    Cao, Jiaqing; Han, Xinyou; Qi, Xin; Jin, Xiangyun; Li, Xiaolin

    2017-01-01

    lncRNA-TUG1 (Taurine upregulated 1) is up regulated and highly correlated with poor prognosis and disease status in osteosarcoma. TUG1 knockdown inhibits osteosarcoma cell proliferation, migration and invasion, and promotes apoptosis. However, its mechanism of action has not been well addressed. Growing evidence documented that lncRNA works as competing endogenous (ce)RNAs to modulate the expression and biological functions of miRNA. As a putative combining target of TUG1, miR-144-3p has been...

  11. Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

    Science.gov (United States)

    Ferrari, D; Lichtler, A C; Pan, Z Z; Dealy, C N; Upholt, W B; Kosher, R A

    1998-05-01

    During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior me