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Sample records for addition expression levels

  1. Additional Warning System for Cross Level

    NASA Astrophysics Data System (ADS)

    Lewiński, Andrzej; Bester, Lucyna

    The paper contains an analysis of the safety level crossing equipped with an additional warning system for drivers that are within the level crossing before the approaching train. The proposed system is based on wireless data standard, WiMax and sensor networks WSN, placed an additional warning helps to improve safety at unguarded railway crossings. Mathematical analysis was carried out for unguarded level crossing model, and then for system with signaling the level crossing ssp and for system equipped with additional warning system for drivers. For the analysis presented models used stochastic Markov processes which allowed estimating the indicators of probabilistic studied systems.

  2. Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level

    PubMed Central

    Yu, Shi; Pilot, Guillaume

    2014-01-01

    Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants. PMID:25477887

  3. 47. MAIN WAREHOUSE SECOND LEVEL ADDITION Second level was ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    47. MAIN WAREHOUSE - SECOND LEVEL ADDITION Second level was added in 1941. Note the variety of building materials used in the wall: cement, bricks and finally cement blocks, with wood topping the entire wall. - Hovden Cannery, 886 Cannery Row, Monterey, Monterey County, CA

  4. Depression and Pain: Independent and Additive Relationships to Anger Expression

    DTIC Science & Technology

    2013-10-01

    Naval Health Research Center Depression and Pain : Independent and Additive Relationships to Anger Expression Marcus K. Taylor Gerald E...AND SUBTITLE Depression and Pain : Independent and Additive Relationships to Anger Expression 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM...expression are understudied. OBJECTIVE: This study was designed to examine the independent and additive relationships of depression and pain to anger

  5. The Expression of Additive and Nonadditive Genetic Variation under Stress

    PubMed Central

    Blows, M. W.; Sokolowski, M. B.

    1995-01-01

    Experimental lines of Drosophila melanogaster derived from a natural population, which had been isolated in the laboratory for ~70 generations, were crossed to determine if the expression of additive, dominance and epistatic genetic variation in development time and viability was associated with the environment. No association was found between the level of additive genetic effects and environmental value for either trait, but nonadditive genetic effects increased at both extremes of the environmental range for development time. The expression of high levels of dominance and epistatic genetic variation at environmental extremes may be a general expectation for some traits. The disruption of the epistatic gene complexes in the parental lines resulted in hybrid breakdown toward faster development and there was some indication of hybrid breakdown toward higher viability. A combination of genetic drift and natural selection had therefore resulted in different epistatic gene complexes being selected after ~70 generations from a common genetic base. After crossing, the hybrid populations were observed for 10 generations. Epistasis contributed on average 12 hr in development time. Fluctuating asymmetry in sternopleural bristle number also evolved in the hybrid populations, decreasing by >18% in the first seven generations after hybridization. PMID:7672585

  6. Congruence of additive and non-additive effects on gene expression estimated from pedigree and SNP data.

    PubMed

    Powell, Joseph E; Henders, Anjali K; McRae, Allan F; Kim, Jinhee; Hemani, Gibran; Martin, Nicholas G; Dermitzakis, Emmanouil T; Gibson, Greg; Montgomery, Grant W; Visscher, Peter M

    2013-05-01

    There is increasing evidence that heritable variation in gene expression underlies genetic variation in susceptibility to disease. Therefore, a comprehensive understanding of the similarity between relatives for transcript variation is warranted--in particular, dissection of phenotypic variation into additive and non-additive genetic factors and shared environmental effects. We conducted a gene expression study in blood samples of 862 individuals from 312 nuclear families containing MZ or DZ twin pairs using both pedigree and genotype information. From a pedigree analysis we show that the vast majority of genetic variation across 17,994 probes is additive, although non-additive genetic variation is identified for 960 transcripts. For 180 of the 960 transcripts with non-additive genetic variation, we identify expression quantitative trait loci (eQTL) with dominance effects in a sample of 339 unrelated individuals and replicate 31% of these associations in an independent sample of 139 unrelated individuals. Over-dominance was detected and replicated for a trans association between rs12313805 and ETV6, located 4MB apart on chromosome 12. Surprisingly, only 17 probes exhibit significant levels of common environmental effects, suggesting that environmental and lifestyle factors common to a family do not affect expression variation for most transcripts, at least those measured in blood. Consistent with the genetic architecture of common diseases, gene expression is predominantly additive, but a minority of transcripts display non-additive effects.

  7. Equipment boxes (later addition) one level down from antenna cab, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Equipment boxes (later addition) one level down from antenna cab, looking southwest. - Western Union Telegraph Company, Jennerstown Relay, Laurel Summit Road off U.S. 30, Laughlintown, Westmoreland County, PA

  8. 38. VIEW SOUTHWEST OF STREET LEVEL IN SHOWROOM ADDITION (NOW ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    38. VIEW SOUTHWEST OF STREET LEVEL IN SHOWROOM ADDITION (NOW USED AS FINISHING ROOM) WITH FRESHLY PAINTED DORY BOATS ON DISPLAY. 2X4 STUD WALL AND ROOF FRAMING CAN BE SEEN. - Lowell's Boat Shop, 459 Main Street, Amesbury, Essex County, MA

  9. 113. Stage level floor structure. In addition to the movable ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    113. Stage level floor structure. In addition to the movable sections, there were hinged slots that could be opened in the stage floor (see sheet 4 of 9, note 4; sheet 5 of 9, note 2; and sheet 7 of 9, note 1). A remaining cast iron bracket is visible in the left foreground of the photograph. The actual structure for a hinged section is visible in the background, to the right of center. The hydraulic ram (type D) visible below the floor level is the south ram in the middle row; the view is facing north. - Auditorium Building, 430 South Michigan Avenue, Chicago, Cook County, IL

  10. 45. VIEW OF UPPER LEVEL CRUSHER ADDITION FROM CRUSHED OXIDIZED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    45. VIEW OF UPPER LEVEL CRUSHER ADDITION FROM CRUSHED OXIDIZED ORE BIN. 18 INCH BELT CONVEYOR BIN FEED, LOWER CENTER, WITH STEPHENS-ADAMSON 25 TON/HR ELEVATOR SPLIT DISCHARGE (OXIDIZED/UNOXIDIZED) IN CENTER. CRUDE ORE BINS AND MACHINE SHOP BEYOND. NOTE TOP OF CRUSHED OXIDIZED ORE BIN IS BELOW TOP OF CRUDE ORE BINS. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  11. 41. VIEW NORTH OF UPPER LEVEL OF CRUSHER ADDITION. DINGS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    41. VIEW NORTH OF UPPER LEVEL OF CRUSHER ADDITION. DINGS MAGNETIC PULLEY AT CENTER. ALSO SHOWS 100-TON CRUSHED UNOXIDIZED ORE BIN (RIGHT), PULLEY FORM 18 INCH BELT CONVEYOR CRUSHED OXIDIZED ORE BIN FEED AND STEPHENSADAMSON 25 TON/HR BUCKET ELEVATOR (UPPER CENTER). THE UPPER PORTION OF THE SAMPLING ELEVATOR IS ABOVE THE MAGNETIC PULLEY (CENTER LEFT) WITH THE ROUTE OF THE 16 INCH BELT CONVEYOR FINES FEED TO CRUSHED OXIDIZED ORE BIN TO ITS LEFT. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  12. A Comparison of Artificial Subtle Expressions with Human-like Expressions on Expressing Confidence Level

    NASA Astrophysics Data System (ADS)

    Komatsu, Takanori; Kobayashi, Kazuki; Yamada, Seiji; Funakoshi, Kotaro; Nakano, Mikio

    Expressing the confidence level of a system's suggestions by using speech sounds is an important cue to users of the system for perceiving how likely it is for the suggestions to be correct. We assume that expressing confidence levels by using human-like expressions would cause users to have a poorer impression of the systems than if artificial subtle expressions (ASEs) were used when the quality of the presented information does not match the expressed confidence level. We confirmed that this assumption was correct by conducting a psychological experiment.

  13. Genome-wide transcript analysis of maize hybrids: allelic additive gene expression and yield heterosis.

    PubMed

    Guo, Mei; Rupe, Mary A; Yang, Xiaofeng; Crasta, Oswald; Zinselmeier, Christopher; Smith, Oscar S; Bowen, Ben

    2006-09-01

    Heterosis, or hybrid vigor, has been widely exploited in plant breeding for many decades, but the molecular mechanisms underlying the phenomenon remain unknown. In this study, we applied genome-wide transcript profiling to gain a global picture of the ways in which a large proportion of genes are expressed in the immature ear tissues of a series of 16 maize hybrids that vary in their degree of heterosis. Key observations include: (1) the proportion of allelic additively expressed genes is positively associated with hybrid yield and heterosis; (2) the proportion of genes that exhibit a bias towards the expression level of the paternal parent is negatively correlated with hybrid yield and heterosis; and (3) there is no correlation between the over- or under-expression of specific genes in maize hybrids with either yield or heterosis. The relationship of the expression patterns with hybrid performance is substantiated by analysis of a genetically improved modern hybrid (Pioneer hybrid 3394) versus a less improved older hybrid (Pioneer hybrid 3306) grown at different levels of plant density stress. The proportion of allelic additively expressed genes is positively associated with the modern high yielding hybrid, heterosis and high yielding environments, whereas the converse is true for the paternally biased gene expression. The dynamic changes of gene expression in hybrids responding to genotype and environment may result from differential regulation of the two parental alleles. Our findings suggest that differential allele regulation may play an important role in hybrid yield or heterosis, and provide a new insight to the molecular understanding of the underlying mechanisms of heterosis.

  14. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  15. Emotional expression in music: contribution, linearity, and additivity of primary musical cues.

    PubMed

    Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

    2013-01-01

    The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77-89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0-8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010).

  16. Emotional expression in music: contribution, linearity, and additivity of primary musical cues

    PubMed Central

    Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

    2013-01-01

    The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77–89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0–8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010). PMID:23908642

  17. A promoter-level mammalian expression atlas

    PubMed Central

    2015-01-01

    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. PMID:24670764

  18. A promoter-level mammalian expression atlas.

    PubMed

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

    2014-03-27

    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.

  19. Analysis of baseline gene expression levels from ...

    EPA Pesticide Factsheets

    The use of gene expression profiling to predict chemical mode of action would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in gene expression. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques. The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selectiv

  20. Selenoprotein expression is regulated at multiple levels in prostate cells.

    PubMed

    Rebsch, Cheryl M; Penna, Frank J; Copeland, Paul R

    2006-12-01

    Selenium supplementation in a population with low basal blood selenium levels has been reported to decrease the incidence of several cancers including prostate cancer. Based on the clinical findings, it is likely that the antioxidant function of one or more selenoproteins is responsible for the chemopreventive effect, although low molecular weight seleno-compounds have also been posited to selectively induce apoptosis in transformed cells. To address the effects of selenium supplementation on selenoprotein expression in prostate cells, we have undertaken an analysis of antioxidant selenoprotein expression as well as selenium toxicity in non-tumorigenic prostate epithelial cells (RWPE-1) and prostate cancer cells (LNCaP and PC-3). Our results show that two of the glutathione peroxidase family members (GPX1 and GPX4) are highly induced by supplemental selenium in prostate cancer cells but only slightly induced in RWPE-1 cells. In addition, GPX1 levels are dramatically lower in PC-3 cells as compared to RWPE-1 or LNCaP cells. GPX2 protein and mRNA, however, are only detectable in RWPE-1 cells. Of the three selenium compounds tested (sodium selenite, sodium selenate and selenomethionine), only sodium selenite shows toxicity in a physiological range of selenium concentrations. Notably and in contrast to previous studies, RWPE-1 cells were significantly more sensitive to selenite than either of the prostate cancer cell lines. These results demonstrate that selenoproteins and selenium metabolism are regulated at multiple levels in prostate cells.

  1. Influence of HLA-C Expression Level on HIV Control

    PubMed Central

    Apps, Richard; Qi, Ying; Carlson, Jonathan M.; Chen, Haoyan; Gao, Xiaojiang; Thomas, Rasmi; Yuki, Yuko; Del Prete, Greg Q.; Goulder, Philip; Brumme, Zabrina L.; Brumme, Chanson J.; John, Mina; Mallal, Simon; Nelson, George; Bosch, Ronald; Heckerman, David; Stein, Judy L.; Soderberg, Kelly A.; Moody, M. Anthony; Denny, Thomas N.; Zeng, Xue; Fang, Jingyuan; Moffett, Ashley; Lifson, Jeffrey D.; Goedert, James J.; Buchbinder, Susan; Kirk, Gregory D.; Fellay, Jacques; McLaren, Paul; Deeks, Steven G.; Pereyra, Florencia; Walker, Bruce; Michael, Nelson L.; Weintrob, Amy; Wolinsky, Steven; Liao, Wilson; Carrington, Mary

    2013-01-01

    A variant upstream of human leukocyte antigen C (HLA-C) shows the most significant genome-wide effect on HIV control in European Americans and is also associated with the level of HLA-C expression. We characterized the differential cell surface expression levels of all common HLA-C allotypes and tested directly for effects of HLA-C expression on outcomes of HIV infection in 5243 individuals. Increasing HLA-C expression was associated with protection against multiple outcomes independently of individual HLA allelic effects in both African and European Americans, regardless of their distinct HLA-C frequencies and linkage relationships with HLA-B and HLA-A. Higher HLA-C expression was correlated with increased likelihood of cytotoxic T lymphocyte responses and frequency of viral escape mutation. In contrast, high HLA-C expression had a deleterious effect in Crohn’s disease, suggesting a broader influence of HLA expression levels in human disease. PMID:23559252

  2. Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression patterns in the F1 hybrid.

    PubMed

    Stupar, Robert M; Springer, Nathan M

    2006-08-01

    Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent. Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.

  3. Circulating irisin levels and muscle FNDC5 mRNA expression are independent of IL-15 levels in mice.

    PubMed

    Quinn, LeBris S; Anderson, Barbara G; Conner, Jennifer D; Wolden-Hanson, Tami

    2015-11-01

    Interleukin-15 (IL-15) and irisin are exercise-induced myokines that exert favorable effects on energy expenditure and metabolism. IL-15 can induce PGC-1α expression, which in turn induces expression of irisin and its precursor, FNDC5. Therefore, the present study tested the hypothesis that increases in circulating irisin levels and muscle FNDC5 mRNA expression are dependent on IL-15. Circulating irisin levels and gastrocnemius muscle FNDC5 mRNA expression were examined following acute exercise in control and IL-15-deleted (IL-15 KO) mice, following injection of IL-15 into IL-15 KO mice, and in transgenic mice with elevated circulating IL-15 levels (IL-15 Tg mice). Circulating IL-15 levels and muscle PGC-1α and PPARδ mRNA expressions were determined as positive controls. No effect of IL-15 deletion on post-exercise serum irisin levels or muscle FNDC5 mRNA expression was detected. While serum IL-15 levels and muscle PGC-1α expression were elevated post-exercise in control mice, both serum irisin levels and muscle FNDC5 expression decreased shortly after exercise in both control and IL-15 KO mice. A single injection of recombinant IL-15 into IL-15 KO mice that significantly increased muscle PPARδ and PGC-1α mRNA expressions had no effect on circulating irisin release, but modestly induced muscle FNDC5 expression. Additionally, serum irisin and gastrocnemius muscle FNDC5 expression in IL-15 Tg mice were similar to those of control mice. Muscle FNDC5 mRNA expression and irisin release are not IL-15-dependent in mice.

  4. The Level of Expressed Emotion Scale: A Useful Measure of Expressed Emotion in Adolescents?

    ERIC Educational Resources Information Center

    Nelis, Sharon M.; Rae, Gordon; Liddell, Christine

    2011-01-01

    Research has suggested that self-report measures of expressed emotion (EE) may be employed as a proxy measure of environmental stress in the home. The appropriateness of the Level of Expressed Emotion scale as a measure of perceived expressed emotion was examined in a sample of adolescents. Participants were 239 male and 422 female adolescents…

  5. Sequence determinants of prokaryotic gene expression level under heat stress.

    PubMed

    Xiong, Heng; Yang, Yi; Hu, Xiao-Pan; He, Yi-Ming; Ma, Bin-Guang

    2014-11-01

    Prokaryotic gene expression is environment-dependent and temperature plays an important role in shaping the gene expression profile. Revealing the regulation mechanisms of gene expression pertaining to temperature has attracted tremendous efforts in recent years particularly owning to the yielding of transcriptome and proteome data by high-throughput techniques. However, most of the previous works concentrated on the characterization of the gene expression profile of individual organism and little effort has been made to disclose the commonality among organisms, especially for the gene sequence features. In this report, we collected the transcriptome and proteome data measured under heat stress condition from recently published literature and studied the sequence determinants for the expression level of heat-responsive genes on multiple layers. Our results showed that there indeed exist commonness and consistent patterns of the sequence features among organisms for the differentially expressed genes under heat stress condition. Some features are attributed to the requirement of thermostability while some are dominated by gene function. The revealed sequence determinants of bacterial gene expression level under heat stress complement the knowledge about the regulation factors of prokaryotic gene expression responding to the change of environmental conditions. Furthermore, comparisons to thermophilic adaption have been performed to reveal the similarity and dissimilarity of the sequence determinants for the response to heat stress and for the adaption to high habitat temperature, which elucidates the complex landscape of gene expression related to the same physical factor of temperature.

  6. Influence of temperature, mixing, and addition of microcystin-LR on microcystin gene expression in Microcystis aeruginosa.

    PubMed

    Scherer, Pia I; Raeder, Uta; Geist, Juergen; Zwirglmaier, Katrin

    2017-02-01

    Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change-related factors. In this study, a new RT-qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin-LR (10 μg L(-1) or 60 μg L(-1) ) significantly altered mcyB and mcyD expression. The expression levels of mcyB and mcyD were correlated but not identical.

  7. Analysis of gelsolin expression pattern in developing chicken embryo reveals high GSN expression level in tissues of neural crest origin.

    PubMed

    Mazur, Antonina Joanna; Morosan-Puopolo, Gabriela; Makowiecka, Aleksandra; Malicka-Błaszkiewicz, Maria; Nowak, Dorota; Brand-Saberi, Beate

    2016-01-01

    Gelsolin is one of the most intensively studied actin-binding proteins. However, in the literature comprehensive studies of GSN expression during development have not been performed yet in all model organisms. In zebrafish, gelsolin is a dorsalizing factor that modulates bone morphogenetic proteins signaling pathways, whereas knockout of the gelsolin coding gene, GSN is not lethal in murine model. To study the role of gelsolin in development of higher vertebrates, it is crucial to estimate GSN expression pattern during development. Here, we examined GSN expression in the developing chicken embryo. We applied numerous methods to track GSN expression in developing embryos at mRNA and protein level. We noted a characteristic GSN expression pattern. Although GSN transcripts were present in several cell types starting from early developmental stages, a relatively high GSN expression was observed in eye, brain vesicles, midbrain, neural tube, heart tube, and splanchnic mesoderm. In older embryos, we observed a high GSN expression in the cranial ganglia and dorsal root ganglia. A detailed analysis of 10-day-old chicken embryos revealed high amounts of gelsolin especially within the head region: in the olfactory and optic systems, meninges, nerves, muscles, presumptive pituitary gland, and pericytes, but not oligodendrocytes in the brain. Obtained results suggest that GSN is expressed at high levels in some tissues of ectodermal origin including all neural crest derivatives. Additionally, we describe that silencing of GSN expression in brain vesicles leads to altered morphology of the mesencephalon. This implies gelsolin is crucial for chicken brain development.

  8. How to achieve high-level expression of microbial enzymes

    PubMed Central

    Liu, Long; Yang, Haiquan; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field. PMID:23686280

  9. The Effectiveness of an Additional Stretching Exercise Program in Improving Flexibility Level among Preschool Boys

    ERIC Educational Resources Information Center

    Lee, Wee Akina Sia Seng; Rengasamy, Shabeshan A/L; Raju, Subramaniam A/L

    2014-01-01

    This study was conducted to examine the effectiveness of a two minutes' additional stretching exercise program in a 30 minutes games teaching lesson in improving the flexibility level of 6 year old preschool boys (M = 5.92, SD = 0.27) in a preschool in Malaysia. Fifty (50) preschool boys were selected for the study based on the intact sampling…

  10. Calcium regulates caveolin-1 expression at the transcriptional level

    SciTech Connect

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  11. Gene expression levels in normal human lymphoblasts with variable sensitivities to arsenite: Identification of GGT1 and NFKBIE expression levels as possible biomarkers of susceptibility

    SciTech Connect

    Komissarova, Elena V.; Li Ping; Uddin, Ahmed N.; Chen, Xuyan; Nadas, Arthur; Rossman, Toby G.

    2008-01-15

    Drinking arsenic-contaminated water is associated with increased risk of neoplasias of the skin, lung, bladder and possibly other sites, as well as other diseases. Earlier, we showed that human lymphoblast lines from different normal unexposed donors showed variable sensitivities to the toxic effects of arsenite. In the present study, we used microarray analysis to compare the basal gene expression profiles between two arsenite-resistant (GM02707, GM00893) and two arsenite-sensitive lymphoblast lines (GM00546, GM00607). A number of genes were differentially expressed in arsenite-sensitive and arsenite-resistant cells. Among these, {gamma}-glutamyltranspeptidase 1 (GGT1) and NF{kappa}B inhibitor-epsilon (NFKBIE) showed higher expression levels in arsenite-resistant cells. RT-PCR analysis with gene-specific primers confirmed these results. Reduction of GGT1 expression level in arsenite-resistant lymphoblasts with GGT1-specific siRNA resulted in increased cell sensitivity to arsenite. In conclusion, we have demonstrated for the first time that expression levels of GGT1 and possibly NFKBIE might be useful as biomarkers of genetic susceptibility to arsenite. Expression microarrays can thus be exploited for identifying additional biomarkers of susceptibility to arsenite and to other toxicants.

  12. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    EPA Science Inventory

    Normal Nasal Gene Expression Levels Using cDNA Array Technology.

    The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  13. High VEGFR1/2 expression levels are predictors of poor survival in patients with cervical cancer

    PubMed Central

    Dang, Yun-Zhi; Zhang, Ying; Li, Jian-Ping; Hu, Jing; Li, Wei-Wei; Li, Pei; Wei, Li-Chun; Shi, Mei

    2017-01-01

    Abstract The aim of the study to evaluate the prognostic significance of vascular endothelial growth factor receptor 1 and 2 (VEGFR1/2) expression levels and to correlate these levels with clinicopathological parameters in patients with cervical cancer. Forty-two patients with International Federation of Gynecology and Obstetrics Stage IIB–IVB cervical cancer were analyzed between January 2011 and December 2012. RNA expression levels of VEGFR1/2 were assessed by branched DNA-liquidchip technology and immunohistochemistry. Associations between RNA expression levels, important clinicopathological parameters, and patient survival were statistically evaluated. Higher VEGFR1/2 expression levels were predictive of poor overall survival (P = 0.009 and P = 0.024, respectively). Patients with higher VEGFR1 expression levels were associated with poorer progression-free survival than those with lower VEGFR1 expression levels (P = 0.043). In addition, patients with higher VEGFR1 expression levels were more likely to develop distant metastases than those with lower VEGFR1 expression levels (P = 0.049). Higher VEGFR2 expression levels were associated with larger tumor size (P = 0.037). VEGFR1/2 expression levels were prognostic factors for patients with cervical cancer. Higher VEGFR1/2 expression levels were also predictive of poor overall survival. PMID:28072723

  14. Peripheral blood gene expression profiles linked to monoamine metabolite levels in cerebrospinal fluid

    PubMed Central

    Luykx, J J; Olde Loohuis, L M; Neeleman, M; Strengman, E; Bakker, S C; Lentjes, E; Borgdorff, P; van Dongen, E P A; Bruins, P; Kahn, R S; Horvath, S; de Jong, S; Ophoff, R A

    2016-01-01

    The blood–brain barrier separates circulating blood from the central nervous system (CNS). The scope of this barrier is not fully understood which limits our ability to relate biological measurements from peripheral to central phenotypes. For example, it is unknown to what extent gene expression levels in peripheral blood are reflective of CNS metabolism. In this study, we examine links between central monoamine metabolite levels and whole-blood gene expression to better understand the connection between peripheral systems and the CNS. To that end, we correlated the prime monoamine metabolites in cerebrospinal fluid (CSF) with whole-genome gene expression microarray data from blood (N=240 human subjects). We additionally applied gene-enrichment analysis and weighted gene co-expression network analyses (WGCNA) to identify modules of co-expressed genes in blood that may be involved with monoamine metabolite levels in CSF. Transcript levels of two genes were significantly associated with CSF serotonin metabolite levels after Bonferroni correction for multiple testing: THAP7 (P=2.8 × 10−8, β=0.08) and DDX6 (P=2.9 × 10−7, β=0.07). Differentially expressed genes were significantly enriched for genes expressed in the brain tissue (P=6.0 × 10−52). WGCNA revealed significant correlations between serotonin metabolism and hub genes with known functions in serotonin metabolism, for example, HTR2A and COMT. We conclude that gene expression levels in whole blood are associated with monoamine metabolite levels in the human CSF. Our results, including the strong enrichment of brain-expressed genes, illustrate that gene expression profiles in peripheral blood can be relevant for quantitative metabolic phenotypes in the CNS. PMID:27959337

  15. Nitrogen Addition Significantly Affects Forest Litter Decomposition under High Levels of Ambient Nitrogen Deposition

    PubMed Central

    Chen, Gang; Peng, Yong; Xiao, Yin-long; Hu, Ting-xing; Zhang, Jian; Li, Xian-wei; Liu, Li; Tang, Yi

    2014-01-01

    Background Forest litter decomposition is a major component of the global carbon (C) budget, and is greatly affected by the atmospheric nitrogen (N) deposition observed globally. However, the effects of N addition on forest litter decomposition, in ecosystems receiving increasingly higher levels of ambient N deposition, are poorly understood. Methodology/Principal Findings We conducted a two-year field experiment in five forests along the western edge of the Sichuan Basin in China, where atmospheric N deposition was up to 82–114 kg N ha–1 in the study sites. Four levels of N treatments were applied: (1) control (no N added), (2) low-N (50 kg N ha–1 year–1), (3) medium-N (150 kg N ha–1 year–1), and (4) high-N (300 kg N ha–1 year–1), N additions ranging from 40% to 370% of ambient N deposition. The decomposition processes of ten types of forest litters were then studied. Nitrogen additions significantly decreased the decomposition rates of six types of forest litters. N additions decreased forest litter decomposition, and the mass of residual litter was closely correlated to residual lignin during the decomposition process over the study period. The inhibitory effect of N addition on litter decomposition can be primarily explained by the inhibition of lignin decomposition by exogenous inorganic N. The overall decomposition rate of ten investigated substrates exhibited a significant negative linear relationship with initial tissue C/N and lignin/N, and significant positive relationships with initial tissue K and N concentrations; these relationships exhibited linear and logarithmic curves, respectively. Conclusions/Significance This study suggests that the expected progressive increases in N deposition may have a potential important impact on forest litter decomposition in the study area in the presence of high levels of ambient N deposition. PMID:24551152

  16. [Therapeutic and toxic theophylline levels in asthma attacks--is there a need for additional theophylline?].

    PubMed

    Zeidman, A; Gardyn, J; Fradin, Z; Fink, G; Mittelman, M

    1997-07-01

    Although first-line therapy for bronchial asthma has changed over the past decade to anti-inflammatory medication such as inhaled corticosteroids and cromolyn with possible addition of beta-agonists, theophylline is still useful and therefore widely used. However, several studies have raised serious questions regarding its efficacy in acute asthmatic exacerbations. These studies, the narrow therapeutic range of the drug, the frequency of side effects and interactions with common drugs, and individual variation in clearance and metabolism, have prompted its reevaluation in the management of asthma. Therapeutic serum levels of theophylline are between 10 to 20 mcg/ml. Most adults achieve these concentrations with daily slow-release oral theophylline preparations, 200-400 mg (approximately 10 mg/Kg) twice a day. However, when such a patient presents to the emergency room (ER) in an asthmatic attack, immediate intravenous theophylline is often given, regardless of maintenance treatment. Since the rationale for this common therapeutic approach has been challenged, the current study was undertaken. Serum theophylline levels were measured in 23 consecutive asthmatics presenting to the ER in an acute attack. 15 (68%) had therapeutic levels (above 10 mcg/ml) and 2 had toxic levels (above 20 mcg/ml), prior to receiving the standard intravenous theophylline dose given for an attack. These data indicate that most patients with bronchial asthma on oral maintenance theophylline do not require additional intravenous theophylline when in an attack. It probably will not benefit them and may even induce serious theophylline toxicity.

  17. Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli.

    PubMed

    Svensson, Johan; Andersson, Christer; Reseland, Janne E; Lyngstadaas, Petter; Bülow, Leif

    2006-07-01

    Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E. coli cells expressing the histidine tagged amelogenin rp(H)M180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin.

  18. Plasma levels of procalcitonin and eight additional inflammatory molecules in febrile neutropenic patients

    PubMed Central

    Neuenschwander, Letícia Carvalho; Bittencourt, Henrique; Tibúrcio Ribeiro, Ana Flávia; Teixeira, Antônio Lúcio; Teixeira, Mauro M.; Teixeira, Jairo Cerqueira; Nobre, Vandack

    2011-01-01

    OBJECTIVE: This study aimed to examine the association between different inflammatory markers and specific clinical endpoints in patients with febrile neutropenia. METHOD: We prospectively evaluated the expression of procalcitonin (PCT), interleukin 8 (IL-8), induced protein-10, tumor necrosis factor alpha (TNF-α), two soluble TNF-α receptors (sTNF-R I and sTNF-R II), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 alpha, and eotaxin in 37 episodes of febrile neutropenia occurring in 31 hospitalized adult onco-hematologic patients. Peripheral blood samples were collected in the morning at inclusion (day of fever onset) and on days 1, 3, and 7 after the onset of fever. Approximately 2–3 ml of plasma was obtained from each blood sample and stored at -80°C. RESULTS: The sTNF-R II level at inclusion (day 1), the PCT level on the day of fever onset, and the change (day 3 - day 1) in the IL-8 and eotaxin levels were significantly higher in patients who died during the 28-day follow-up. A requirement for early adjustment of antimicrobial treatment was associated with higher day 3 levels of IL-8, sTNF-R II, PCT, and MCP-1. CONCLUSION: Procalcitonin, sTNF-R II, IL-8, MCP-1, and eotaxin could potentially be used to assess the risk of death and the requirement for early adjustment of antimicrobial treatment in febrile, neutropenic onco-hematologic patients. The levels of the other markers showed no association with any of the evaluated endpoints. PMID:22012040

  19. Power of screening tests for colorectal cancer enhanced by high levels of M2-PK in addition to FOBT.

    PubMed

    Zaccaro, Cristina; Saracino, Ilaria Maria; Fiorini, Giulia; Figura, Natale; Holton, John; Castelli, Valentina; Pesci, Valeria; Gatta, Luigi; Vaira, Dino

    2017-02-02

    Colorectal cancer (CRC) is a multistep process that involves adenoma-carcinoma sequence. CRC can be prevented by routine screening, which can detect precancerous lesions. The aim of this study is to clarify whether faecal occult blood test (i-FOBT), tumor M2 pyruvate kinase (t-M2-PK), and endocannabinoid system molecules (cannabinoid receptors type 1-CB1, type 2-CB2, and fatty acid amide hydrolase-FAAH) might represent better diagnostic tools, alone or in combination, for an early diagnosis of CRC. An immunochemical FOB test (i-FOBT) and quantitative ELISA stool test for t-M2-PK were performed in 127 consecutive patients during a 12 month period. Endocannabinoid system molecules and t-M2-PK expression were detected by immunostaining in healthy tissues and normal mucosa surrounding adenomatous and cancerous colon lesions. i-FOBT and t-M2-PK combination leads to a better diagnostic accuracy for pre-neoplastic and neoplastic colon lesions. T-M2-PK quantification in stool samples and in biopsy samples (immunostaining) correlates with tumourigenesis stages. CB1 and CB2 are well expressed in healthy tissues, and their expression decreases in the presence of advanced stages of carcinogenesis and disappears in CRC. FAAH signal is well expressed in normal mucosa and low-risk adenoma, and increased in high-risk adenoma and carcinoma adjacent tissues. This study shows that high levels of t-M2-PK in addition to FOBT enhance the power of a CRC screening test. Endocannabinoid system molecule expression correlates with colon carcinogenesis stages. Developing future faecal tests for their quantification must be undertaken to obtain a more accurate early non-invasive diagnosis for CRC.

  20. Expression QTL analysis of top loci from GWAS meta-analysis highlights additional schizophrenia candidate genes.

    PubMed

    de Jong, Simone; van Eijk, Kristel R; Zeegers, Dave W L H; Strengman, Eric; Janson, Esther; Veldink, Jan H; van den Berg, Leonard H; Cahn, Wiepke; Kahn, René S; Boks, Marco P M; Ophoff, Roel A

    2012-09-01

    There is genetic evidence that schizophrenia is a polygenic disorder with a large number of loci of small effect on disease susceptibility. Genome-wide association studies (GWASs) of schizophrenia have had limited success, with the best finding at the MHC locus at chromosome 6p. A recent effort of the Psychiatric GWAS consortium (PGC) yielded five novel loci for schizophrenia. In this study, we aim to highlight additional schizophrenia susceptibility loci from the PGC study by combining the top association findings from the discovery stage (9394 schizophrenia cases and 12 462 controls) with expression QTLs (eQTLs) and differential gene expression in whole blood of schizophrenia patients and controls. We examined the 6192 single-nucleotide polymorphisms (SNPs) with significance threshold at P<0.001. eQTLs were calculated for these SNPs in a sample of healthy controls (n=437). The transcripts significantly regulated by the top SNPs from the GWAS meta-analysis were subsequently tested for differential expression in an independent set of schizophrenia cases and controls (n=202). After correction for multiple testing, the eQTL analysis yielded 40 significant cis-acting effects of the SNPs. Seven of these transcripts show differential expression between cases and controls. Of these, the effect of three genes (RNF5, TRIM26 and HLA-DRB3) coincided with the direction expected from meta-analysis findings and were all located within the MHC region. Our results identify new genes of interest and highlight again the involvement of the MHC region in schizophrenia susceptibility.

  1. Hsp70 level in progeny of aging grasshoppers from variously polluted habitats and additionally exposed to zinc during diapause.

    PubMed

    Augustyniak, Maria; Tarnawska, Monika; Babczyńska, Agnieszka; Augustyniak, Michał

    2009-08-01

    The hsp70 level in the bodies of 1st instars of grasshoppers Chorthippus brunneus from unpolluted (Pilica) and polluted (Olkusz, Szopienice) sites and additionally exposed to various doses of zinc during diapause and embryonic development prior to hatching were measured by Western blotting. The main aim of our work was to assess the relationship between the age of female grasshoppers originating from variously polluted habitat and the hsp70 level in their progeny. Possible reasons for population variation in hsp70 levels were discussed. The hsp70 level in the offspring's body depended on the place of origin. The strongest expression of hsp70 was found in the bodies of larvae hatching from the eggs laid by young females from Pilica (reference site). In contrast, a low initial level of hsp70 in larvae from polluted sites, especially in young females' progeny, was observed. The application of zinc during diapause influenced the hsp70 level in grasshopper larvae; however, the direction of the changes depended on the insects' place of origin. In larvae from the reference site, and also (but to a lesser degree) from Olkusz, the increase in the hsp70 level after zinc treatment was most pronounced. Whereas in grasshoppers from Szopienice, zinc (in 100microg g(-1) dry weight of sand) did not change the hsp70 level, or (in 500microg g(-1) dry weight of sand) caused a reduction in hsp70. The differences may result from maternal effects; however, possible adaptation also cannot be excluded. To confirm this statement further studies are needed.

  2. The expression levels of the sirtuins in patients with BCC.

    PubMed

    Temel, Metin; Koç, Mustafa Nihat; Ulutaş, Saffet; Göğebakan, Bülent

    2016-05-01

    Basal cell carcinoma (BCC) is the most common tumor in humans. Reduced expression of sirtuins interferes with DNA repair, which may cause mutations and genomic instability, and eventually leads to tumor development. In the present study, we investigate the expression levels of SIRT genes in non-tumoral and tumor tissues of patients with BCC. A total of 27 patients (16 males, 11 females) with BCC were included in the study; the mean age was 65.40 ± 10.74 years and mean follow-up was 2.5 ± 0.5 years. There were multiple synchronous lesions in six patients, and the remaining 21 patients had a single lesion. Tumor and non-tumoral tissue samples were collected from all patients, and mRNA expression levels of SIRT1-7 (Sirt1.1, Sirt1.2, Sirt2, Sirt3, Sirt4, Sirt5, Sirt6, and Sirt7) were examined by real-time PCR. The results showed that expressions of SIRT1.1, SIRT1.2, SIRT4, SIRT5, SIRT6, and SIRT7 mRNAs were unchanged in tumor tissues of BCC patients compared with non-tumoral tissue samples. Importantly, the expressions of SIRT2 and SIRT3 mRNAs were significantly reduced in tumor tissue samples from BCC patients compared with non-tumoral tissues (P = 0.02 and P = 0.03, respectively). In light of the previous reports that have demonstrated a link between SIRT proteins and cancer, our findings suggest that SIRT2 and SIRT3 may plan important roles in BCC pathogenesis and could be candidate prognostic biomarkers for BCC.

  3. Additive relationship between serum fibroblast growth factor 21 level and coronary artery disease

    PubMed Central

    2013-01-01

    Background Expression and activity of the fibroblast growth factor (FGF) 21 hormone-like protein are associated with development of several metabolic disorders. This study was designed to investigate whether serum FGF21 level was also associated with the metabolic syndrome-related cardiovascular disease, atherosclerosis, and its clinical features in a Chinese cohort. Methods Two-hundred-and-fifty-three subjects visiting the Cardiology Department (Sixth People's Hospital affiliated to Shanghai JiaoTong University) were examined by coronary arteriography (to diagnose coronary artery disease (CAD)) and hepatic ultrasonography (to diagnose non-alcoholic fatty liver disease (NAFLD)). Serum FGF21 level was measured by enzyme-linked immunosorbent assay and analyzed for correlation to subject and clinical characteristics. The independent factors of CAD were determined by multivariate logistic regression analysis. Results Subjects with NAFLD showed significantly higher serum FGF21 than those without NAFLD (388.0 pg/mL (253.0-655.4) vs. 273.3 pg/mL (164.9-383.7), P < 0.01). Subjects with CAD showed significantly higher serum FGF21, regardless of NAFLD diagnosis (P < 0.05). Serum FGF21 level significantly elevated with the increasing number of metabolic disorders (P for trend < 0.01). After adjustment of age, sex, and BMI, FGF21 was positively correlated with total cholesterol (P < 0.05) and triglyceride (P < 0.01). FGF21 was identified as an independent factor of CAD (odds ratio = 2.984, 95% confidence interval: 1.014-8.786, P < 0.05). Conclusions Increased level of serum FGF21 is associated with NAFLD, metabolic disorders and CAD. PMID:23981342

  4. Comparison of additive image fusion vs. feature-level image fusion techniques for enhanced night driving

    NASA Astrophysics Data System (ADS)

    Bender, Edward J.; Reese, Colin E.; Van Der Wal, Gooitzen S.

    2003-02-01

    The Night Vision & Electronic Sensors Directorate (NVESD) has conducted a series of image fusion evaluations under the Head-Tracked Vision System (HTVS) program. The HTVS is a driving system for both wheeled and tracked military vehicles, wherein dual-waveband sensors are directed in a more natural head-slewed imaging mode. The HTVS consists of thermal and image-intensified TV sensors, a high-speed gimbal, a head-mounted display, and a head tracker. A series of NVESD field tests over the past two years has investigated the degree to which additive (A+B) image fusion of these sensors enhances overall driving performance. Additive fusion employs a single (but user adjustable) fractional weighting for all the features of each sensor's image. More recently, NVESD and Sarnoff Corporation have begun a cooperative effort to evaluate and refine Sarnoff's "feature-level" multi-resolution (pyramid) algorithms for image fusion. This approach employs digital processing techniques to select at each image point only the sensor with the strongest features, and to utilize only those features to reconstruct the fused video image. This selection process is performed simultaneously at multiple scales of the image, which are combined to form the reconstructed fused image. All image fusion techniques attempt to combine the "best of both sensors" in a single image. Typically, thermal sensors are better for detecting military threats and targets, while image-intensified sensors provide more natural scene cues and detect cultural lighting. This investigation will address the differences between additive fusion and feature-level image fusion techniques for enhancing the driver's overall situational awareness.

  5. Additive white Gaussian noise level estimation in SVD domain for images.

    PubMed

    Liu, Wei; Lin, Weisi

    2013-03-01

    Accurate estimation of Gaussian noise level is of fundamental interest in a wide variety of vision and image processing applications as it is critical to the processing techniques that follow. In this paper, a new effective noise level estimation method is proposed on the basis of the study of singular values of noise-corrupted images. Two novel aspects of this paper address the major challenges in noise estimation: 1) the use of the tail of singular values for noise estimation to alleviate the influence of the signal on the data basis for the noise estimation process and 2) the addition of known noise to estimate the content-dependent parameter, so that the proposed scheme is adaptive to visual signals, thereby enabling a wider application scope of the proposed scheme. The analysis and experiment results demonstrate that the proposed algorithm can reliably infer noise levels and show robust behavior over a wide range of visual content and noise conditions, and that is outperforms relevant existing methods.

  6. Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.

    PubMed

    Gungabeesoon, Jeremy; Tremblay, Michel L; Uetani, Noriko

    2016-07-01

    The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 β-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels.

  7. [Influence Additional Cognitive Tasks on EEG Beta Rhythm Parameters during Forming and Testing Set to Perception of the Facial Expression].

    PubMed

    Yakovenko, I A; Cheremushkin, E A; Kozlov, M K

    2015-01-01

    The research of changes of a beta rhythm parameters on condition of working memory loading by extension of a interstimuli interval between the target and triggering stimuli to 16 sec is investigated on 70 healthy adults in two series of experiments with set to a facial expression. In the second series at the middle of this interval for strengthening of the load was entered the additional cognitive task in the form of conditioning stimuli like Go/NoGo--circles of blue or green color. Data analysis of the research was carried out by means of continuous wavelet-transformation on the basis of "mather" complex Morlet-wavelet in the range of 1-35 Hz. Beta rhythm power was characterized by the mean level, maxima of wavelet-transformation coefficient (WLC) and latent periods of maxima. Introduction of additional cognitive task to pause between the target and triggering stimuli led to essential increase in absolute values of the mean level of beta rhythm WLC and relative sizes of maxima of beta rhythm WLC. In the series of experiments without conditioning stimulus subjects with large number of mistakes (from 6 to 40), i.e. rigid set, in comparison with subjects with small number of mistakes (to 5), i.e. plastic set, at the forming stage were characterized by higher values of the mean level of beta rhythm WLC. Introduction of the conditioning stimuli led to smoothing of intergroup distinctions throughout the experiment.

  8. Ground-Level Ozone Following Astrophysical Ionizing Radiation Events: An Additional Biological Hazard?

    PubMed

    Thomas, Brian C; Goracke, Byron D

    2016-01-01

    Astrophysical ionizing radiation events such as supernovae, gamma-ray bursts, and solar proton events have been recognized as a potential threat to life on Earth, primarily through depletion of stratospheric ozone and subsequent increase in solar UV radiation at Earth's surface and in the upper levels of the ocean. Other work has also considered the potential impact of nitric acid rainout, concluding that no significant threat is likely. Not yet studied to date is the potential impact of ozone produced in the lower atmosphere following an ionizing radiation event. Ozone is a known irritant to organisms on land and in water and therefore may be a significant additional hazard. Using previously completed atmospheric chemistry modeling, we examined the amount of ozone produced in the lower atmosphere for the case of a gamma-ray burst and found that the values are too small to pose a significant additional threat to the biosphere. These results may be extended to other ionizing radiation events, including supernovae and extreme solar proton events.

  9. Elite sport is not an additional source of distress for adolescents with high stress levels.

    PubMed

    Gerber, Markus; Holsboer-Trachsler, Edith; Pühse, Uwe; Brand, Serge

    2011-04-01

    This study examined whether participation in elite sport interacts with stress in decreasing or increasing symptoms of depression and anxiety among adolescents, and further, whether the interplay between participation in high-performance sport and stress is related to the perceived quality of sleep. 434 adolescents (278 girls, 156 boys; age: M = 17.2 yr.) from 15 "Swiss Olympic Sport Classes" and 9 conventional classes answered a questionnaire and completed a 7-day sleep log. Analyses of covariance showed that heightened stress was related to more depressive symptoms and higher scores for trait-anxiety. Moreover, those classified as having poor sleep by a median split cutoff reported higher levels of depressive symptoms. No significant (multivariate) main effects were found for high-performance sport athletes. Similarly, no significant two- or three-way interaction effects were found. These results caution against exaggerated expectations concerning sport participation as a stress buffer. Nevertheless, participation in high-performance sport was not found to be an additional source of distress for adolescents who reported high stress levels despite prior research that has pointed toward such a relationship.

  10. Genome-wide meta-analysis of maize heterosis reveals the potential role of additive gene expression at pericentromeric loci

    PubMed Central

    2014-01-01

    Background The identification of QTL involved in heterosis formation is one approach to unravel the not yet fully understood genetic basis of heterosis - the improved agronomic performance of hybrid F1 plants compared to their inbred parents. The identification of candidate genes underlying a QTL is important both for developing markers and determining the molecular genetic basis of a trait, but remains difficult owing to the large number of genes often contained within individual QTL. To address this problem in heterosis analysis, we applied a meta-analysis strategy for grain yield (GY) of Zea mays L. as example, incorporating QTL-, hybrid field-, and parental gene expression data. Results For the identification of genes underlying known heterotic QTL, we made use of tight associations between gene expression pattern and the trait of interest, identified by correlation analyses. Using this approach genes strongly associated with heterosis for GY were discovered to be clustered in pericentromeric regions of the complex maize genome. This suggests that expression differences of sequences in recombination-suppressed regions are important in the establishment of heterosis for GY in F1 hybrids and also in the conservation of heterosis for GY across genotypes. Importantly functional analysis of heterosis-associated genes from these genomic regions revealed over-representation of a number of functional classes, identifying key processes contributing to heterosis for GY. Based on the finding that the majority of the analyzed heterosis-associated genes were addtitively expressed, we propose a model referring to the influence of cis-regulatory variation on heterosis for GY by the compensation of fixed detrimental expression levels in parents. Conclusions The study highlights the utility of a meta-analysis approach that integrates phenotypic and multi-level molecular data to unravel complex traits in plants. It provides prospects for the identification of genes relevant for

  11. Additional shear resistance from fault roughness and stress levels on geometrically complex faults

    NASA Astrophysics Data System (ADS)

    Fang, Zijun; Dunham, Eric M.

    2013-07-01

    The majority of crustal faults host earthquakes when the ratio of average background shear stress τb to effective normal stress σeff is τb/σeff≈0.6. In contrast, mature plate-boundary faults like the San Andreas Fault (SAF) operate at τb/σeff≈0.2. Dynamic weakening, the dramatic reduction in frictional resistance at coseismic slip velocities that is commonly observed in laboratory experiments, provides a leading explanation for low stress levels on mature faults. Strongly velocity-weakening friction laws permit rupture propagation on flat faults above a critical stress level τpulse/σeff≈0.25. Provided that dynamic weakening is not restricted to mature faults, the higher stress levels on most faults are puzzling. In this work, we present a self-consistent explanation for the relatively high stress levels on immature faults that is compatible with low coseismic frictional resistance, from dynamic weakening, for all faults. We appeal to differences in structural complexity with the premise that geometric irregularities introduce resistance to slip in addition to frictional resistance. This general idea is quantified for the special case of self-similar fractal roughness of the fault surface. Natural faults have roughness characterized by amplitude-to-wavelength ratios α between 10-3 and 10-2. Through a second-order boundary perturbation analysis of quasi-static frictionless sliding across a band-limited self-similar interface in an ideally elastic solid, we demonstrate that roughness induces an additional shear resistance to slip, or roughness drag, given by τdrag=8π3α2G∗Δ/λmin, for G∗=G/(1-ν) with shear modulus Gand Poisson's ratio ν, slip Δ, and minimum roughness wavelength λmin. The influence of roughness drag on fault mechanics is verified through an extensive set of dynamic rupture simulations of earthquakes on strongly rate-weakening fractal faults with elastic-plastic off-fault response. The simulations suggest that fault rupture, in

  12. Probe-Level Analysis of Expression Microarrays Characterizes Isoform-Specific Degradation during Mouse Oocyte Maturation

    PubMed Central

    Salisbury, Jesse; Hutchison, Keith W.; Wigglesworth, Karen; Eppig, John J.; Graber, Joel H.

    2009-01-01

    Background Gene expression microarrays have provided many insights into changes in gene expression patterns between different tissue types, developmental stages, and disease states. Analyses of these data focused primarily measuring the relative abundance of transcripts of a gene, while treating most or all transcript isoforms as equivalent. Differences in the selection between transcript isoforms can, however, represent critical changes to either the protein product or the posttranscriptional regulation of the transcript. Novel analyses on existing microarray data provide fresh insights and new interpretations into transcriptome-wide changes in expression. Methodology A probe-level analysis of existing gene expression arrays revealed differences in mRNA processing, primarily affecting the 3′-untranslated region. Working with the example of microarrays drawn from a transcriptionally silent period of mouse oocyte development, probe-level analysis (implemented here as rmodel) identified genes whose transcript isoforms have differing stabilities. Comparison of micorarrays measuring cDNA generated from oligo-dT and random primers revealed further differences in the polyadenylation status of some transcripts. Additional analysis provided evidence for sequence-targeted cleavage, including putative targeting sequences, as one mechanism of degradation for several hundred transcripts in the maturing oocyte. Conclusions The capability of probe-level analysis to elicit novel findings from existing expression microarray data was demonstrated. The characterization of differences in stability between transcript isoforms in maturing mouse oocytes provided some mechanistic details of degradation. Similar analysis of existing archives of expression microarray data will likely provide similar discoveries. PMID:19834616

  13. Arrays in rays: terminal addition in echinoderms and its correlation with gene expression.

    PubMed

    Mooi, Rich; David, Bruno; Wray, Gregory A

    2005-01-01

    The echinoderms are deuterostomes that superimpose radial symmetry upon bilateral larval morphology. Consequently, they are not the first animals that come to mind when the concepts of segmentation and terminal addition are being discussed. However, it has long been recognized that echinoderms have serial elements along their radii formed in accordance with the ocular plate rule (OPR). The OPR is a special case of terminal growth, forming elements of the ambulacra that define the rays in echinoderms. New elements are added at the terminus of the ray, which may or may not be marked by a calcified element called the terminal plate (the "ocular" of sea urchins). The OPR operates in every echinoderm, from the occasionally bizarre fossils of the Cambrian to the most familiar extant taxa. Using the OPR and other criteria of recognition, echinoderm body wall can be divided into two main regions: extraxial components are associated with the somatocoels, axial components (formed in accordance with the OPR) with the hydrocoel. We compare patterns of development in axial regions of echinoderms with those found in the anterior-posterior axes of the earliest echinoderms as well as other invertebrates. Although axial and extraxial skeletons appear to be composed of the same biomineral matrix, the genes involved in patterning these two skeletal components are likely distinct. During development of the axial skeleton, for instance, the genes engrailed and orthodenticle are expressed in spatial and temporal patterns consistent with the OPR. Other genes such as distal-less seem to demarcate early ontogenetic boundaries between the axial rudiment and the extraxial larval body. There is a complex and pervasive reorganization of gene expression domains to produce the highly divergent morphologies seen in the Echinodermata. We integrate morphological and genetic information, particularly with respect to the origins of radial symmetry in the rudiment, and the concomitant development of

  14. Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation

    NASA Technical Reports Server (NTRS)

    1980-01-01

    An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.

  15. Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

    PubMed

    Péladeau, Christine; Ahmed, Aatika; Amirouche, Adel; Crawford Parks, Tara E; Bronicki, Lucas M; Ljubicic, Vladimir; Renaud, Jean-Marc; Jasmin, Bernard J

    2016-01-01

    Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and β-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

  16. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    PubMed

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  17. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  18. Shades of Emotion: What the Addition of Sunglasses or Masks to Faces Reveals about the Development of Facial Expression Processing

    ERIC Educational Resources Information Center

    Roberson, Debi; Kikutani, Mariko; Doge, Paula; Whitaker, Lydia; Majid, Asifa

    2012-01-01

    Three studies investigated developmental changes in facial expression processing, between 3 years-of-age and adulthood. For adults and older children, the addition of sunglasses to upright faces caused an equivalent decrement in performance to face inversion. However, younger children showed "better" classification of expressions of faces wearing…

  19. A comparison of brain gene expression levels in domesticated and wild animals.

    PubMed

    Albert, Frank W; Somel, Mehmet; Carneiro, Miguel; Aximu-Petri, Ayinuer; Halbwax, Michel; Thalmann, Olaf; Blanco-Aguiar, Jose A; Plyusnina, Irina Z; Trut, Lyudmila; Villafuerte, Rafael; Ferrand, Nuno; Kaiser, Sylvia; Jensen, Per; Pääbo, Svante

    2012-09-01

    Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.

  20. A Comparison of Brain Gene Expression Levels in Domesticated and Wild Animals

    PubMed Central

    Albert, Frank W.; Somel, Mehmet; Carneiro, Miguel; Aximu-Petri, Ayinuer; Halbwax, Michel; Thalmann, Olaf; Blanco-Aguiar, Jose A.; Trut, Lyudmila; Villafuerte, Rafael; Ferrand, Nuno; Kaiser, Sylvia; Jensen, Per; Pääbo, Svante

    2012-01-01

    Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30–75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different. PMID:23028369

  1. Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

    PubMed Central

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

  2. Levels of lycopene β-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

    PubMed

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.

  3. Surface L-type Ca2+ channel expression levels are increased in aged hippocampus

    PubMed Central

    Núñez-Santana, Félix Luis; Oh, Myongsoo Matthew; Antion, Marcia Diana; Lee, Amy; Hell, Johannes Wilhelm; Disterhoft, John Francis

    2014-01-01

    Age-related increase in L-type Ca2+ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca2+ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age-related cognitive deficits. Here, using specific antibodies against Cav1.2 and Cav1.3 subunits of LTCCs, we systematically re-examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Cav1.2 and Cav1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Cav1.2 in the CA1 and CA3 regions and of Cav1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Cav1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Cav1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age-related cognitive deficits cannot be attributed to a global change in L-type channel expression nor to the level of phosphorylation of Cav1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age-related increase in L-type Ca2+ channel activity in CA1 pyramidal neurons. PMID:24033980

  4. Simulation of uphill/downhill running on a level treadmill using additional horizontal force.

    PubMed

    Gimenez, Philippe; Arnal, Pierrick J; Samozino, Pierre; Millet, Guillaume Y; Morin, Jean-Benoit

    2014-07-18

    Tilting treadmills allow a convenient study of biomechanics during uphill/downhill running, but they are not commonly available and there is even fewer tilting force-measuring treadmill. The aim of the present study was to compare uphill/downhill running on a treadmill (inclination of ± 8%) with running on a level treadmill using additional backward or forward pulling forces to simulate the effect of gravity. This comparison specifically focused on the energy cost of running, stride frequency (SF), electromyographic activity (EMG), leg and foot angles at foot strike, and ground impact shock. The main results are that SF, impact shock, and leg and foot angle parameters determined were very similar and significantly correlated between the two methods, the intercept and slope of the linear regression not differing significantly from zero and unity, respectively. The correlation of oxygen uptake (V̇O2) data between both methods was not significant during uphill running (r=0.42; P>0.05). V̇O2 data were correlated during downhill running (r=0.74; P<0.01) but there was a significant difference between the methods (bias=-2.51 ± 1.94 ml min(-1) kg(-1)). Linear regressions for EMG of vastus lateralis, biceps femoris, gastrocnemius lateralis, soleus and tibialis anterior were not different from the identity line but the systematic bias was elevated for this parameter. In conclusion, this method seems appropriate for the study of SF, leg and foot angle, impact shock parameters but is less applicable for physiological variables (EMG and energy cost) during uphill/downhill running when using a tilting force-measuring treadmill is not possible.

  5. Expression levels of B7-H3 and TLT-2 in human oral squamous cell carcinoma

    PubMed Central

    ZHANG, SHAN-SHAN; TANG, JING; YU, SHU-YI; MA, LI; WANG, FENG; XIE, SHU-LE; JIN, LONG; YANG, HONG-YU

    2015-01-01

    The aim of the present study was to investigate the role of immune regulatory molecules B7-H3 [also known as cluster of differentiation 276] and triggering receptor expressed on myeloid cell-like transcript-2 (TLT-2) in patients with oral squamous cell carcinoma (OSCC). Human OSCC samples were obtained from 76 patients (female, 32; male, 44; age range, 23–81 years; median age, 50.9 years) that underwent resection for OSCC at Peking University Shenzhen Hospital (Shenzhen, China) between 2007 and 2010. In addition, control oral mucosal samples were obtained from 76 healthy individuals (female, 36; male, 40; age range, 21–62 years; median age, 45.3 years) during wisdom tooth extraction. Protein and gene expression levels of B7-H3 and TLT-2 were determined by immunohistochemical analysis and reverse transcription-polymerase chain reaction (RT-PCR). In the healthy oral mucosa samples, B7-H3 expression was identified to be weak, while the expression of TLT-2 was only detected sporadically in the cell membrane and cytoplasm. By contrast, the two regulatory molecules were widely expressed in the aforementioned localizations in human OSCC specimens upon immunohistochemical examination. Furthermore, quantitative RT-PCR confirmed the presence of significantly higher B7-H3 and TLT-2 expression levels in OSCC specimens compared with the oral mucosa of healthy individuals. The significantly higher expression levels of B7-H3 and TLT-2 in human OSCC specimens may indicate an inhibitory role of these molecules in the antitumoral immune response. To investigate interactions between these two molecules and individual antitumoral immune response in OSCC patients, prospective clinical studies with an adequate sample size are required. PMID:26622626

  6. High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

    PubMed

    Shen, Wei; Shu, Min; Ma, Lixin; Ni, Hong; Yan, Hong

    2016-03-01

    The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest.

  7. Amplification of steroid-mediated SP-B expression by physiological levels of caffeine.

    PubMed

    Fehrholz, Markus; Hütten, Matthias; Kramer, Boris W; Speer, Christian P; Kunzmann, Steffen

    2014-01-01

    Factors positively influencing surfactant homeostasis in general and surfactant protein B (SP-B) expression in particular are considered of clinical importance regarding an improvement of lung function in preterm infants. The objective of this study was to identify effects of physiological levels of caffeine on glucocorticoid-mediated SP-B expression in vitro and in vivo. Levels of SP-B and pepsinogen C were quantified by quantitative real-time RT-PCR or immunoblotting in NCI-H441 cells daily exposed to caffeine and/or dexamethasone (DEX). In vivo, SP-B expression was analyzed in bronchoalveolar lavage (BAL) of preterm sheep exposed to antenatal DEX and/or postnatal caffeine. If DEX and caffeine were continuously present, SP-B mRNA and protein levels were increased for up to 6 days after induction (P < 0.05). Additionally, caffeine enhanced SP-B mRNA expression in DEX-pretreated cells (P < 0.05). Moreover, caffeine amplified DEX-induced pepsinogen C mRNA expression (P < 0.05). After short-term treatment with caffeine in vivo, only slightly higher SP-B levels could be detected in BAL of preterm sheep following antenatal DEX, combined with an increase of arterial oxygen partial pressure (P < 0.01). Our data demonstrated that the continuous presence of caffeine in vitro is able to amplify DEX-mediated SP-B expression. In contrast, short-term improvement of lung function in vivo is likely to be independent of altered SP-B transcription and translation. An impact of caffeine on release of surfactant reservoirs from lamellar bodies could, however, quickly affect SP-B content in BAL, which has to be further investigated. Our findings indicate that caffeine is able to amplify main effects of glucocorticoids that result from changes in surfactant production, maturation, and release.

  8. Food additives: Sodium benzoate, potassium sorbate, azorubine, and tartrazine modify the expression of NFκB, GADD45α, and MAPK8 genes.

    PubMed

    Raposa, B; Pónusz, R; Gerencsér, G; Budán, F; Gyöngyi, Z; Tibold, A; Hegyi, D; Kiss, I; Koller, Á; Varjas, T

    2016-09-01

    It has been reported that some of the food additives may cause sensitization, inflammation of tissues, and potentially risk factors in the development of several chronic diseases. Thus, we hypothesized that expressions of common inflammatory molecules - known to be involved in the development of various inflammatory conditions and cancers - are affected by these food additives. We investigated the effects of commonly used food preservatives and artificial food colorants based on the expressions of NFκB, GADD45α, and MAPK8 (JNK1) from the tissues of liver. RNA was isolated based on Trizol protocol and the activation levels were compared between the treated and the control groups. Tartrazine alone could elicit effects on the expressions of NFκB (p = 0.013) and MAPK8 (p = 0.022). Azorubine also resulted in apoptosis according to MAPK8 expression (p = 0.009). Preservatives were anti-apoptotic in high dose. Sodium benzoate (from low to high doses) dose-dependently silenced MAPK8 expression (p = 0.004 to p = 0.002). Addition of the two preservatives together elicited significantly greater expression of MAPK8 at half-fold dose (p = 0.002) and at fivefold dose (p = 0.008). This study suggests that some of the food preservatives and colorants can contribute to the activation of inflammatory pathways.

  9. Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors.

    PubMed

    Sgourou, Argyro; Routledge, Samantha; Spathas, Dionysios; Athanassiadou, Aglaia; Antoniou, Michael N

    2009-08-20

    Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

  10. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  11. Effect of Greens and Soil Type, Sulfur Addition and Lithium Level on Leaf Constituents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A greenhouse experiment was conducted near Weslaco, Texas (Lat. 26o 8' N, Long. 97o 57' W) between Dec. 2006 and Feb 2007 to evaluate the effect of soil type, added sulfur and lithium level on the growth and leaf nutrients, particularly biofortified levels of Li and S, in spinach and mustard gree...

  12. Spinach and mustard greens response to soil type, sulfur addition and lithium level

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A greenhouse experiment was conducted near Weslaco, Texas (Lat. 26o 8' N, Long. 97o 57' W) between Dec. 2006 and Feb 2007 to evaluate the effect of soil type, added sulfur and lithium level on the growth and leaf nutrients, particularly biofortified levels of Li and S, in spinach and mustard gree...

  13. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

  14. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments.

  15. Clinical impact of L1CAM expression measured on the transcriptome level in ovarian cancer

    PubMed Central

    Azim, Samira Abdel; Duggan-Peer, Michaela; Sprung, Susanne; Reimer, Daniel; Fiegl, Heidi; Soleiman, Afschin; Marth, Christian; Zeimet, Alain G.

    2016-01-01

    Background High expression of L1 cell adhesion molecules (L1CAM) has been repeatedly shown to be associated with aggressive disease behavior, which translates in poor clinical outcome in various cancer entities. However, in ovarian cancer results based either on immunohistochemistry or cytosolic protein quantifications remained conflicting regarding clinical behavior. In the present work we assessed L1CAM expression on the transcriptome level with the highly sensitive quantitative real-time PCR (qRT-PCR) to define its relevance in ovarian cancer biology. Results There was a significant difference in L1CAM high and low mRNA expressing cancers with regard to disease-free (p=0.002) and overall survival (p=0.008). L1CAM proofed to be an independent predictor for disease progression (HR 1.8, p=0.01) and overall survival (HR 1.6, p=0.04). Furthermore, a significant positive correlation between the level of L1CAM and the grade of tumor differentiation (p=0.04), the FIGO stage (p=0.025) as well as the histological subtype (p= 0.002) was found. Methods This study included fresh frozen tissue samples of 138 patients with FIGO I-IV stage ovarian cancer. L1CAM mRNA expression was determined using qRT-PCR. In the calculations special attention was put on the various histological subtypes. In survival analysis median L1CAM mRNA expression obtained in the entire cohort of ovarian cancer samples was used as a cut-off to distinguish between high and low L1CAM mRNA expression. Conclusion L1CAM mRNA expression appears to play a substantial role in the pathophysiology of ovarian cancer that is translated into poor clinical outcome. Additionally humanized L1CAM antibodies, which can serve as potential future treatment options are under testing. PMID:27174921

  16. Observation of an additional electronic level of the EL2 defect

    NASA Astrophysics Data System (ADS)

    Stiévenard, D.; Delerue, C.; von Bardeleben, H. J.; Bourgoin, J. C.; Guillot, G.; Brémond, G.; Azoulay, R.

    1991-07-01

    Using deep-level transient spectroscopy (DLTS), we have studied the properties of the EL2 defect in the alloy system Ga1-xAlxAs grown by metal-organic chemical-vapor deposition, with x=0.145. We have observed the stable state of the defect, i.e., its quench under a 1.18-eV illumination and a different DLTS peak, associated with EL2. The study of the behavior of this peak versus the illumination and thermal treatment allows us to associate this peak with a different electronic level of EL2: the (-/0) level if EL2 is an isolated antisite AsGa or the (0/+) level if EL2 is associated with the (AsGa-As+i) pair.

  17. Solving Additive Problems at Pre-Elementary School Level with the Support of Graphical Representation

    ERIC Educational Resources Information Center

    Selva, Ana Coelho Vieira; Falcao, Jorge Tarcisio da Rocha; Nunes, Terezinha

    2005-01-01

    This research offers empirical evidence of the importance of supplying diverse symbolic representations in order to support concept development in mathematics. Graphical representation can be a helpful symbolic tool for concept development in the conceptual field of additive structures. Nevertheless, this symbolic tool has specific difficulties…

  18. Expression levels of TWIST1 are associated with the clinicopathological stage of B-cell non-Hodgkin lymphoma

    PubMed Central

    JIA, CUNDONG; LIANG, LIPING; YANG, LILI; ZHAO, FENG; BAI, JINGPING

    2014-01-01

    The aim of the present study was to investigate the expression level of TWIST1 in B-cell non-Hodgkin lymphoma (BNHL) and its association with the clinicopathological characteristics of BNHL. Expression levels of TWIST1 were analyzed in patients with BNHL (n=45) and lymphadenosis (n=21) using immunohistochemical staining and western blot analysis. In addition, the mRNA expression levels of TWIST1 in the peripheral blood were detected by fluorescent quantitative polymerase chain reaction. The positive rate of TWIST1 expression in the BNHL tissue was 82.2%, which was significantly higher compared with the lymphadenosis tissue (5%; P<0.05). In addition, the protein expression level of TWIST1 in the BNHL tissue was higher compared with the lymphadenosis tissue. TWIST1 expression was also higher in stage III/IV BNHL tissues than in stage I/II tissues (P<0.05). The tissues were staged following the Ann Arbor system. Furthermore, the mRNA expression level of TWIST1 in the peripheral blood of the BNHL tissue (3.03±0.03) was higher compared with the lymphadenosis tissue, and the mRNA expression level of TWIST1 was higher in stage III/IV (4.41±0.12) tissues than in stage I/II BNHL (2.03±0.08) tissues. In conclusion, TWIST1 expression was higher in the tissue and peripheral blood of patients with BNHL when compared with those with lymphadenosis. Thus, TWIST1 expression was associated with the clinicopathological stage of BNHL. PMID:25289047

  19. The genetic basis of evolutionary change in gene expression levels

    PubMed Central

    Emerson, J. J.; Li, Wen-Hsiung

    2010-01-01

    The regulation of gene expression is an important determinant of organismal phenotype and evolution. However, the widespread recognition of this fact occurred long after the synthesis of evolution and genetics. Here, we give a brief sketch of thoughts regarding gene regulation in the history of evolution and genetics. We then review the development of genome-wide studies of gene regulatory variation in the context of the location and mode of action of the causative genetic changes. In particular, we review mapping of the genetic basis of expression variation through expression quantitative trait locus studies and measuring the cis/trans component of expression variation in allele-specific expression studies. We conclude by proposing a systematic integration of ideas that combines global mapping studies, cis/trans tests and modern population genetics methodologies, in order to directly estimate the forces acting on regulatory variation within and between species. PMID:20643748

  20. A Pilot Study to Examine the Effect of Additional Structured Outdoor Playtime on Preschoolers' Physical Activity Levels

    ERIC Educational Resources Information Center

    Alhassan, Sofiya; Nwaokelemeh, Ogechi; Lyden, Kate; Goldsby, TaShauna; Mendoza, Albert

    2013-01-01

    The impact of additional structured outdoor playtime on preschoolers'; physical activity (PA) level is unclear. The purpose of this pilot study was to explore the effects of increasing structured outdoor playtime on preschoolers'; PA levels. Eight full-day classrooms (n = 134 children) from two preschool programmes were randomised into a treatment…

  1. Addition by Subtraction: The Relation between Dropout Rates and School-Level Academic Achievement

    ERIC Educational Resources Information Center

    Glennie, Elizabeth; Bonneau, Kara; vanDellen, Michelle; Dodge, Kenneth A.

    2012-01-01

    Background/Context: Efforts to improve student achievement should increase graduation rates. However, work investigating the effects of student-level accountability has consistently demonstrated that increases in the standards for high school graduation are correlated with increases in dropout rates. The most favored explanation for this finding…

  2. Nonlinear responses of coastal salt marshes to nutrient additions and sea level rise

    EPA Science Inventory

    Increasing nutrients and accelerated sea level rise (SLR) can cause marsh loss in some coastal systems. Responses to nutrients and SLR are complex and vary with soil matrix, marsh elevation, sediment inputs, and hydroperiod. We describe field and greenhouse studies examining sing...

  3. Addition by Subtraction: The Relation Between Dropout Rates and School-Level Academic Achievement

    PubMed Central

    GLENNIE, ELIZABETH; BONNEAU, KARA; VANDELLEN, MICHELLE; DODGE, KENNETH A.

    2013-01-01

    Background/Context Efforts to improve student achievement should increase graduation rates. However, work investigating the effects of student-level accountability has consistently demonstrated that increases in the standards for high school graduation are correlated with increases in dropout rates. The most favored explanation for this finding is that high-stakes testing policies that mandate grade repetition and high school exit exams may be the tipping point for students who are already struggling academically. These extra demands may, in fact, push students out of school. Purpose/Objective/Focus This article examines two hypotheses regarding the relation between school-level accountability and dropout rates. The first posits that improvements in school performance lead to improved success for everyone. If school-level accountability systems improve a school for all students, then the proportion of students performing at grade level increases, and the dropout rate decreases. The second hypothesis posits that schools facing pressure to improve their overall accountability score may pursue this increase at the cost of other student outcomes, including dropout rate. Research Design Our approach focuses on the dynamic relation between school-level academic achievement and dropout rates over time—that is, between one year’s achievement and the subsequent year’s dropout rate, and vice versa. This article employs longitudinal data of records on all students in North Carolina public schools over an 8-year period. Analyses employ fixed-effects models clustering schools and districts within years and controls each year for school size, percentage of students who were free/reduced-price lunch eligible, percentage of students who are ethnic minorities, and locale. Findings/Results This study finds partial evidence that improvements in school-level academic performance will lead to improvements (i.e., decreases) in school-level dropout rates. Schools with improved

  4. Bimodal distribution of RNA expression levels in human skeletal muscle tissue

    PubMed Central

    2011-01-01

    Background Many human diseases and phenotypes are related to RNA expression, levels of which are influenced by a wide spectrum of genetic and exposure-related factors. In a large genome-wide study of muscle tissue expression, we found that some genes exhibited a bimodal distribution of RNA expression, in contrast to what is usually assumed in studies of a single healthy tissue. As bimodality has classically been considered a hallmark of genetic control, we assessed the genome-wide prevalence, cause, and association of this phenomenon with diabetes-related phenotypes in skeletal muscle tissue from 225 healthy Pima Indians using exon array expression chips. Results Two independent batches of microarrays were used for bimodal assessment and comparison. Of the 17,881 genes analyzed, eight (GSTM1, HLA-DRB1, ERAP2, HLA-DRB5, MAOA, ACTN3, NR4A2, and THNSL2) were found to have bimodal expression replicated in the separate batch groups, while 24 other genes had evidence of bimodality in only one group. Some bimodally expressed genes had modest associations with pre-diabetic phenotypes, of note ACTN3 with insulin resistance. Most of the other bimodal genes have been reported to be involved with various other diseases and characteristics. Association of expression with cis genetic variation in a subset of 149 individuals found all but one of the confirmed bimodal genes and nearly half of all potential ones to be highly significant expression quantitative trait loci (eQTL). The rare prevalence of these bimodally expressed genes found after controlling for batch effects was much lower than the prevalence reported in other studies. Additional validation in data from separate muscle expression studies confirmed the low prevalence of bimodality we observed. Conclusions We conclude that the prevalence of bimodal gene expression is quite rare in healthy muscle tissue (<0.2%), and is much lower than limited reports from other studies. The major cause of these clearly bimodal

  5. Additional reduction in serum phosphorus levels by pulverized lanthanum carbonate chewable in hemodialysis patients.

    PubMed

    Yamashita, Tetsuri; Ogawa, Tetsuya; Takahashi, Masaki; Mitsuhashi, Tetsuya; Shizuku, Junichi; Takahashi, Naoshi; Ohba, Takashi; Miyajima, Sayako; Kabaya, Takashi; Otsuka, Kuniaki; Nitta, Kosaku

    2013-04-01

    Lanthanum carbonate (LC) is one of the relatively new phosphate binders. The general LC dosage form is a chewable pharmaceutical preparation. This investigation was targeted to subjects who do not chew LC chewable preparations adequately, for the purpose of studying the clinical efficacy of changing to pulverized prescriptions, such as changes in serum phosphorus levels (P levels). The study took place at Minamisenju Hospital in October 2011, with 41 subjects on maintenance hemodialysis. We pulverized all of the LC chewable medicines of the LC insufficient mastication group (non-chewing: NC group, n = 18) using a crusher, and changed them to pulverized prescriptions. The testing period was set at 10 weeks. In the NC group, there was a significant lowering of P levels from 5.86 ± 1.31 mg/dL before pulverization of the LC chewable preparation (week 0) to 5.38 ± 1.26 mg/dL after 2 weeks of administration of the pulverized medication (P = 0.0310), 5.20 ± 1.25 mg/dL after 4 weeks (P = 0.0077), and 5.12 ± 1.34 mg/dL after 6 weeks (P = 0.0167). P levels in other patients than NC group showed no significant change. In this study, the P levels in the NC group was lowered significantly by changing the LC chewable to the pulverized prescription, and the residual LC images on the abdominal X-rays disappeared to the point where they could barely be confirmed.

  6. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  7. Priming effect in agricultural and forest soils depending on glucose level and N addition

    NASA Astrophysics Data System (ADS)

    Splettstoesser, Thomas; Kumar, Amit; Sun, Yue

    2015-04-01

    Growing plants continuously release easily available organic compounds into the rhizosphere. By their interactions with soil microbial biomass (MB) these compounds result in changes of organic matter turnover rates. The understanding of this priming effect (PE) is important for the estimation of climate change impacts on different land use systems. In order to investigate the PE, we conducted a soil incubation experiment under laboratory conditions with two loamy soils: one under cropland and the second under a deciduous forest near Göttingen. 13C and 14C Glucose were added in four levels reaching from 10% to 300% of MB-C. Furthermore two nitrogen levels were established in order to investigate the effects of fertilization on PE. During the whole experiment CO2 release was monitored by trapping in a NaOH solution. Nitrogen mineralization rate, activity of enzymes, and composition of MB were analyzed at the start, after one day, after one week and at the end of the experiment. The results on priming effects induced in agricultural and forest soils depending on N and glucose levels will be presented.

  8. Evaluation of alternative chemical additives for high-level waste vitrification feed preparation processing

    SciTech Connect

    Seymour, R.G.

    1995-06-07

    During the development of the feed processing flowsheet for the Defense Waste Processing Facility (DWPF) at the Savannah River Site (SRS), research had shown that use of formic acid (HCOOH) could accomplish several processing objectives with one chemical addition. These objectives included the decomposition of tetraphenylborate, chemical reduction of mercury, production of acceptable rheological properties in the feed slurry, and controlling the oxidation state of the glass melt pool. However, the DEPF research had not shown that some vitrification slurry feeds had a tendency to evolve hydrogen (H{sub 2}) and ammonia (NH{sub 3}) as the result of catalytic decomposition of CHOOH with noble metals (rhodium, ruthenium, palladium) in the feed. Testing conducted at Pacific Northwest Laboratory and later at the Savannah River Technical Center showed that the H{sub 2} and NH{sub 3} could evolve at appreciable rates and quantities. The explosive nature of H{sub 2} and NH{sub 3} (as ammonium nitrate) warranted significant mitigation control and redesign of both facilities. At the time the explosive gas evolution was discovered, the DWPF was already under construction and an immediate hardware fix in tandem with flowsheet changes was necessary. However, the Hanford Waste Vitrification Plant (HWVP) was in the design phase and could afford to take time to investigate flowsheet manipulations that could solve the problem, rather than a hardware fix. Thus, the HWVP began to investigate alternatives to using HCOOH in the vitrification process. This document describes the selection, evaluation criteria, and strategy used to evaluate the performance of the alternative chemical additives to CHOOH. The status of the evaluation is also discussed.

  9. The effects of laughter on post-prandial glucose levels and gene expression in type 2 diabetic patients.

    PubMed

    Hayashi, Takashi; Murakami, Kazuo

    2009-07-31

    This report mainly summarizes the results of our study in which the physiological effects of laughter--as a positive emotional expression--were analyzed with respect to gene expression changes to demonstrate the hypothesis that the mind and genes mutually influence each other. We observed that laughter suppressed 2-h postprandial blood glucose level increase in patients with type 2 diabetes and analyzed gene expression changes. Some genes showed specific changes in their expression. In addition, we revealed that laughter decreased the levels of prorenin in blood; prorenin is involved in the onset of diabetic complications. Further, laughter normalized the expression of the prorenin receptor gene on peripheral blood leukocytes, which had been reduced in diabetic patients; this demonstrated that the inhibitory effects of laughter on the onset/deterioration of diabetic complications at the gene-expression level. In a subsequent study, we demonstrated the effects of laughter by discriminating 14 genes, related to natural killer (NK) cell activity, to exhibit continuous increases in expression as a result of laughter. Our results supported NK cell-mediated improvement in glucose tolerance at the gene-expression level. In this report, we also review other previous studies on laughter.

  10. Impacts of calcium addition and different oil types and levels on in vitro rumen fermentation and digestibility.

    PubMed

    Gülşen, Nurettin; Umucalilar, Huzur Derya; Inal, Fatma; Hayirli, Armagan

    2006-12-01

    This in vitro study was designed to investigate the effects of calcium addition to substrates differing in source and level of oil on fermentation, gas production, and digestibility parameters. Substrates were made from basal mixtures containing three levels of calcium salt (0, 1, and 2% CaCl2) to contain three levels (3, 6, and 9%) of two types (sunflower and soy) of oil. After collecting from two Holstein bulls and mixing with buffer, rumen fluid was used to incubate the resulting 18 mixtures in duplicate. Ionizable calcium, pH and NH3-N concentration were measured during incubation. Gas production was measured at 6, 12, 24, and 48 h after incubation. Kinetics parameters of gas production and in vitro dry matter digestibility (IVDMD) were calculated from regression coefficients of an exponential equation and a linear equation, respectively. Data were analysed using 3-way ANOVA with repeated measure option in which the parameter time was a subplot. Oil type did not affect pH and ionizable calcium concentration. There were linear increases and decreases in pH and ionizable calcium concentration in response to increasing oil and calcium levels, respectively. However, with increasing oil levels there were no interactions between calcium addition and oil level on pH and ionizable calcium concentration. None of the treatments affected NH3-N concentration. The amount of gas produced from substrates containing sunflower oil was greater than soy oil (41.7 vs. 40.5 ml). Cumulative gas production and amount of gas production from insoluble but slowly fermentable portion of the supplemental mixtures linearly decreased and linearly increased as oil and calcium levels increased in the substrates, respectively. However, interactions of calcium addition and oil level on gas production and kinetics of gas production were lacking. Oil type did not affect IVDMD. Despite lacking main effects, interaction of calcium addition and oil level indicated that increasing calcium level

  11. Enhanced critical currents in (Gd,Y)Ba2Cu3Ox superconducting tapes with high levels of Zr addition

    SciTech Connect

    Selvamanickam, V; Chen, Y; Shi, T; Liu, Y; Khatri, ND; Liu, J; Yao, Y; Xiong, X; Lei, C; Soloveichik, S; Galstyan, E; Majkic, G

    2013-01-21

    The critical current and structural properties of (Gd,Y)BaCuO tapes made by metal organic chemical vapor deposition (MOCVD) with Zr addition levels up to 30 at.% have been investigated. The reduction in critical current beyond the previously optimized Zr addition level of 7.5 at.% was found to be due to structural deterioration of the (Gd,Y)Ba2Cu3Ox film. By a modified MOCVD process,enhanced critical current densities have been achieved with high levels of Zr addition,including 3.83 MA cm(-2) in 15 at.% Zr- added 1.1 mu m thick film at 77 K in zero magnetic field. Critical currents as high as 1072 A/ 12 mm have been reached in (Gd,Y) BaCuO tapes with 15 at.% Zr addition at 30 K in a field of 3 T applied perpendicular to the tape,corresponding to a pinning force value of 268 GN m(-3). The enhanced critical currents achievable with a high density of nanoscale defects by employing high levels of second- phase additions enable the performance targets needed for the use of HTS tapes in coil applications involving high magnetic fields at temperatures below 50 K to be met.

  12. Enhanced critical currents in (Gd,Y)Ba2Cu3Ox superconducting tapes with high levels of Zr addition

    NASA Astrophysics Data System (ADS)

    Selvamanickam, V.; Chen, Y.; Shi, T.; Liu, Y.; Khatri, N. D.; Liu, J.; Yao, Y.; Xiong, X.; Lei, C.; Soloveichik, S.; Galstyan, E.; Majkic, G.

    2013-03-01

    The critical current and structural properties of (Gd,Y)BaCuO tapes made by metal organic chemical vapor deposition (MOCVD) with Zr addition levels up to 30 at.% have been investigated. The reduction in critical current beyond the previously optimized Zr addition level of 7.5 at.% was found to be due to structural deterioration of the (Gd,Y)Ba2Cu3Ox film. By a modified MOCVD process, enhanced critical current densities have been achieved with high levels of Zr addition, including 3.83 MA cm-2 in 15 at.% Zr-added 1.1 μm thick film at 77 K in zero magnetic field. Critical currents as high as 1072 A/12 mm have been reached in (Gd,Y)BaCuO tapes with 15 at.% Zr addition at 30 K in a field of 3 T applied perpendicular to the tape, corresponding to a pinning force value of 268 GN m-3. The enhanced critical currents achievable with a high density of nanoscale defects by employing high levels of second-phase additions enable the performance targets needed for the use of HTS tapes in coil applications involving high magnetic fields at temperatures below 50 K to be met.

  13. Formation of C-C Bonds via Ruthenium Catalyzed Transfer Hydrogenation: Carbonyl Addition from the Alcohol or Aldehyde Oxidation Level.

    PubMed

    Shibahara, Fumitoshi; Krische, Michael J

    2008-01-01

    Under the conditions of ruthenium catalyzed transfer hydrogenation employing isopropanol as terminal reductant, π-unsaturated compounds (1,3-dienes, allenes, 1,3-enynes and alkynes) reductively couple to aldehydes to furnish products of carbonyl addition. In the absence of isopropanol, π-unsaturated compounds couple directly from the alcohol oxidation level to form identical products of carbonyl addition. Such "alcohol-unsaturate C-C couplings" enable carbonyl allylation, propargylation and vinylation from the alcohol oxidation level in the absence of stoichiometric organometallic reagents or metallic reductants. Thus, direct catalytic C-H functionalization of alcohols at the carbinol carbon is achieved.

  14. Addition of a Gastrointestinal Microbiome Modulator to Metformin Improves Metformin Tolerance and Fasting Glucose Levels

    PubMed Central

    Burton, Jeffrey H.; Johnson, Matthew; Johnson, Jolene; Hsia, Daniel S.; Greenway, Frank L.; Heiman, Mark L.

    2015-01-01

    Background: Adverse effects of metformin are primarily related to gastrointestinal (GI) intolerance that could limit titration to an efficacious dose or cause discontinuation of the medication. Because some metformin side effects may be attributable to shifts in the GI microbiome, we tested whether a GI microbiome modulator (GIMM) used in combination with metformin would ameliorate the GI symptoms. Methods: A 2-period crossover study design was used with 2 treatment sequences, either placebo in period 1 followed by GIMM in period 2 or vice versa. Study periods lasted for 2 weeks, with a 2-week washout period between. During the first week, type 2 diabetes patients (T2D) who experienced metformin GI intolerance took 500 mg metformin along with their assigned NM504 (GIMM) or placebo treatment with breakfast and with dinner. In the second week, the 10 subjects took 500 mg metformin (t.i.d.), with GIMM or placebo consumed with the first and third daily metformin doses. Subjects were permitted to discontinue metformin dosing if it became intolerable. Results: The combination of metformin and GIMM treatment produced a significantly better tolerance score to metformin than the placebo combination (6.78 ± 0.65 [mean ± SEM] versus 4.45 ± 0.69, P = .0006). Mean fasting glucose levels were significantly (P < .02) lower with the metformin–GIMM combination (121.3 ± 7.8 mg/dl) than with metformin-placebo (151.9 ± 7.8 mg/dl). Conclusion: Combining a GI microbiome modulator with metformin might allow the greater use of metformin in T2D patients and improve treatment of the disease. PMID:25802471

  15. Quantifying the Effect of DNA Packaging on Gene Expression Level

    NASA Astrophysics Data System (ADS)

    Kim, Harold

    2010-10-01

    Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.

  16. High-level expression of two thermophilic β-mannanases in Yarrowialipolytica.

    PubMed

    YaPing, Wang; Ben, Rao; Ling, Zhang; Lixin, Ma

    2017-02-22

    Two thermophilic β-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.

  17. Impact of mass addition on extreme water level statistics during storms along the coast of the Mediterranean Sea

    NASA Astrophysics Data System (ADS)

    Lionello, Piero; Conte, Dario; Marzo, Luigi; Scarascia, Luca

    2015-04-01

    In the Mediterranean Sea there are two contrasting factors affecting the maximum level that water will reach during a storm in the next decades: the increase of mean sea level and the decrease of storminess. Future reduction of storminess, which is associated with a decreased intensity of the Mediterranean branch on the north hemisphere storm track, will determine a reduction of maxima of wind wave height and storm surge levels. Changes of mean sea level are produced by regional steric effects and by net mass addition. While it is possible to compute the steric effects with regional models, mass addition is ultimately the consequence of a remote cause: the melting of Greenland and Antarctica ice caps. This study considers four indicators of extreme water levels, which, ranked in order of increasing values: the average of the 10 largest annual maxima (wlind10), the largest annual maximum (wlind1), the 5 (rv5) and 50 (rv50) year return level. The analysis is based on a coordinated set of wave and storm surge simulation forced by inputs provided by regional climate model simulations that were carried out in the CIRCE EU-fp7 and cover the period 1951-2050. Accounting for all affecting factors but the mass addition, in about 60% of the Mediterranean coast reduced storminess and steric expansion will compensate each other and produce no significant change of maximum water level statistics. The remaining 40% of the coastline is almost equally divided between significant positive and negative changes. However, if a supplementary sea level increase, representing the effect of water mass addition, is added, the fraction of the coast with significant positive/negative changes increase/decrease quickly. If mass addition would contribute 10cm, there will be no significant negative changes and for any indicator. With a 20cm addition the increase would be significant for wlind10, wlind1, rv5 along more than 75% of the Mediterranean coastline. With a 35cm addition the increase

  18. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  19. Tumor endothelial cells express high pentraxin 3 levels.

    PubMed

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

    2016-12-01

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

  20. Survey of residue levels of organic solvents in "existing food additives" and health food materials by head-space GC.

    PubMed

    Uematsu, Yoko; Ogimoto, Mami; Suzuki, Kumi; Kabashima, Junichirou; Ito, Koichi; Nakazato, Mitsuo

    2008-01-01

    Organic solvent residue levels in "Existing Food Additives" (n=145), health food materials (n=23), and commercial health food products (n=19) were surveyed. Ethanol was the dominant solvent found in the samples, suggesting its use in the manufacturing process. Methanol, acetone, 2-propanol and ethyl acetate was also found. No residual solvent exceeded the limits set by the Food Sanitation Law.

  1. Learning Achievement and the Efficiency of Learning the Concept of Vector Addition at Three Different Grade Levels

    ERIC Educational Resources Information Center

    Gubrud, Allan R.; Novak, Joseph D.

    1973-01-01

    Empirical data relate to Bruner's and Ausubel's theories of learning concepts at different age levels. The concept of vector addition was taught to eighth, ninth, and tenth grade students. The concept was learned and retained by high ability ninth and all tenth grade students. (PS)

  2. Choosing channel quantization levels and viterbi decoding for space diversity reception over the additive white Guassian noise channel

    NASA Technical Reports Server (NTRS)

    Kalson, S.

    1986-01-01

    Previous work in the area of choosing channel quantization levels for a additive white Gaussian noise channel composed of one receiver-demodulator is reviewed, and how this applies to the Deep Space Network composed of several receiver-demodulators (space diversity reception) is shown. Viterbi decoding for the resulting quantized channel is discussed.

  3. Interactive effects of nitrogen addition, warming and invasion across organizational levels in an old-field plant community.

    PubMed

    Gornish, Elise S

    2014-10-08

    Response to global change is dependent on the level of biological organization (e.g. the ecologically relevant spatial scale) in which species are embedded. For example, individual responses can affect population-level responses, which, in turn, can affect community-level responses. Although relationships are known to exist among responses to global change across levels of biological organization, formal investigations of these relationships are still uncommon. I conducted an exploratory analysis to identify how nitrogen addition and warming by open top chambers might affect plants across spatial scales by estimating treatment effect size at the leaf level, the plant level and the community level. Moreover, I investigated if the presence of Pityopsis aspera, an experimentally introduced plant species, modified the relationship between spatial scale and effect size across treatments. I found that, overall, the spatial scale significantly contributes to differences in effect size, supporting previous work which suggests that mechanisms driving biotic response to global change are scale dependent. Interestingly, the relationship between spatial scale and effect size in both the absence and presence of experimental invasion is very similar for nitrogen addition and warming treatments. The presence of invasion, however, did not affect the relationship between spatial scale and effect size, suggesting that in this system, invasion may not exacerbate or attenuate climate change effects. This exercise highlights the value of moving beyond integration and scaling to the practice of directly testing for scale effects within single experiments.

  4. Ecotoxicological effects of a veterinary food additive, copper sulphate, on antioxidant enzymes and mRNA expression in earthworms.

    PubMed

    Xiong, Wenguang; Ding, Xueyao; Zhang, Yiming; Sun, Yongxue

    2014-01-01

    The present study was aimed to investigate the effect of the veterinary food additive copper sulphate (CuSO₄) on the eco-toxicological responses of earthworms Eisenia fetida (E. fetida). The following biomarkers were measured: catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activities. Gene expression analyses such as metallothionein (MT) and heat shock protein 70 (Hsp70) were also examined. A time-dependent increase of CAT activity was found at 400 mg/kg and SOD activity at 200 and 400 mg/kg. The highest expression of Hsp70 (4.4-fold) was observed at day 15 at 400 mg/kg. Our results indicated that the measured antioxidant enzymes (except GST) had the ability to provide antioxidant defenses against the stressor; and compared to expression of MT, expression of Hsp70 could be more reliable molecular tools with predictive possibilities to monitor the eco-toxicity of stressors such as feed additive CuSO₄.

  5. Repeated forced swim stress has additive effects in anxiety behavior and in cathecolamine levels of adult rats exposed to deltamethrin.

    PubMed

    Habr, Soraya F; Macrini, Daclé J; Florio, Jorge C; Bernardi, Maria M

    2014-01-01

    Deltamethrin (DTM) is a type II pyrethroid insecticide that elicits autonomic and neuroendocrine responses that indicate high levels of stress, presumably caused by the neurotoxic effect of the insecticide. This study investigated the effect of DTM exposure (10 mg/kg, p.o.) and an additional stress induced in the forced swim test (FST) in behavioral tasks related to anxiety, serum corticosterone levels, and striatal neurotransmitter levels. Open field behavior and social interaction were evaluated after DTM administration (10 mg kg(-1), p.o). DTM per se reduced rearing frequency in the open field, but no alterations in locomotion frequency or immobility duration were detected. Stress increased immobility duration compared with non-stressed animals. DTM reduced social interaction and increased corticosterone levels, and these effects were enhanced in stressed animals. Mainly stress affected dopaminergic and serotoninergic activity. In anxiety behavior and in both neurotransmitters and metabolites levels it was observed an additive effect of stress in DTM treated rat data. These results indicate that DTM enhanced the anxiogenic responses and stress had an additive effect over the DTM stress. The neurochemical data did not indicate an interaction between stress and DTM exposure. The present results maybe important for implementing pyrethroid insecticide safety standards.

  6. Use of heat stress responsive gene expression levels for early selection of heat tolerant cabbage (Brassica oleracea L.).

    PubMed

    Park, Hyun Ji; Jung, Won Yong; Lee, Sang Sook; Song, Jun Ho; Kwon, Suk-Yoon; Kim, Hyeran; Kim, Chulwook; Ahn, Jun Cheul; Cho, Hye Sun

    2013-06-04

    Cabbage is a relatively robust vegetable at low temperatures. However, at high temperatures, cabbage has disadvantages, such as reduced disease tolerance and lower yields. Thus, selection of heat-tolerant cabbage is an important goal in cabbage breeding. Easier or faster selection of superior varieties of cabbage, which are tolerant to heat and disease and have improved taste and quality, can be achieved with molecular and biological methods. We compared heat-responsive gene expression between a heat-tolerant cabbage line (HTCL), "HO", and a heat-sensitive cabbage line (HSCL), "JK", by Genechip assay. Expression levels of specific heat stress-related genes were increased in response to high-temperature stress, according to Genechip assays. We performed quantitative RT-PCR (qRT-PCR) to compare expression levels of these heat stress-related genes in four HTCLs and four HSCLs. Transcript levels for heat shock protein BoHsp70 and transcription factor BoGRAS (SCL13) were more strongly expressed only in all HTCLs compared to all HSCLs, showing much lower level expressions at the young plant stage under heat stress (HS). Thus, we suggest that expression levels of these genes may be early selection markers for HTCLs in cabbage breeding. In addition, several genes that are involved in the secondary metabolite pathway were differentially regulated in HTCL and HSCL exposed to heat stress.

  7. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass.

    PubMed

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten; Pilegaard, Henriette; Bangsbo, Jens

    2005-07-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.

  8. Very low levels of direct additive genetic variance in fitness and fitness components in a red squirrel population

    PubMed Central

    McFarlane, S Eryn; Gorrell, Jamieson C; Coltman, David W; Humphries, Murray M; Boutin, Stan; McAdam, Andrew G

    2014-01-01

    A trait must genetically correlate with fitness in order to evolve in response to natural selection, but theory suggests that strong directional selection should erode additive genetic variance in fitness and limit future evolutionary potential. Balancing selection has been proposed as a mechanism that could maintain genetic variance if fitness components trade off with one another and has been invoked to account for empirical observations of higher levels of additive genetic variance in fitness components than would be expected from mutation–selection balance. Here, we used a long-term study of an individually marked population of North American red squirrels (Tamiasciurus hudsonicus) to look for evidence of (1) additive genetic variance in lifetime reproductive success and (2) fitness trade-offs between fitness components, such as male and female fitness or fitness in high- and low-resource environments. “Animal model” analyses of a multigenerational pedigree revealed modest maternal effects on fitness, but very low levels of additive genetic variance in lifetime reproductive success overall as well as fitness measures within each sex and environment. It therefore appears that there are very low levels of direct genetic variance in fitness and fitness components in red squirrels to facilitate contemporary adaptation in this population. PMID:24963372

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

    PubMed

    Lin, Yi-Lung; Lin, Shih-Chang

    2017-02-01

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

  10. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

    PubMed Central

    Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

    2015-01-01

    Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

  11. Correlation between liver cancer pain and the HIF-1 and VEGF expression levels

    PubMed Central

    Zhang, Geng; Feng, Gui-Yin; Guo, Yan-Ru; Liang, Dong-Qi; Yuan, Yuan; Wang, Hai-Lun

    2017-01-01

    A possible correlation between liver cancer pain and the hypoxia-inducible factor (HIF)-1 and vascular endothelial growth factor (VEGF) expression levels was examined. From January, 2015 to January, 2016, 30 patients suffering from liver cancer with pain, 30 patients with liver cancer without pain and 30 hepatitis patients with pain were enrolled in the study. Pain level was evaluated by visual analogue scale (VAS), the expression levels of HIF-1 and VEGF mRNA were determined by RT-PCR and the expression levels of HIF-1 and VEGF proteins were examined by ELISA. Before intervention, the VAS in the hepatitis group was significantly higher than that of the liver cancer pain group. However, after intervention the VAS in the two groups was reduced. HIF-1 and VEGF mRNA expression levels in the liver cancer pain group were significantly higher than those in the liver cancer group before and after intervention. The expression levels of HIF-1 and VEGF mRNA in the hepatitis group were the lowest. The expression levels of HIF-1 and VEGF mRNA in the liver cancer pain group considerably increased after intervention. The expression levels of HIF-1 and VEGF mRNA in the other two groups showed no changes before or after intervention. Before and after the intervention, VAS in the liver cancer pain group was positively correlated to the expression levels of HIF-1 and VEGF. Thus, pain occurrence and the pain level in liver cancer patients were correlated with the expression levels of HIF-1 and VEGF. As the regular three-step medicine analgesic ladder is ineffective in these cases, verification of HIF-1 and VEGF expression levels may be considered the new target for pain release. PMID:28123525

  12. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

    PubMed Central

    Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

  13. Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

    PubMed

    Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

    2017-01-01

    Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

  14. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  15. Gene expression levels in small specimens from patients detected using oligonucleotide arrays.

    PubMed

    Hoffmann, Katrin; Firth, Martin J; Freitas, Joseph R; de Klerk, Nicholas H; Kees, Ursula R

    2005-01-01

    Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.

  16. Dietary soya protein intake and exercise training have an additive effect on skeletal muscle fatty acid oxidation enzyme activities and mRNA levels in rats.

    PubMed

    Morifuji, Masashi; Sanbongi, Chiaki; Sugiura, Katsumi

    2006-09-01

    Exercise training and regular physical activity increase oxidation of fat. Enhanced oxidation of fat is important for preventing lifestyle diseases such as hypertension and obesity. The aim of the present study in rats was to determine whether intake of dietary soya protein and exercise training have an additive effect on the activity and mRNA expression of enzymes involved in skeletal muscle fatty acid oxidation. Male Sprague-Dawley rats (n 32) were assigned randomly into four groups (eight rats per group) and then divided further into sedentary or exercise-trained groups fed either casein or soya protein diets. Rats in the exercise groups were trained for 2 weeks by swimming for 120 min/d, 6 d/week. Exercise training decreased hepatic triacylglycerol levels and retroperitoneal adipose tissue weight and increased skeletal muscle carnitine palmitoyltransferase 1 (CPT1) activity and mRNA expression of CPT1, beta-hydroxyacyl-CoA dehydrogenase (HAD), acyl-CoA oxidase, PPARgamma coactivator 1alpha (PGC1alpha) and PPARalpha. Soya protein significantly decreased hepatic triacylglycerol levels and epididymal adipose tissue weight and increased skeletal muscle CPT1 activity and CPT1, HAD, acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase, PGC1alpha and PPARalpha mRNA levels. Furthermore, skeletal muscle HAD activity was the highest in exercise-trained rats fed soya protein. We conclude that exercise training and soya protein intake have an important additive role on induction of PPAR pathways, leading to increased activity and mRNA expression of enzymes involved in fatty acid oxidation in skeletal muscle and reduced accumulation of body fat.

  17. Relationship between Legible Handwriting and Level of Success of Third Grade Students in Written Expression

    ERIC Educational Resources Information Center

    Bayat, Seher; Küçükayar, Hasan

    2016-01-01

    This study aims to identify third-grade students' performance levels for written expression and handwriting and to find the relationship between these performances. The study is based on relational screening model. It is carried out with 110 third grade students. Students' levels of success in handwriting and in written expression are evaluated…

  18. Effects of maternally exposed coloring food additives on receptor expressions related to learning and memory in rats.

    PubMed

    Ceyhan, Betul Mermi; Gultekin, Fatih; Doguc, Duygu Kumbul; Kulac, Esin

    2013-06-01

    Exposure to artificial food colors and additives (AFCAs) has been implicated in the induction and severity of some childhood behavioral and learning disabilities. N-methyl-D-aspartate receptors (NMDARs) and nicotinic acetylcholine receptors (nACHRs) are thought to be effective in the learning and memory-generating process. In this study, we investigated the effects of intrauterine exposure to AFCAs on subunit concentrations of NMDARs and nAChRs isoforms in rats. We administered a mixture of AFCAs (Eritrosin, Ponceau 4R, Allura Red AC, Sunset Yellow FCF, Tartrazin, Amaranth, Brilliant Blue, Azorubin and Indigotin) to female rats before and during gestation. The concentration of NR2A and NR2B subunits and nAChR α7, α4β2 isoforms in their offspring's hippocampi were measured by Western Blotting. Expressions of NR2B and nAChR β2 were significantly increased (17% and 6.70%, respectively), whereas expression of nAChR α4 was significantly decreased (5.67%) in male experimental group compared to the male control group (p<0.05). In the female experimental group, AFCAs caused a 14% decrease in NR2B expression when compared to the female control group (p<0.05). Our results indicate that exposure to AFCAs during the fetal period may lead to alterations in expressions of NMDARs and nAChRs in adulthood. These alterations were different between male and female genders.

  19. Effect of Various Food Additives on the Levels of 4(5)-Methylimidazole in a Soy Sauce Model System.

    PubMed

    Lee, Sumin; Lee, Jung-Bin; Hwang, Junho; Lee, Kwang-Geun

    2016-01-01

    In this study, the effect of food additives such as iron sulfate, magnesium sulfate, zinc sulfate, citric acid, gallic acid, and ascorbic acid on the reduction of 4(5)-methylimidazole (4(5)-MI) was investigated using a soy sauce model system. The concentration of 4(5)-MI in the soy sauce model system with 5% (v/v) caramel colorant III was 1404.13 μg/L. The reduction rate of 4(5)-MI level with the addition of 0.1M additives followed in order: iron sulfate (81%) > zinc sulfate (61%) > citric acid (40%) > gallic acid (38%) > ascorbic acid (24%) > magnesium sulfate (13%). Correlations between 4(5)-MI levels and the physicochemical properties of soy sauce, including the amount of caramel colorant, pH value, and color differences, were determined. The highest correlations were found between 4(5)-MI levels and the amount of caramel colorant and pH values (r(2) = 0.9712, r(2) = 0.9378). The concentration of caramel colorants in 8 commercial soy sauces were estimated, and ranged from 0.01 to 1.34% (v/v).

  20. TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

    PubMed

    Wang, Feng; Hou, Hongyan; Wu, Shiji; Tang, Qing; Liu, Weiyong; Huang, Min; Yin, Botao; Huang, Jing; Mao, Lie; Lu, Yanfang; Sun, Ziyong

    2015-10-01

    Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T-cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK-cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN-γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK-cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.

  1. Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

    PubMed Central

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn; Bendixen, Christian; Wang, Xuefei; Conley, Lene Nagstrup; Berget, Ingunn; Tajet, Håvard; Grindflek, Eli

    2007-01-01

    Background: Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes. Results: Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between

  2. STAT4 Associates with SLE Through Two Independent Effects that Correlate with Gene Expression and Act Additively with IRF5 to Increase Risk

    PubMed Central

    Abelson, Anna-Karin; Delgado-Vega, Angélica M.; Kozyrev, Sergey V.; Sánchez, Elena; Velázquez-Cruz, Rafael; Eriksson, Niclas; Wojcik, Jerome; Reddy, Prasad Linga; Lima, Guadalupe; D’Alfonso, Sandra; Migliaresi, Sergio; Baca, Vicente; Orozco, Lorena; Witte, Torsten; Ortego-Centeno, Norberto; Abderrahim, Hadi; Pons-Estel, Bernardo A.; Gutiérrez, Carmen; Suárez, Ana; González-Escribano, Maria Francisca; Martin, Javier; Alarcón-Riquelme, Marta E.

    2013-01-01

    Objectives To confirm and define the genetic association of STAT4 and systemic lupus erythematosus, investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5. Methods 30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in 5 new sets of cases and controls for replication. STAT4 cDNA was analyzed by 5’-RACE PCR and sequencing. Expression levels were measured by quantitative PCR. Results In the fine-mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. We also detected transcription of alternative tissue-specific exons 1, indicating presence of tissue-specific promoters of potential importance in the expression of STAT4. No interaction with associated SNPs of IRF5 was observed using regression analysis. Conclusions These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. Our results also indicate that both genes STAT4 and IRF5 act additively to increase risk for SLE. PMID:19019891

  3. Expression of neurexin and neuroligin in the enteric nervous system and their down-regulated expression levels in Hirschsprung disease.

    PubMed

    Zhang, Qiangye; Wang, Jian; Li, Aiwu; Liu, Hongzhen; Zhang, Wentong; Cui, Xinhai; Wang, Kelai

    2013-04-01

    To investigate the expression levels of neurexins and neuroligins in the enteric nervous system (ENS) in Hirschsprung Disease (HSCR). Longitudinal muscles with adherent mesenteric plexus were obtained by dissection of the fresh gut wall of mice, guinea pigs, and humans. Double labeling of neurexin I and Hu (a neuron marker), neuroligin 1 and Hu, neurexin I and synaptophysin (a presynaptic marker), and neuroligin 1 and PSD95 (a postsynaptic marker) was performed by immunofluorescence staining. Images were merged to determine the relative localizations of the proteins. Expression levels of neurexin and neuroligin in different segments of the ENS in HSCR were investigated by immunohistochemistry. Neurexin and neuroligin were detected in the mesenteric plexus of mice, guinea pigs, and humans with HSCR. Neurexin was located in the presynapse, whereas neuroligin was located in the postsynapse. Expression levels of neurexin and neuroligin were significant in the ganglionic colonic segment of HSCR, moderate in the transitional segment, and negative in the aganglionic colonic segment. The expressions of neurexin and neuroligin in the transitional segments were significantly down-regulated compared with the levels in the normal segments (P < 0.05). Expression levels of neurexin and neuroligin in ENS are significantly down-regulated in HSCR, which may be involved in the pathogenesis of HSCR.

  4. Porcine lactoferrin expression in transgenic rice and its effects as a feed additive on early weaned piglets.

    PubMed

    Lee, Tzu-Tai; Chang, Chi-Chung; Juang, Rung-Shian; Chen, Ray-Bin; Yang, Hsiu-Ya; Chu, Li-Wei; Wang, Shih-Rong; Tseng, Tung-Hai; Wang, Chang-Sheng; Chen, Liang-Jwu; Yu, Bi

    2010-04-28

    The purpose of this study is to determine the growth performance and immune characteristics of early weaned piglets receiving rice bran expressing porcine lactoferrin as a feed additive. Full-length cDNA encoding porcine lactoferrin (LF) driven by a rice actin promoter was transformed into rice plants, and its integration into the rice genome was verified by Southern blot analysis. The expression of recombinant LF (rLF) in whole grains and rice bran was also confirmed, and the amount of rLF accumulated in rice bran was estimated by immunoblot assay to be approximately 0.1% of rice bran weight. An iron-binding assay showed that the rLF retained iron-binding activity and the binding capacity of 1 mg/mL rLF would be saturated by 100 microM of FeCl(3). Thirty-six early weaned piglets at 21 days old were randomly selected into two groups and fed a diet containing 5% transgenic rice bran containing 50 mg/kg rLF (rLF group) and 5% rice bran (control group) to investigate the piglets' growth performance and immune characteristics. The results showed no significant difference in growth performance between the groups during the feeding period. However, the aerobic bacteria, anaerobic bacteria, and coliform counts in the cecal contents of the rLF-fed group were significantly lower than those of the control group. Additional immune characteristics such as the IgG concentration in the rLF group was higher than the control group at the 28th day, but leukocyte counts and the peripheral lymphocyte ratio remained similar. In summary, porcine LF expressed in rice bran, a byproduct of rice, can be used as a functional additive to improve antimicrobial capabilities and IgG concentration of early weaned piglets.

  5. The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

    PubMed Central

    Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

    2015-01-01

    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression

  6. The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

    PubMed

    Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

    2015-12-01

    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression

  7. IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing.

    PubMed

    Deonovic, Benjamin; Wang, Yunhao; Weirather, Jason; Wang, Xiu-Jie; Au, Kin Fai

    2016-11-28

    Allele-specific expression (ASE) is a fundamental problem in studying gene regulation and diploid transcriptome profiles, with two key challenges: (i) haplotyping and (ii) estimation of ASE at the gene isoform level. Existing ASE analysis methods are limited by a dependence on haplotyping from laborious experiments or extra genome/family trio data. In addition, there is a lack of methods for gene isoform level ASE analysis. We developed a tool, IDP-ASE, for full ASE analysis. By innovative integration of Third Generation Sequencing (TGS) long reads with Second Generation Sequencing (SGS) short reads, the accuracy of haplotyping and ASE quantification at the gene and gene isoform level was greatly improved as demonstrated by the gold standard data GM12878 data and semi-simulation data. In addition to methodology development, applications of IDP-ASE to human embryonic stem cells and breast cancer cells indicate that the imbalance of ASE and non-uniformity of gene isoform ASE is widespread, including tumorigenesis relevant genes and pluripotency markers. These results show that gene isoform expression and allele-specific expression cooperate to provide high diversity and complexity of gene regulation and expression, highlighting the importance of studying ASE at the gene isoform level. Our study provides a robust bioinformatics solution to understand ASE using RNA sequencing data only.

  8. Association of MAPT haplotypes with Alzheimer’s disease risk and MAPT brain gene expression levels

    PubMed Central

    2014-01-01

    Introduction MAPT encodes for tau, the predominant component of neurofibrillary tangles that are neuropathological hallmarks of Alzheimer’s disease (AD). Genetic association of MAPT variants with late-onset AD (LOAD) risk has been inconsistent, although insufficient power and incomplete assessment of MAPT haplotypes may account for this. Methods We examined the association of MAPT haplotypes with LOAD risk in more than 20,000 subjects (n-cases = 9,814, n-controls = 11,550) from Mayo Clinic (n-cases = 2,052, n-controls = 3,406) and the Alzheimer’s Disease Genetics Consortium (ADGC, n-cases = 7,762, n-controls = 8,144). We also assessed associations with brain MAPT gene expression levels measured in the cerebellum (n = 197) and temporal cortex (n = 202) of LOAD subjects. Six single nucleotide polymorphisms (SNPs) which tag MAPT haplotypes with frequencies greater than 1% were evaluated. Results H2-haplotype tagging rs8070723-G allele associated with reduced risk of LOAD (odds ratio, OR = 0.90, 95% confidence interval, CI = 0.85-0.95, p = 5.2E-05) with consistent results in the Mayo (OR = 0.81, p = 7.0E-04) and ADGC (OR = 0.89, p = 1.26E-04) cohorts. rs3785883-A allele was also nominally significantly associated with LOAD risk (OR = 1.06, 95% CI = 1.01-1.13, p = 0.034). Haplotype analysis revealed significant global association with LOAD risk in the combined cohort (p = 0.033), with significant association of the H2 haplotype with reduced risk of LOAD as expected (p = 1.53E-04) and suggestive association with additional haplotypes. MAPT SNPs and haplotypes also associated with brain MAPT levels in the cerebellum and temporal cortex of AD subjects with the strongest associations observed for the H2 haplotype and reduced brain MAPT levels (β = -0.16 to -0.20, p = 1.0E-03 to 3.0E-03). Conclusions These results confirm the previously reported MAPT H2 associations with LOAD risk in two large series, that this haplotype has the strongest

  9. Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

    PubMed Central

    2012-01-01

    Background The predominant model for regulation of gene expression through DNA methylation is an inverse association in which increased methylation results in decreased gene expression levels. However, recent studies suggest that the relationship between genetic variation, DNA methylation and expression is more complex. Results Systems genetic approaches for examining relationships between gene expression and methylation array data were used to find both negative and positive associations between these levels. A weighted correlation network analysis revealed that i) both transcriptome and methylome are organized in modules, ii) co-expression modules are generally not preserved in the methylation data and vice-versa, and iii) highly significant correlations exist between co-expression and co-methylation modules, suggesting the existence of factors that affect expression and methylation of different modules (i.e., trans effects at the level of modules). We observed that methylation probes associated with expression in cis were more likely to be located outside CpG islands, whereas specificity for CpG island shores was present when methylation, associated with expression, was under local genetic control. A structural equation model based analysis found strong support in particular for a traditional causal model in which gene expression is regulated by genetic variation via DNA methylation instead of gene expression affecting DNA methylation levels. Conclusions Our results provide new insights into the complex mechanisms between genetic markers, epigenetic mechanisms and gene expression. We find strong support for the classical model of genetic variants regulating methylation, which in turn regulates gene expression. Moreover we show that, although the methylation and expression modules differ, they are highly correlated. PMID:23157493

  10. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution

    PubMed Central

    Erickson, Keesha E.; Otoupal, Peter B.

    2017-01-01

    ABSTRACT Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment

  11. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution.

    PubMed

    Erickson, Keesha E; Otoupal, Peter B; Chatterjee, Anushree

    2017-01-01

    Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress

  12. Modification of the levels of polyphenols in wort and beer by addition of hexamethylenetetramine or sulfite during mashing.

    PubMed

    Andersen, M L; Skibsted, L H

    2001-11-01

    The effects of addition of hexamethylenetetramine (HMT) or sulfite during mashing on the polyphenol content and oxidative stability of wort and beer have been evaluated in a series of laboratory mashings and pilot brews. HMT reduced the concentration of catechin, prodelphinidin B-3, and procyanidin B-3 in wort and beer, whereas the concentration of ferulic acid was unaffected. Sulfite had only a minor effect on the concentration of phenolics in wort and beer. Addition of HMT or sulfite during mashing increased the oxidative stability of the beer slightly as judged by the tendency of formation of radicals (ESR spin trapping technique), although sensory analysis gave identical flavor acceptance scores to beers produced from untreated and HMT-treated wort and lower scores to beer from sulfite-treated wort. No difference in the oxidative stability of the differently treated sweet worts could be detected as judged by the rate of formation of radicals. HMT addition during mashing has thus been demonstrated to be a valuable experimental tool to control the level of polyphenols in wort and for producing brews with various levels of polyphenols from a single malt.

  13. Hypoxia disrupts the expression levels of circadian rhythm genes in hepatocellular carcinoma.

    PubMed

    Yu, Chao; Yang, Sheng-Li; Fang, Xiefan; Jiang, Jian-Xin; Sun, Cheng-Yi; Huang, Tao

    2015-05-01

    Disturbance in the expression of circadian rhythm genes is a common feature in certain types of cancer, however the mechanisms mediating this disturbance remain to be elucidated. The present study aimed to investigate the effect of hypoxia on the expression of circadian rhythm genes in liver cancer cells and to identify the mechanisms underlying this effect in hepatocellular carcinoma (HCC). The HCC cell line, PLC/PRF/5. was treated with either a vehicle control or CoCl2 at 50, 100 or 200 µΜ for 24 h. Following treatment, the protein expression levels of hypoxia‑inducible factor (HIF)‑1α and HIF‑2α were detected by western blotting and the mRNA expression levels of circadian rhythm genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Arnt‑like 1 (Bmal1), period (Per)1, Per2, Per3, cryptochrome (Cry)1, Cry2 and casein kinase Iε (CKIε), were detected by reverse transcription quantitative polymerase chain reaction (RT‑qPCR). Expression plasmids containing HIF‑1α or HIF‑2α were transfected into the PLC/PRF/5 cells using liposomes and RT‑qPCR was used to determine the effects of the transfections on the expression levels of circadian rhythm genes. Following treatment with CoCl2, the protein expression levels of HIF‑1α and HIF‑2α were upregulated in a CoCl2 concentration‑dependent manner. The mRNA expression levels of Clock, Bmal1 and Cry2 were increased, and the mRNA expression levels of Per1, Per2, Per3, Cry1 and CKIε were decreased following CoCl2 treatment (P<0.05). In the PLC/PRF/5 cells transfected with the plasmid containing HIF‑1α, the mRNA expression levels of Clock, Bmal1 and Cry2 were increased, and the mRNA expression levels of Per1, Per2, Per3, Cry1 and CKIε were decreased. In the PLC/PRF/5 cells transfected with the plasmid containing HIF‑2α, the mRNA expression levels of Clock, Bmal1, Per1, Cry1, Cry2 and CKIε were upregulated, and the mRNA expression levels of Per2 and Per3 were

  14. HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant

    PubMed Central

    Growdon, Whitfield B.; Groeneweg, Jolijn; Byron, Virginia; DiGloria, Celeste; Borger, Darrell R.; Tambouret, Rosemary; Foster, Rosemary; Chenna, Ahmed; Sperinde, Jeff; Winslow, John; Rueda, Bo R.

    2015-01-01

    Background Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa. Materials and Methods With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000-2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression. Results We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8 RF/mm2 was observed in 53% (n = 54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels (p < 0.001). Conclusions: These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa. PMID:25602714

  15. RNA-Binding Protein AUF1 Promotes Myogenesis by Regulating MEF2C Expression Levels

    PubMed Central

    Panda, Amaresh C.; Abdelmohsen, Kotb; Yoon, Je-Hyun; Martindale, Jennifer L.; Yang, Xiaoling; Curtis, Jessica; Mercken, Evi M.; Chenette, Devon M.; Zhang, Yongqing; Schneider, Robert J.; Becker, Kevin G.; de Cabo, Rafael

    2014-01-01

    The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation (RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3′ untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1. PMID:24891619

  16. Criteria for acceptable levels of the Shinkansen Super Express train noise and vibration in residential areas

    NASA Astrophysics Data System (ADS)

    Yamanaka, K.; Nakagawa, T.; Kobayashi, F.; Kanada, S.; Tanahashi, M.; Muramatsu, T.; Yamada, S.

    1982-10-01

    A survey of 1187 housewives living in 18 areas along the Shinkansen Super Express (bullet train) railway was conducted by means of a self-administered health questionnaire (modified Cornell Medical Index). In addition, geographically corresponding measurements of noise level and vibration intensity were taken. The relationship of noise and vibration to positive responses (health complaints) related to bodily symptoms, illness and emotional disturbances was analyzed. The factors which correlated with an increase in the average number of positive responses included noise, vibration, age and health status. Such factors as marital status, educational level, part time work, duration of inhabitancy and occupation of the head of the houshold correlated poorly with the number of positive responses. Unhealthy respondents compared to healthy respondents are more frequently affected by noise and vibration. The rate of positive responses in the visual, respiratory, cardiovascular, digestive and nervous systems, sleep disturbances and emotional disturbances increased accordingly as noise and vibration increased. Combined effects of noise and vibration stimuli on the total number of positive responses (an indicator of general health) were found. This study has produced results indicating that the maximum permissible noise level should not exceed 70 dB(A) in the residential areas along the Shinkansen railway.

  17. Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor.

    PubMed

    Khongphinitbunjong, Kitiphong; de Guzman, Lilia I; Tarver, Matthew R; Rinderer, Thomas E; Chen, Yanping; Chantawannakul, Panuwan

    2015-01-01

    The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 10(3) or 10(5) fold the levels of their uninfested brood. In contrast, POL (10(2) or 10(4) fold) and RHB (10(2) or l0(4) fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism.

  18. Additional value of CSF amyloid-beta 40 levels in the differentiation between FTLD and control subjects.

    PubMed

    Verwey, Nicolaas A; Kester, Maartje I; van der Flier, Wiesje M; Veerhuis, Robert; Berkhof, Hans; Twaalfhoven, Harry; Blankenstein, Marinus A; Scheltens And, Philip; Pijnenburg, Yolande A L

    2010-01-01

    To determine the additional value of cerebrospinal fluid (CSF)amyloid-beta1-40 (Abeta40) next to amyloid-beta1-42 (beta42), total tau (Tau), and tau phosphorylated at threonine-181 (pTau) to distinguish patients with frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and controls, we measured CSF levels of Abeta40, Abeta42, pTau, and Tau in 55 patients with FTLD, 60 with AD, and 40 control subjects. Logistic regression was used to identify biomarkers that best distinguished the groups. Additionally, a decision tree (cost=test method; Matlab 7.7) was used to predict diagnosis selecting the best set of biomarkers with the optimal cut-off. Logistic regression showed that Abeta42 and pTau CSF levels provided optimal distinction between AD and FTLD. A combination of Abeta42, Tau, and Abeta40 optimally discriminated FTLD from controls and AD from controls. The decision tree used Abeta42 (cut-off 578 pg/ml) to identify AD (positive predictive value (PPV) 97%), followed by Tau(cut-off 336 pg/ml) to identify FTLD (PPV 67%), and in the last step,Abeta40 (cut-off 10 ng/ml) was used to differentiate controls (PPV68%). Applying CSF Abeta40 levels in the model, the PPV of diagnosis increased to 75% as opposed to 70% when only Abeta42 and Tau were used. CSF Abeta40 levels added to the conventional CSF biomarkers increases the potential to discriminate subjects with dementia from controls. Our findings favor the implementation of CSF Abeta40 in differential diagnosis between FTLD, AD, and control subjects.

  19. High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B

    PubMed Central

    Trusca, Violeta G; Mihai, Adina D; Fuior, Elena V; Fenyo, Ioana M; Gafencu, Anca V

    2016-01-01

    AIM: To investigate the effect of high homocysteine (Hcy) levels on apolipoprotein E (apoE) expression and the signaling pathways involved in this gene regulation. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to assess apoE expression in cells treated with various concentrations (50-500 μmol/L) of Hcy. Calcium phosphate-transient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2 (ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcy-mediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKβ. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) or nuclear factor of activated T cells (NFAT). Chromatin immunoprecipitation (ChIP) assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 μmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region -100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy (250-750 μmol/L) induced a 2-3 fold decrease in apoE mRNA levels in HEK-293 cells, while apoE gene expression was not significantly affected by treatment with lower concentrations of Hcy (100 μmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter

  20. Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

    PubMed Central

    Hu, Hong; Gao, Jie; He, Jun; Yu, Bing; Zheng, Ping; Huang, Zhiqing; Mao, Xiangbing; Yu, Jie; Han, Guoquan; Chen, Daiwen

    2013-01-01

    The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases. PMID:23472192

  1. Low-level laser therapy promotes dendrite growth via upregulating brain-derived neurotrophic factor expression

    NASA Astrophysics Data System (ADS)

    Meng, Chengbo; He, Zhiyong; Xing, Da

    2014-09-01

    Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via the increase of both BDNF mRNA and protein expression. In addition, dendrite growth was improved after LLLT, characterized by upregulation of PSD95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT can ameliorate Aβ-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against Aβ-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of Alzheimer's disease.

  2. Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells.

    PubMed

    Chen, Nianhang; Reith, Maarten E A

    2007-04-01

    The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.

  3. Criteria for high-level expression of a fungal laccase gene in transgenic maize.

    PubMed

    Hood, Elizabeth E; Bailey, Michele R; Beifuss, Katherine; Magallanes-Lundback, Maria; Horn, Michael E; Callaway, Evelyn; Drees, Carol; Delaney, Donna E; Clough, Richard; Howard, John A

    2003-03-01

    Expression of industrial enzymes in transgenic plants offers an alternative system to fungal fermentation for large-scale production. Very high levels of expression are required to make the enzymes cost-effective. We tested several parameters to determine the best method for achieving high levels of expression for a fungal laccase gene. Transgenic maize plants were generated using an Agrobacterium-mediated system. The molecular parameters that induced the highest expression were the maize embryo-preferred globulin 1 promoter and targeting of the protein to the cell wall. Two independent transgenic events that yielded multiple clonal plants were characterized in detail. Independent transgenic events 01 and 03 contained two or one copies of T-DNA, respectively. Plants derived from a single transgenic event varied in expression level, and the variation in expression levels was heritable. Within the seed, expression in these plants was primarily within the embryo, and was associated with seed browning and limited germination. High oil germplasm was used to increase germination, as well as to assist in increasing expression 20-fold in five generations through breeding and selection.

  4. Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato gene arrays were used to investigate how high levels of transgenic peroxidase expression and feeding by the corn earworm, Helicoverpa zea, affected expression of defensive and other genes. High peroxidase activity significantly upregulated proteinase inhibitors and a few other defensive gene...

  5. HLA-G expression levels influence the tolerogenic activity of human DC-10

    PubMed Central

    Amodio, Giada; Comi, Michela; Tomasoni, Daniela; Gianolini, Monica Emma; Rizzo, Roberta; LeMaoult, Joël; Roncarolo, Maria-Grazia; Gregori, Silvia

    2015-01-01

    Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule with known immune-modulatory functions. Our group identified a subset of human dendritic cells, named DC-10, that induce adaptive interleukin-10-producing T regulatory type 1 (Tr1) cells via the interleukin-10-dependent HLA-G/ILT4 pathway. In this study we aimed at defining the role of HLA-G in DC-10-mediated Tr1 cell differentiation. We analyzed phenotype, functions, and genetic variations in the 3′ untranslated region of the HLA-G locus of in vitro-differentiated DC-10 from 67 healthy donors. We showed that HLA-G expression on DC-10 is donor-dependent. Functional studies demonstrated that DC-10, independently of HLA-G expression, secrete interleukin-10 and negligible levels of interleukin-12. Interestingly, DC-10 with high HLA-G promote allo-specific anergic T cells that contain a significantly higher frequency of Tr1 cells, defined as interleukin-10-producing (P=0.0121) or CD49b+LAG-3+ (P=0.0031) T cells, compared to DC-10 with low HLA-G. We found that the HLA-G expression on DC-10 is genetically imprinted, being associated with specific variations in the 3′ untranslated region of the gene, and it may be finely tuned by microRNA-mediated post-transcriptional regulation. These data highlight the important role of HLA-G in boosting DC-10 tolerogenic activity and confirm that interleukin-10 production by DC-10 is necessary but not sufficient to promote Tr1 cells at high frequency. These new insights into the role of HLA-G in DC-10-mediated induction of Tr1 cells provide additional information for clinical use in Tr1- or DC-10-based cell therapy approaches. PMID:25661445

  6. HLA-G expression levels influence the tolerogenic activity of human DC-10.

    PubMed

    Amodio, Giada; Comi, Michela; Tomasoni, Daniela; Gianolini, Monica Emma; Rizzo, Roberta; LeMaoult, Joël; Roncarolo, Maria-Grazia; Gregori, Silvia

    2015-04-01

    Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule with known immune-modulatory functions. Our group identified a subset of human dendritic cells, named DC-10, that induce adaptive interleukin-10-producing T regulatory type 1 (Tr1) cells via the interleukin-10-dependent HLA-G/ILT4 pathway. In this study we aimed at defining the role of HLA-G in DC-10-mediated Tr1 cell differentiation. We analyzed phenotype, functions, and genetic variations in the 3' untranslated region of the HLA-G locus of in vitro-differentiated DC-10 from 67 healthy donors. We showed that HLA-G expression on DC-10 is donor-dependent. Functional studies demonstrated that DC-10, independently of HLA-G expression, secrete interleukin-10 and negligible levels of interleukin-12. Interestingly, DC-10 with high HLA-G promote allo-specific anergic T cells that contain a significantly higher frequency of Tr1 cells, defined as interleukin-10-producing (P=0.0121) or CD49b(+)LAG-3(+) (P=0.0031) T cells, compared to DC-10 with low HLA-G. We found that the HLA-G expression on DC-10 is genetically imprinted, being associated with specific variations in the 3' untranslated region of the gene, and it may be finely tuned by microRNA-mediated post-transcriptional regulation. These data highlight the important role of HLA-G in boosting DC-10 tolerogenic activity and confirm that interleukin-10 production by DC-10 is necessary but not sufficient to promote Tr1 cells at high frequency. These new insights into the role of HLA-G in DC-10-mediated induction of Tr1 cells provide additional information for clinical use in Tr1- or DC-10-based cell therapy approaches.

  7. Activity levels and expression of antioxidant enzymes in the ascorbate-glutathione cycle in artificially aged rice seed.

    PubMed

    Yin, Guangkun; Xin, Xia; Song, Chao; Chen, Xiaoling; Zhang, Jinmei; Wu, Shuhua; Li, Ruifang; Liu, Xu; Lu, Xinxiong

    2014-07-01

    Reactive oxygen species are the main contributors to seed deterioration. In order to study scavenging systems for reactive oxygen species in aged seed, we performed analyses using western blotting, real-time quantitative reverse-transcription polymerase chain reaction, high-performance liquid chromatography, and antioxidant enzyme activity analyses in artificially aged rice seeds (Oryza sativa L. cv. wanhua no.11). Aging seeds by storing them at 50 °C for 1, 9, or 17 months increased the superoxide radical and hydrogen peroxide levels and reduced the germination percentage from 99% to 92%, 55%, and 2%, respectively. The activity levels of superoxide dismutase (SOD), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) did not change in aged seeds. In contrast, the activity levels of catalase (CAT), ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) were significantly decreased in aged seeds, as were the expression of catalase and cytosolic ascorbate peroxidase protein. Transcript accumulation analysis showed that specific expression patterns were complex for each of the antioxidant enzyme types in the rice embryos. Overall, the expression of most genes was down-regulated, along with their protein expression. In addition, the reduction in the amount of ascorbate and glutathione was associated with the reduction in scavenging enzymes activity in aged rice embryos. Our data suggest that the depression of the antioxidant system, especially the reduction in the expression of CAT1, APX1 and MDHAR1, may be responsible for the accumulation of reactive oxygen species in artificially aged seed embryos, leading to a loss of seed vigor.

  8. Pitch Estimation, Voicing Decision, and Noise Spectrum Estimation for Speech Corrupted by High Levels of Additive Noise.

    NASA Astrophysics Data System (ADS)

    Krubsack, David Allan

    1990-01-01

    This dissertation presents two algorithms that extract parameters which are important to speech processing in high levels of noise. The first algorithm determines whether a signal containing noise corrupted human speech is voiced or not and estimates the fundamental frequency (pitch) of voiced speech. The second algorithm produces an estimate of the additive noise which is corrupting the speech. Previous research related to the voicing decision and pitch estimation has been concentrated at signal-to -noise ratios (SNRs) above 0 dB. Consequently, speech processing requiring the extraction of these parameters in higher levels of noise could not be performed with much success. The research presented in this dissertation concentrates on SNRs around and below 0 dB. Although the algorithm, based on the autocorrelation function, is designed to work well for high levels of noise, good results for the no noise case have been maintained. The idea of a confidence measure for parameter estimation is introduced. Confidence measures are defined and developed for both the voicing decision and the pitch estimation algorithms. Estimation of noise that is corrupting a speech signal has been motivated by the need to enhance the corrupted speech. Previous research has concentrated on speech which is band limited to about 3500 Hz. Therefore, the estimation of the noise corrupting high frequency speech had not been considered. The noise estimation algorithm presented in this dissertation considers the effects of high frequency speech on the noise estimate in addition to the effects of low frequency speech. A new spectral averaging method is introduced which significantly reduces the corrupting effect of the speech components on the noise estimate for SNRs above 0 dB. The algorithm is tested for stationary white noise, stationary non-white noise, and non-stationary white noise.

  9. Predicting Gene Expression Level from Relative Codon Usage Bias: An Application to Escherichia coli Genome

    PubMed Central

    Roymondal, Uttam; Das, Shibsankar; Sahoo, Satyabrata

    2009-01-01

    We present an expression measure of a gene, devised to predict the level of gene expression from relative codon bias (RCB). There are a number of measures currently in use that quantify codon usage in genes. Based on the hypothesis that gene expressivity and codon composition is strongly correlated, RCB has been defined to provide an intuitively meaningful measure of an extent of the codon preference in a gene. We outline a simple approach to assess the strength of RCB (RCBS) in genes as a guide to their likely expression levels and illustrate this with an analysis of Escherichia coli (E. coli) genome. Our efforts to quantitatively predict gene expression levels in E. coli met with a high level of success. Surprisingly, we observe a strong correlation between RCBS and protein length indicating natural selection in favour of the shorter genes to be expressed at higher level. The agreement of our result with high protein abundances, microarray data and radioactive data demonstrates that the genomic expression profile available in our method can be applied in a meaningful way to the study of cell physiology and also for more detailed studies of particular genes of interest. PMID:19131380

  10. FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype.

    PubMed

    Arsenault, Jason; Gholizadeh, Shervin; Niibori, Yosuke; Pacey, Laura K; Halder, Sebok K; Koxhioni, Enea; Konno, Ayumu; Hirai, Hirokazu; Hampson, David R

    2016-12-01

    Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector-based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35-115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5-6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long-term curative

  11. Generation of CD8+ T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy

    PubMed Central

    Höfflin, Sandra; Prommersberger, Sabrina; Uslu, Ugur; Schuler, Gerold; Schmidt, Christopher W; Lennerz, Volker; Dörrie, Jan; Schaft, Niels

    2015-01-01

    Adoptive T-cell therapy of cancer often fails due to the tumor cells' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8+ T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed “CCT6Am TCR.” These CD8+ T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6Am TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8+ T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach. PMID:26178065

  12. Alternating expression levels of WWOX tumor suppressor and cancer-related genes in patients with bladder cancer

    PubMed Central

    PŁUCIENNIK, ELŻBIETA; NOWAKOWSKA, MAGDALENA; STĘPIEN, ANNA; WOŁKOWICZ, MATEUSZ; STAWIŃSKI, ADAM; RÓŻAŃSKI, WALDEMAR; LIPIŃSKI, MAREK; BEDNAREK, ANDRZEJ K

    2014-01-01

    The aim of the present study was to determine the roles of the WWOX tumor suppressor and cancer-related genes in bladder tumor carcinogenesis. Reverse transcription-quantitative polymerase chain reaction was used to analyze the status of WWOX promoter methylation (using MethylScreen™ technology) and loss of heterozygosity (LOH) in papillary urothelial cancer tissues. The associations between the expression levels of the following tumorigenesis-related genes were also assessed: The WWOX tumor suppressor gene, the MKI67 proliferation gene, the BAX, BCL2 and BIRC5 apoptotic genes, the EGFR signal transduction gene, the VEGF vascular endothelial growth factor gene, and the CCND1 and CCNE1 cell cycle genes. The results reveal a high frequency of LOH in intron 1 in the WWOX gene, as well as an association between reduced WWOX expression levels and increased promoter methylation. In addition, the present study demonstrates that in bladder tumors, apoptosis is inhibited by increased expression levels of the BCL2 gene. A correlation between the proliferation indices of the MKI67 and the BIRC5 genes was also revealed. Furthermore, the expression levels of VEGF were identified to be positively associated with those of the EGFR gene. PMID:25295115

  13. Development of a standard operating procedure (SOP) for the precise quantification of transgene expression levels in rice plants.

    PubMed

    James, Victoria A.; Worland, Barbara; Snape, John W.; Vain, Philippe

    2004-04-01

    Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. It is important to quantify and eliminate this unwanted variation as much as possible in order to attain precision in transgenic studies. Large-scale transgenic studies could, by their design, generate additional variation. The influence of different plant growth, sampling and analysis strategies in generating spurious variation in transgene expression level quantification in rice plant populations was assessed. The use of multiple independent plant phenotypic analyses (enzymatic assays in this study) was identified as the major source of spurious variation (doubling or tripling the variation). The quantification of transgene expression levels was also found to be significantly influenced by plant age, the choice of leaf sampled and leaf size. All of these factors reduced the precision of molecular genetic studies and generated artefactual results in transgenic studies. Identification of the sources of extraneous variation allowed the development of a new standard operating procedure (SOP) for rice, designed to control spurious variation. SOP allowed the influence of differences in growth period and independent phenotypic analyses to be minimized. The coefficient of variation in transgene expression levels, between and within genetically identical rice plants, was reduced to approximately 10 to 15% using SOP. Adoption of quality assurance (QA) criteria such as SOP is key to improving the reproducibility of transgenic studies.

  14. Secondary Data Analytics of Aquaporin Expression Levels in Glioblastoma Stem-Like Cells

    PubMed Central

    Isokpehi, Raphael D; Wollenberg Valero, Katharina C; Graham, Barbara E; Pacurari, Maricica; Sims, Jennifer N; Udensi, Udensi K; Ndebele, Kenneth

    2015-01-01

    Glioblastoma is the most common brain tumor in adults in which recurrence has been attributed to the presence of cancer stem cells in a hypoxic microenvironment. On the basis of tumor formation in vivo and growth type in vitro, two published microarray gene expression profiling studies grouped nine glioblastoma stem-like (GS) cell lines into one of two groups: full (GSf) or restricted (GSr) stem-like phenotypes. Aquaporin-1 (AQP1) and aquaporin-4 (AQP4) are water transport proteins that are highly expressed in primary glial-derived tumors. However, the expression levels of AQP1 and AQP4 have not been previously described in a panel of 92 glioma samples. Therefore, we designed secondary data analytics methods to determine the expression levels of AQP1 and AQP4 in GS cell lines and glioblastoma neurospheres. Our investigation also included a total of 2,566 expression levels from 28 Affymetrix microarray probe sets encoding 13 human aquaporins (AQP0–AQP12); CXCR4 (the receptor for stromal cell derived factor-1 [SDF-1], a potential glioma stem cell therapeutic target]); and PROM1 (gene encoding CD133, the widely used glioma stem cell marker). Interactive visual representation designs for integrating phenotypic features and expression levels revealed that inverse expression levels of AQP1 and AQP4 correlate with distinct phenotypes in a set of cell lines grouped into full and restricted stem-like phenotypes. Discriminant function analysis further revealed that AQP1 and AQP4 expression are better predictors for tumor formation and growth types in glioblastoma stem-like cells than are CXCR4 and PROM1. Future investigations are needed to characterize the molecular mechanisms for inverse expression levels of AQP1 and AQP4 in the glioblastoma stem-like neurospheres. PMID:26279619

  15. Mouse lymphomyeloid cells can function with significantly decreased expression levels of cytochrome C.

    PubMed

    Shilov, E S; Kislyakov, I V; Gorshkova, E A; Zvartsev, R V; Drutskaya, M S; Mufazalov, I A; Skulachev, V P; Nedospasov, S A

    2014-12-01

    Cytochrome c is an indispensable electron carrier in the mitochondrial respiratory chain and also an important mediator of the internal pathway triggering apoptosis. Mice with a complete deficiency of the Cycs gene encoding the somatic cytochrome c die during the embryogenesis. Using the technology of LoxP-cre-dependent tissue-specific recombination, we obtained some mouse strains with significantly reduced expression of cytochrome c in certain cell types ("conditional genetic knockdown"). This knockdown was achieved by abrogation of the normal splicing of the Cycs locus pre-mRNA due to an additional acceptor site inside the stop-cassette neo(r). Previously, we observed embryonic lethality in homozygous mice with the same knockdown of cytochrome c in all cells of the organism. In the present work we studied two novel mouse strains with conditional knockdown of the Cycs gene in T lymphocytes and macrophages. Somewhat surprisingly, the mice of these two strains under normal conditions were not phenotypically different from the wild-type mice, either on the whole organism level or on the level of activity of individual target cells. Thus, the amount of cytochrome c in lymphomyeloid cells does not affect their development and normal functioning.

  16. Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions.

    PubMed

    Radniecki, Tyler S; Semprini, Lewis; Dolan, Mark E

    2009-02-01

    The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl2 achieving maximum concentrations from 3 to 90 microM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up-regulation was 45-fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 microM CuSO4 or 2250 microM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 inhibition is irreversible and amoA was up-regulated at 30 and 90 microM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up-regulation of merA during exposure to non-inhibitory Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition.

  17. CCR5 Expression Levels in HIV-Uninfected Women Receiving Hormonal Contraception.

    PubMed

    Sciaranghella, Gaia; Wang, Cuiwei; Hu, Haihong; Anastos, Kathryn; Merhi, Zaher; Nowicki, Marek; Stanczyk, Frank Z; Greenblatt, Ruth M; Cohen, Mardge; Golub, Elizabeth T; Watts, D Heather; Alter, Galit; Young, Mary A; Tsibris, Athe M N

    2015-11-01

    Human immunodeficiency virus (HIV) infectivity increases as receptor/coreceptor expression levels increase. We determined peripheral CD4, CCR5, and CXCR4 expression levels in HIV-uninfected women who used depot medroxyprogesterone acetate (DMPA; n = 32), the levonorgestrel-releasing intrauterine device (LNG-IUD; n = 27), oral contraceptive pills (n = 32), or no hormonal contraception (n = 33). The use of LNG-IUD increased the proportion of CD4(+) and CD8(+) T cells that expressed CCR5; increases in the magnitude of T-cell subset CCR5 expression were observed with DMPA and LNG-IUD use (P < .01 for all comparisons). LNG-IUD and, to a lesser extent, DMPA use were associated with increased peripheral T-cell CCR5 expression.

  18. CCR5 Expression Levels in HIV-Uninfected Women Receiving Hormonal Contraception

    PubMed Central

    Sciaranghella, Gaia; Wang, Cuiwei; Hu, Haihong; Anastos, Kathryn; Merhi, Zaher; Nowicki, Marek; Stanczyk, Frank Z.; Greenblatt, Ruth M.; Cohen, Mardge; Golub, Elizabeth T.; Watts, D. Heather; Alter, Galit; Young, Mary A.; Tsibris, Athe M. N.

    2015-01-01

    Human immunodeficiency virus (HIV) infectivity increases as receptor/coreceptor expression levels increase. We determined peripheral CD4, CCR5, and CXCR4 expression levels in HIV-uninfected women who used depot medroxyprogesterone acetate (DMPA; n = 32), the levonorgestrel-releasing intrauterine device (LNG-IUD; n = 27), oral contraceptive pills (n = 32), or no hormonal contraception (n = 33). The use of LNG-IUD increased the proportion of CD4+ and CD8+ T cells that expressed CCR5; increases in the magnitude of T-cell subset CCR5 expression were observed with DMPA and LNG-IUD use (P < .01 for all comparisons). LNG-IUD and, to a lesser extent, DMPA use were associated with increased peripheral T-cell CCR5 expression. PMID:25895986

  19. Effects of cell-cycle-dependent expression on random fluctuations in protein levels

    PubMed Central

    Soltani, Mohammad

    2016-01-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression. PMID:28083102

  20. Effects of cell-cycle-dependent expression on random fluctuations in protein levels.

    PubMed

    Soltani, Mohammad; Singh, Abhyudai

    2016-12-01

    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

  1. Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies.

    PubMed

    Yan, Jenny; Anderson, Caitlin; Viets, Kayla; Tran, Sang; Goldberg, Gregory; Small, Stephen; Johnston, Robert J

    2017-03-01

    How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. Here, we find that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ('dark' shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback and redundancy to generate cell type-specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.

  2. Effects of a phytogenic feed additive on growth performance, susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin.

    PubMed

    Peterson, Brian C; Peatman, E; Ourth, D D; Waldbieser, G C

    2015-05-01

    A study was conducted to investigate the effect of a phytogenic feed additive (Digestarom® P.E.P. MGE; containing the essential oils carvacrol, thymol, anethol, and limonene) on growth performance and disease susceptibility to Edwardsiella ictaluri. Two hundred and fifty juvenile channel catfish, Ictalurus punctatus (7.2 ± 0.1 g) were allotted into the following treatments: Control (floating diet) and EO (floating diet supplemented with essential oils). The fish were fed their respective diets for 6 weeks. At the end of the study, all fish were exposed to virulent E. ictaluri by bath immersion (1.9 × 10(7) cfu/mL; final concentration). Plasma and tissue samples were taken to quantify protein and mRNA expression levels of mannose binding lectin (MBL). Weight gain and food conversion ratio were similar between treatments. After exposing fish to virulent E. ictaluri and monitoring mortality for 21 days, survival was 43% higher (69.5 vs 48.4%) in fish fed EO compared to fish not treated with EO (P < 0.05). One day after challenge, plasma MBL levels were down-regulated in the non-treated fish compared to non-challenged fish. In the EO fish, MBL levels were similar to non-challenged fish but significantly higher than non-treated fed fish (P < 0.001). By d 7, plasma MBL levels increased in non-treated fed fish to levels observed in the EO and non-challenged fish. On d 14, MBL mRNA levels were upregulated 15-fold in fish fed EO compared to non-treated fed fish and non-challenged fish (P < 0.001). The results demonstrate that essential oils improved survival of channel catfish challenged with E. ictaluri. Mechanisms through which essential oils improve survival may involve MBL.

  3. Fertilizer addition lessens the flux of microbial carbon to higher trophic levels in soil food webs of grassland.

    PubMed

    Lemanski, Kathleen; Scheu, Stefan

    2014-10-01

    Roots and root-derived C compounds are increasingly recognised as important resources for soil animal food webs. We used (13)C-labelled glucose as a model C compound representing root exudates to follow the incorporation of root-derived C into the soil animal food web of a temperate grassland over a period of 52 weeks. We investigated variations in glucose C incorporation with fertilizer addition and sward composition, i.e. variations in plant functional groups. The approach allowed the differentiation of trophic chains based on primary decomposers feeding on litter and phytophagous species feeding on roots (i.e. not incorporating glucose C) from those based on secondary decomposers feeding on microorganisms (thereby assimilating glucose C). Each of the studied soil animal species incorporated glucose C, indicating that the majority of grassland soil animal species rely on microorganisms as food resources with microorganisms being fuelled by root exudates. However, incorporation of glucose C into soil animal species varied markedly with species identity, suggesting that detritivorous microarthropods complement each other in channelling microbial C through soil food webs. Fertilizer addition markedly reduced the concentration of glucose C in most soil animal species as well as the absolute transfer of glucose C into oribatid mites as major secondary decomposers. The results suggest that fertilizer addition shifts the basis of the decomposer food web towards the use of unlabelled resources, presumably roots, i.e. towards a herbivore system, thereby lessening the link between microorganisms and microbial grazers and hampering the propagation of microbial C to higher trophic levels.

  4. Neuromodulation independently determines correlated channel expression and conductance levels in motor neurons of the stomatogastric ganglion.

    PubMed

    Temporal, Simone; Desai, Mohati; Khorkova, Olga; Varghese, Gladis; Dai, Aihua; Schulz, David J; Golowasch, Jorge

    2012-01-01

    Neuronal identity depends on the regulated expression of numerous molecular components, especially ionic channels, which determine the electrical signature of a neuron. Such regulation depends on at least two key factors, activity itself and neuromodulatory input. Neuronal electrical activity can modify the expression of ionic currents in homeostatic or nonhomeostatic fashion. Neuromodulators typically modify activity by regulating the properties or expression levels of subsets of ionic channels. In the stomatogastric system of crustaceans, both types of regulation have been demonstrated. Furthermore, the regulation of the coordinated expression of ionic currents and the channels that carry these currents has been recently reported in diverse neuronal systems, with neuromodulators not only controlling the absolute levels of ionic current expression but also, over long periods of time, appearing to modify their correlated expression. We hypothesize that neuromodulators may regulate the correlated expression of ion channels at multiple levels and in a cell-type-dependent fashion. We report that in two identified neuronal types, three ionic currents are linearly correlated in a pairwise manner, suggesting their coexpression or direct interactions, under normal neuromodulatory conditions. In each cell, some currents remain correlated after neuromodulatory input is removed, whereas the correlations between the other pairs are either lost or altered. Interestingly, in each cell, a different suite of currents change their correlation. At the transcript level we observe distinct alterations in correlations between channel mRNA amounts, including one of the cell types lacking a correlation under normal neuromodulatory conditions and then gaining the correlation when neuromodulators are removed. Synaptic activity does not appear to contribute, with one possible exception, to the correlated expression of either ionic currents or of the transcripts that code for the respective

  5. Krüppel expression levels are maintained through compensatory evolution of shadow enhancers

    PubMed Central

    Wunderlich, Zeba; Bragdon, Meghan D.J.; Vincent, Ben J.; White, Jonathan A.; Estrada, Javier; DePace, Angela H.

    2015-01-01

    Summary Many developmental genes are controlled by shadow enhancers, pairs of enhancers that drive overlapping expression patterns. We hypothesized that compensatory evolution can maintain the total expression of a gene while individual shadow enhancers diverge between species. To test this hypothesis, we analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. We find that the expression driven by the pair of enhancers is conserved between these three species, but expression levels driven by the individual enhancers are not. Using sequence analysis and experimental perturbation, we show that each shadow enhancer is activated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation. PMID:26344774

  6. Krüppel Expression Levels Are Maintained through Compensatory Evolution of Shadow Enhancers.

    PubMed

    Wunderlich, Zeba; Bragdon, Meghan D J; Vincent, Ben J; White, Jonathan A; Estrada, Javier; DePace, Angela H

    2015-09-22

    Many developmental genes are controlled by shadow enhancers—pairs of enhancers that drive overlapping expression patterns. We hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. To test this hypothesis, we analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. We found that the expression driven by the pair of enhancers is conserved between these three species, but expression levels driven by the individual enhancers are not. Using sequence analysis and experimental perturbation, we show that each shadow enhancer is regulated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation.

  7. Inheritance of gene expression level and selective constraints on trans- and cis-regulatory changes in yeast.

    PubMed

    Schaefke, Bernhard; Emerson, J J; Wang, Tzi-Yuan; Lu, Mei-Yeh Jade; Hsieh, Li-Ching; Li, Wen-Hsiung

    2013-09-01

    Gene expression evolution can be caused by changes in cis- or trans-regulatory elements or both. As cis and trans regulation operate through different molecular mechanisms, cis and trans mutations may show different inheritance patterns and may be subjected to different selective constraints. To investigate these issues, we obtained and analyzed gene expression data from two Saccharomyces cerevisiae strains and their hybrid, using high-throughput sequencing. Our data indicate that compared with other types of genes, those with antagonistic cis-trans interactions are more likely to exhibit over- or underdominant inheritance of expression level. Moreover, in accordance with previous studies, genes with trans variants tend to have a dominant inheritance pattern, whereas cis variants are enriched for additive inheritance. In addition, cis regulatory differences contribute more to expression differences between species than within species, whereas trans regulatory differences show a stronger association between divergence and polymorphism. Our data indicate that in the trans component of gene expression differences genes subjected to weaker selective constraints tend to have an excess of polymorphism over divergence compared with those subjected to stronger selective constraints. In contrast, in the cis component, this difference between genes under stronger and weaker selective constraint is mostly absent. To explain these observations, we propose that purifying selection more strongly shapes trans changes than cis changes and that positive selection may have significantly contributed to cis regulatory divergence.

  8. MAP17 and SGLT1 Protein Expression Levels as Prognostic Markers for Cervical Tumor Patient Survival

    PubMed Central

    Perez, Marco; Praena-Fernandez, Juan M.; Felipe-Abrio, Blanca; Lopez-Garcia, Maria A.; Lucena-Cacace, Antonio; Garcia, Angel; Lleonart, Matilde; Roncador, Guiovanna; Marin, Juan J.; Carnero, Amancio

    2013-01-01

    MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types. PMID:23418532

  9. Additive enhancement of wound healing in diabetic mice by low level light and topical CoQ10

    NASA Astrophysics Data System (ADS)

    Mao, Zhigang; Wu, Jeffrey H.; Dong, Tingting; Wu, Mei X.

    2016-02-01

    Diabetes, a highly prevalent disease that affects 9.3% of Americans, often leads to severe complications and slow wound healing. Preclinical studies have suggested that low level light therapy (LLLT) can accelerate wound healing in diabetic subjects, but significant improvements must be made to overcome the absence of persuasive evidence for its clinical use. We demonstrate here that LLLT can be combined with topical Coenzyme Q10 (CoQ10) to heal wounds in diabetic mice significantly faster than LLLT alone, CoQ10 alone, or controls. LLLT followed by topical CoQ10 enhanced wound healing by 68~103% in diabetic mice in the first week and more than 24% in the second week compared with untreated controls. All wounds were fully healed in two weeks following the dual treatment, in contrast to only 50% wounds or a fewer being fully healed for single or sham treatment. The accelerated healing was corroborated by at least 50% higher hydroxyproline levels, and tripling cell proliferation rates in LLLT and CoQ10 treated wounds over controls. The beneficial effects on wound healing were probably attributed to additive enhancement of ATP production by LLLT and CoQ10 treatment. The combination of LLLT and topical CoQ10 is safe and convenient, and merits further clinical study.

  10. LV wall segmentation using the variational level set method (LSM) with additional shape constraint for oedema quantification

    NASA Astrophysics Data System (ADS)

    Kadir, K.; Gao, H.; Payne, A.; Soraghan, J.; Berry, C.

    2012-10-01

    In this paper an automatic algorithm for the left ventricle (LV) wall segmentation and oedema quantification from T2-weighted cardiac magnetic resonance (CMR) images is presented. The extent of myocardial oedema delineates the ischaemic area-at-risk (AAR) after myocardial infarction (MI). Since AAR can be used to estimate the amount of salvageable myocardial post-MI, oedema imaging has potential clinical utility in the management of acute MI patients. This paper presents a new scheme based on the variational level set method (LSM) with additional shape constraint for the segmentation of T2-weighted CMR image. In our approach, shape information of the myocardial wall is utilized to introduce a shape feature of the myocardial wall into the variational level set formulation. The performance of the method is tested using real CMR images (12 patients) and the results of the automatic system are compared to manual segmentation. The mean perpendicular distances between the automatic and manual LV wall boundaries are in the range of 1-2 mm. Bland-Altman analysis on LV wall area indicates there is no consistent bias as a function of LV wall area, with a mean bias of -121 mm2 between individual investigator one (IV1) and LSM, and -122 mm2 between individual investigator two (IV2) and LSM when compared to two investigators. Furthermore, the oedema quantification demonstrates good correlation when compared to an expert with an average error of 9.3% for 69 slices of short axis CMR image from 12 patients.

  11. Reference points suitable for evaluation of the additional arch length required for leveling the curve of Spee

    PubMed Central

    Cho, Yong-Hwa; Gang, Sung-Nam

    2016-01-01

    Objective The additional arch length required for leveling (AALL) the curve of Spee (COS) can be estimated by subtracting the two-dimensional (2D) arch circumference, which is the projection of the three-dimensional (3D) arch circumference onto the occlusal plane, from the 3D arch circumference, which represents the arch length after leveling the COS. The purpose of this study was to determine whether the cusp tips or proximal maximum convexities are more appropriate reference points for estimating the AALL. Methods Sixteen model setups of the mandibular arch with COS depths ranging from 0 mm to 4.7 mm were constructed using digital simulation. Arch circumferences in 2D and 3D were measured from the cusp tips and proximal maximum convexities and used to calculate the AALL. The values obtained using the two reference points were compared with the paired t-test. Results Although the 3D arch circumference should be constant regardless of the COS depth, it decreased by 3.8 mm in cusp tip measurements and by 0.4 mm in proximal maximum convexity measurements as the COS deepened to 4.7 mm. AALL values calculated using the cusp tips as reference points were significantly smaller than those calculated using the proximal maximum convexities (p = 0.002). Conclusions The AALL is underestimated when the cusp tips are used as measurement reference points; the AALL can be measured more accurately using the proximal maximum convexities. PMID:27896209

  12. Additive enhancement of wound healing in diabetic mice by low level light and topical CoQ10

    PubMed Central

    Mao, Zhigang; Wu, Jeffrey H.; Dong, Tingting; Wu, Mei X.

    2016-01-01

    Diabetes, a highly prevalent disease that affects 9.3% of Americans, often leads to severe complications and slow wound healing. Preclinical studies have suggested that low level light therapy (LLLT) can accelerate wound healing in diabetic subjects, but significant improvements must be made to overcome the absence of persuasive evidence for its clinical use. We demonstrate here that LLLT can be combined with topical Coenzyme Q10 (CoQ10) to heal wounds in diabetic mice significantly faster than LLLT alone, CoQ10 alone, or controls. LLLT followed by topical CoQ10 enhanced wound healing by 68~103% in diabetic mice in the first week and more than 24% in the second week compared with untreated controls. All wounds were fully healed in two weeks following the dual treatment, in contrast to only 50% wounds or a fewer being fully healed for single or sham treatment. The accelerated healing was corroborated by at least 50% higher hydroxyproline levels, and tripling cell proliferation rates in LLLT and CoQ10 treated wounds over controls. The beneficial effects on wound healing were probably attributed to additive enhancement of ATP production by LLLT and CoQ10 treatment. The combination of LLLT and topical CoQ10 is safe and convenient, and merits further clinical study. PMID:26830658

  13. Enhanced expression of glucose-regulated protein 78 correlates with malondialdehyde levels during the formation of liver cirrhosis in rats

    PubMed Central

    ZHANG, YUN; ZHANG, HUIYING; ZHAO, ZHONGFU; LV, MINLI; JIA, JIANTAO; ZHANG, LILI; TIAN, XIAOXIA; CHEN, YUNXIA; LI, BAOHONG; LIU, MINGSHE; HAN, DEWU; JI, CHENG

    2015-01-01

    The aim of the present study was to explore the role of glucose-regulated protein 78 (GRP78) in the development of liver cirrhosis promoted by intestinal endotoxemia in rats. Fifty-one male Wistar rats were randomly divided into the liver cirrhosis 4-week, 6-week and 8-week groups and the normal control group at each time point. Liver cirrhosis was induced by employing multiple pathogenic factors in the rats. Blood and liver tissues were collected. The levels of alanine aminotransferase (ALT), homocysteine, endotoxin and tumor necrosis factor-α (TNF-α) in the plasma, and TNF-α, malondialdehyde (MDA) and procollagen type III peptide (PIIIP) in the liver tissues were determined. The mRNA and protein expression levels of GRP78 in the liver were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Morphological changes were observed through hematoxylin and eosin and van Gieson staining of the liver. Liver cirrhosis caused marked histopathological changes to the livers of the rats. Following significant increases in the levels of ALT, homocysteine, endotoxin and TNF-α in the plasma, and TNF-α, MDA and PIIIP in the liver tissues of all experimental groups with the progression of liver cirrhosis, the mRNA and protein expression levels of GRP78 also gradually increased. In addition, correlation analysis indicated that the enhanced expression of GRP78 correlated with the MDA levels of the rats during the formation of liver cirrhosis. PMID:26668603

  14. Synchronous vitellogenin expression and sexual maturation during migration are negatively correlated with juvenile hormone levels in Mythimna separata

    PubMed Central

    Xiao, Hai-Jun; Fu, Xiao-Wei; Liu, Yong-Qiang; Wu, Kong-Ming

    2016-01-01

    Annual migration of pests between different seasonal habitats can lead to serious crop damage. Reproductive immaturity is generally associated with the migratory process (oogenesis-flight syndrome), but the mechanism of reproductive development during migration varies unpredictably. Here, the vitellogenin gene (MsVg) and three key regulatory enzyme genes (MsJhamt, MsJheh and MsJhe) related to juvenile hormone (JH) synthesis and degradation were identified and characterized in Mythimna separata. The relative expression of MsVg varied significantly in response to seasonal changes and was significantly correlated with stages of ovarian development. The relatively low levels of JH titer did not differ significantly in male moths but slightly increased in female adults during the migratory season, which was consistent with changes in mRNA levels for MsJhamt, MsJheh and MsJhe. JH titer was negatively associated with relative seasonal levels of vitellogenin mRNA transcripts and with ovarian development in migrating M. separata. The synchrony of MsVg expression with sexual maturation highlighted the potential of MsVg transcript levels to serve as an index to monitor the adult reproductive status. In addition, the level of JH and sexual maturity were correlated with the extent of JH in regulating the MsVg expression and reproduction during seasonal northern and southern migration. PMID:27629246

  15. Role of endogenous cortistatin in the regulation of ghrelin system expression at pancreatic level under normal and obese conditions.

    PubMed

    Chanclón, Belén; Luque, Raúl M; Córdoba-Chacón, José; Gahete, Manuel D; Pozo-Salas, Ana I; Castaño, Justo P; Gracia-Navarro, Francisco; Martínez-Fuentes, Antonio J

    2013-01-01

    Ghrelin-system components [native ghrelin, In1-ghrelin, Ghrelin-O-acyltransferase enzyme (GOAT) and receptors (GHS-Rs)] are expressed in a wide variety of tissues, including the pancreas, where they exert different biological actions including regulation of neuroendocrine secretions, food intake and pancreatic function. The expression of ghrelin system is regulated by metabolic conditions (fasting/obesity) and is associated with the progression of obesity and insulin resistance. Cortistatin (CORT), a neuropeptide able to activate GHS-R, has emerged as an additional link in gut-brain interplay. Indeed, we recently reported that male CORT deficient mice (cort-/-) are insulin-resistant and present a clear dysregulation in the stomach ghrelin-system. The present work was focused at analyzing the expression pattern of ghrelin-system components at pancreas level in cort-/- mice and their control littermates (cort +/+) under low- or high-fat diet. Our data reveal that all the ghrelin-system components are expressed at the mouse pancreatic level, where, interestingly, In1-ghrelin was expressed at higher levels than native-ghrelin. Thus, GOAT mRNA levels were significantly lower in cort-/- mice compared with controls while native ghrelin, In1-ghrelin and GHS-R transcript levels remained unaltered under normal metabolic conditions. Moreover, under obese condition, a significant increase in pancreatic expression of native-ghrelin, In1-ghrelin and GHS-R was observed in obese cort+/+ but not in cort-/- mice. Interestingly, insulin expression and release was elevated in obese cort+/+, while these changes were not observed in obese cort-/- mice. Altogether, our results indicate that the ghrelin-system expression is clearly regulated in the pancreas of cort+/+ and cort -/- under normal and/or obesity conditions suggesting that this system may play relevant roles in the endocrine pancreas. Most importantly, our data demonstrate, for the first time, that endogenous CORT is essential

  16. Expression of Dominant-Negative Thyroid Hormone Receptor Alpha1 in Leydig and Sertoli Cells Demonstrates No Additional Defect Compared with Expression in Sertoli Cells Only

    PubMed Central

    Fumel, Betty; Froment, Pascal; Holzenberger, Martin; Livera, Gabriel; Monget, Philippe; Fouchécourt, Sophie

    2015-01-01

    Background In the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial. Methods The TRαAMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter. Findings We showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRαAMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRαAMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRαAMI-ARO males showed normal fertility. This phenotype is similar to TRαAMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRαAMI-ARO. Conclusions/Significance We concluded that the presence of a mutant TRαAMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis. PMID:25793522

  17. Effects of porcine oocytes on the expression levels of transcripts encoding glycolytic enzymes in granulosa cells.

    PubMed

    Matsuno, Yuta; Onuma, Asuka; Fujioka, Yoshie A; Emori, Chihiro; Fujii, Wataru; Naito, Kunihiko; Sugiura, Koji

    2016-09-01

    Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

  18. A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast.

    PubMed

    Ishii, Jun; Izawa, Keiko; Matsumura, Shizuka; Wakamura, Kanako; Tanino, Takanori; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-06-01

    To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.

  19. Gene expression and nucleotide composition are associated with genic methylation level in Oryza sativa

    PubMed Central

    2014-01-01

    Background The methylation of cytosines at CpG dinucleotides, which plays an important role in gene expression regulation, is one of the most studied epigenetic modifications. Thus far, the detection of DNA methylation has been determined mostly by experimental methods, which are not only prone to bench effects and artifacts but are also time-consuming, expensive, and cannot be easily scaled up to many samples. It is therefore useful to develop computational prediction methods for DNA methylation. Our previous studies highlighted the existence of correlations between the GC content of the third codon position (GC3), methylation, and gene expression. We thus designed a model to predict methylation in Oryza sativa based on genomic sequence features and gene expression data. Results We first derive equations to describe the relationship between gene methylation levels, GC3, expression, length, and other gene compositional features. We next assess gene compositional features involving sixmers and their association with methylation levels and other gene level properties. By applying our sixmer-based approach on rice gene expression data we show that it can accurately predict methylation (Pearson’s correlation coefficient r = 0.79) for the majority (79%) of the genes. Matlab code with our model is included. Conclusions Gene expression variation can be used as predictors of gene methylation levels. PMID:24447369

  20. DNA Repair Gene Expression Levels as Indicators of Breast Cancer in the Breast Cancer Family Registry

    PubMed Central

    KAPPIL, MAYA A.; LIAO, YUYAN; TERRY, MARY BETH; SANTELLA, REGINA M.

    2017-01-01

    Aim The expression level of DNA repair-related genes and their association with breast cancer status among participants of the New York site of the Breast Cancer Family Registry was investigated. Materials and Methods RNA from mononuclear cells in 194 sister sets (n=475 women) were assayed for ATM, BRCA1, MSH2, MUTYH and XPC gene expression levels and analyzed using generalized estimating equations (GEE). Results Individuals with decreased ATM and MSH2 expression had significantly higher odds for breast cancer compared to individuals with higher levels of expression (odds ratio (OR)=1.1, 95% confidence interval (CI)=1.02, 1.18) and (OR=1.90, 95% CI=1.21, 2.97), respectively. Upon stratifying the GEE model, reductions in ATM and MSH2 expression levels was heightened among women with an extended family history (FH) of breast cancer. Conclusion Reduced expression of ATM and MSH2 compromises DNA repair capacity and, thereby, increases breast cancer prevalence. PMID:27466510

  1. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    PubMed

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  2. Central expression and anorectic effect of brain-derived neurotrophic factor are regulated by circulating estradiol levels.

    PubMed

    Zhu, Zheng; Liu, Xian; Senthil Kumar, Shiva Priya Dharshan; Zhang, Jing; Shi, Haifei

    2013-03-01

    Estrogens potently suppress food intake. Compelling evidence suggests that estradiol, the primary form of estrogens, reduces food intake by facilitating other anorectic signals. Brain-derived neurotrophic factor (BDNF), like estradiol, appears to suppress food intake by affecting meal size. We hypothesized that estradiol modulates Bdnf expression and the anorectic effect of BDNF. The first goal was to determine whether Bdnf expression was regulated by endogenous estradiol of cycling rats and by cyclic estradiol treatment using ovariectomized rats. Bdnf expression within the ventromedial nucleus of hypothalamus (VMH) was temporally elevated at estrus following the estradiol peak, which coincided with the decline in feeding at this phase of the ovarian cycle. Additionally, food intake and body weight were increased following ovariectomy with a parallel decrease in Bdnf expression in the VMH. All of these alterations were reversed by cyclic estradiol treatment, suggesting that Bdnf expression within the VMH was regulated in an estradiol-dependent manner. The second goal was to determine whether estradiol modulates the anorectic effect of BDNF. Sham-operated estrous rats and ovariectomized rats cyclically treated with estradiol responded to a lower dose of central administration of BDNF to decrease food intake than male rats and oil-treated ovariectomized rats, implying that endogenous estradiol or cyclic estradiol replacement increased the sensitivity to anorectic effect of BDNF. These data indicate that Bdnf expression within the VMH and the anorectic effect of BDNF varied depending on plasma estradiol levels, suggesting that estradiol may regulate BDNF signaling to regulate feeding.

  3. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    PubMed

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

  4. Gene expression levels are correlated with synonymous codon usage, amino acid composition, and gene architecture in the red flour beetle, Tribolium castaneum.

    PubMed

    Williford, Anna; Demuth, Jeffery P

    2012-12-01

    Gene expression levels correlate with multiple aspects of gene sequence and gene structure in phylogenetically diverse taxa, suggesting an important role of gene expression levels in the evolution of protein-coding genes. Here we present results of a genome-wide study of the influence of gene expression on synonymous codon usage, amino acid composition, and gene structure in the red flour beetle, Tribolium castaneum. Consistent with the action of translational selection, we find that synonymous codon usage bias increases with gene expression. However, the correspondence between tRNA gene copy number and optimal codons is weak. At the amino acid level, translational selection is suggested by the positive correlation between tRNA gene numbers and amino acid usage, which is stronger for highly expressed genes. In addition, there is a clear trend for increased use of metabolically cheaper, less complex amino acids as gene expression increases. tRNA gene numbers also correlate negatively with amino acid size/complexity (S/C) score indicating the coupling between translational selection and selection to minimize the use of large/complex amino acids. Interestingly, the analysis of 10 additional genomes suggests that the correlation between tRNA gene numbers and amino acid S/C score is widespread and might be explained by selection against negative consequences of protein misfolding. At the level of gene structure, three major trends are detected: 1) complete coding region length increases across low and intermediate expression levels but decreases in highly expressed genes; 2) the average intron size shows the opposite trend, first decreasing with expression, followed by a slight increase in highly expressed genes; and 3) intron density remains nearly constant across all expression levels. These changes in gene architecture are only in partial agreement with selection favoring reduced cost of biosynthesis.

  5. The expression level of HJURP has an independent prognostic impact and predicts the sensitivity to radiotherapy in breast cancer

    SciTech Connect

    Hu, Zhi; Huang, Ge; Sadanandam, Anguraj; Gu, Shenda; Lenburg, Marc E; Pai, Melody; Bayani, Nora; Blakely, Eleanor A; Gray, Joe W; Mao, Jian-Hua

    2010-06-25

    Introduction: HJURP (Holliday Junction Recognition Protein) is a newly discovered gene reported to function at centromeres and to interact with CENPA. However its role in tumor development remains largely unknown. The goal of this study was to investigate the clinical significance of HJURP in breast cancer and its correlation with radiotherapeutic outcome. Methods: We measured HJURP expression level in human breast cancer cell lines and primary breast cancers by Western blot and/or by Affymetrix Microarray; and determined its associations with clinical variables using standard statistical methods. Validation was performed with the use of published microarray data. We assessed cell growth and apoptosis of breast cancer cells after radiation using high-content image analysis. Results: HJURP was expressed at higher level in breast cancer than in normal breast tissue. HJURP mRNA levels were significantly associated with estrogen receptor (ER), progesterone receptor (PR), Scarff-Bloom-Richardson (SBR) grade, age and Ki67 proliferation indices, but not with pathologic stage, ERBB2, tumor size, or lymph node status. Higher HJURP mRNA levels significantly decreased disease-free and overall survival. HJURP mRNA levels predicted the prognosis better than Ki67 proliferation indices. In a multivariate Cox proportional-hazard regression, including clinical variables as covariates, HJURP mRNA levels remained an independent prognostic factor for disease-free and overall survival. In addition HJURP mRNA levels were an independent prognostic factor over molecular subtypes (normal like, luminal, Erbb2 and basal). Poor clinical outcomes among patients with high HJURP expression werevalidated in five additional breast cancer cohorts. Furthermore, the patients with high HJURP levels were much more sensitive to radiotherapy. In vitro studies in breast cancer cell lines showed that cells with high HJURP levels were more sensitive to radiation treatment and had a higher rate of apoptosis

  6. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    PubMed

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  7. Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions.

    PubMed

    Radniecki, Tyler S; Semprini, Lewis; Dolan, Mark E

    2009-12-01

    The effects of CdSO(4) additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia-monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cultured N. europaea cells. The reactor was fed 50 mM NH(4)+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO(4) were made with increasing maximum concentrations ranging from 1 to 60 microM Cd(2+). The expression of merA was highly correlated with the level of Cd(2+) within the reactor (Rs = 0.90) with significant up-regulation measured at non-inhibitory Cd(2+) concentrations. Cd(2+) appears to target AMO specifically at lower concentrations and caused oxidative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up-regulation of trxA. Since Cd(2+) inhibition is irreversible and amoA was up-regulated in response to Cd(2+) inhibition, it is hypothesized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd(2+) inhibition than previously examined batch cultured cells due to the presence of Mg(2+) and Ca(2+) in the growth media, suggesting that Cd(2+) enters the cell through Mg(2+) and Ca(2+) import channels. The up-regulation of merA during exposure to non-inhibitory Cd(2+) levels indicates that merA is an excellent early warning signal for Cd(2+) inhibition.

  8. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    SciTech Connect

    Chiu Yali; Ouyang Pin . E-mail: ouyang@mail.cgu.edu.tw

    2006-03-10

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.

  9. High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis.

    PubMed

    Hajdu, Anita; Ádám, Éva; Sheerin, David J; Dobos, Orsolya; Bernula, Péter; Hiltbrunner, Andreas; Kozma-Bognár, László; Nagy, Ferenc

    2015-09-01

    Optimal timing of flowering in higher plants is crucial for successful reproduction and is coordinated by external and internal factors, including light and the circadian clock. In Arabidopsis, light-dependent stabilization of the rhythmically expressed CONSTANS (CO) is required for the activation of FLOWERING LOCUS T (FT), resulting in the initiation of flowering. Phytochrome A and cryptochrome photoreceptors stabilize CO in the evening by attenuating the activity of the CONSTITUTIVE PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA-105 1 (COP1-SPA1) ubiquitin ligase complex, which promotes turnover of CO. In contrast, phytochrome B (phyB) facilitates degradation of CO in the morning and delays flowering. Accordingly, flowering is accelerated in phyB mutants. Paradoxically, plants overexpressing phyB also show early flowering, which may arise from an early phase of rhythmic CO expression. Here we demonstrate that overexpression of phyB induces FT transcription at dusk and in the night without affecting the phase or level of CO transcription. This response depends on the light-activated Pfr form of phyB that inhibits the function of the COP1-SPA1 complex by direct interactions. Our data suggest that attenuation of COP1 activity results in the accumulation of CO protein and subsequent induction of FT. We show that phosphorylation of Ser-86 inhibits this function of phyB by accelerating dark reversion and thus depletion of Pfr forms in the night. Our results explain the early flowering phenotype of phyB overexpression and reveal additional features of the molecular machinery by which photoreceptors mediate photoperiodism.

  10. A glucose-insensitive T7 expression system for fully-induced expression of proteins at a subsaturating level of L-arabinose.

    PubMed

    Wang, Zei Wen; Lai, Cheng-Bon; Chang, Chih-Hsiang; Chiang, Chung-Jen; Chao, Yun-Peng

    2011-06-22

    The L-arabinose (Ara)-controlled T7 expression system was previously constructed by creation of an Escherichia coli BL21(BAD) strain. The production of recombinant proteins in this strain was stringently regulated and reached a high level upon induction with Ara. Nevertheless, this system is still associated with inherent problems of interference with glucose and of the all-or-nothing induction profile at a subsaturating level of Ara. In this study, these problems were circumvented by modifying the physiological traits of BL21(BAD) strain. This was followed by deletion of ptsG gene and the araFGH and araBAD operon. The former encodes the glucose transporter while the latter two gene operons produce proteins responsible for Ara uptake and catabolism. In addition, the expression of genomic araE (encodes the Ara transporter) was constitutively enhanced. The resulting strain was designated BAD-5. By expression of the faster degrader GFP(LAA) at a subsaturating level of Ara, 80% of BAD-5 strain was found visually bright in the presence or absence of glucose. A further analysis by flow cytometry showed a uniform distribution of GFP expression for BAD-5 strain. In marked contrast, BL21(BAD) strain exhibiting visual brightness was less than 10% of the cell population and remained dark in the presence of glucose. Moreover, a saturated level of luciferase from Renilla reniformis (Rluc) could be readily obtained in BAD-5 strain at 20 μM Ara regardless of glucose. Rluc in BL21(BAD) strain was produced in an Ara dose-dependent manner, and the protein production became arrested when glucose was present. Overall, it illustrates the usefulness of the improved system for overproduction of recombinant proteins in an efficient, homogeneous, and glucose-insensitive way.

  11. Response of Functional Structure of Soil Microbial Community to Multi-level Nitrogen Additions on the Central Tibetan Plateau

    NASA Astrophysics Data System (ADS)

    Zhang, G.; Yuan, Y.

    2015-12-01

    The use of fossil fuels and fertilizers has increased the amount of biologically reactive nitrogen in the atmosphere over the past century. Tibet is the one of the most threatened regions by nitrogen deposition, thus understanding how its microbial communities function maybe of high importance to predicting microbial responses to nitrogen deposition. Here we describe a short-time nitrogen addition conducted in an alpine steppe ecosystem to investigate the response of functional structure of soil microbial community to multi-level nitrogen addition. Using a GeoChip 4.0, we showed that functional diversities and richness of functional genes were unchanged at low level of nitrogen fertilizer inputs (<20 kg N ha-1 yr-1), but significantly decreased at higher nitrogen fertilizer inputs (>=40 kg N ha-1 yr-1). Detrended correspondence analysis indicated that the functional structure of microbial communities was markedly different across the nitrogen gradients. Most C degradation genes whose abundances significantly increased under elevated N fertilizer were those involved in the degradation of relatively labile C (starch, hemicellulose, cellulose), whereas the abundance of certain genes involved in the degradation of recalcitrant C (i.e. lignin) was largely decreased (such as manganese peroxidase, mnp). The results suggest that the elevated N fertilization rates might significantly accelerate the labile C degradation, but might not spur recalcitrant C degradation. The combined effect of gdh and ureC genes involved in N cycling appeared to shift the balance between ammonia and organic N toward organic N ammonification and hence increased the N mineralization potential. Moreover, Urease directly involved in urea mineralization significantly increased. Lastly, Canonical correspondence analysis showed that soil (TOC+NH4++NO3-+NO2-+pH) and plant (Aboveground plant productivity + Shannon Diversity) variables could explain 38.9% of the variation of soil microbial community

  12. Magnesium modulates the expression levels of calcification-associated factors to inhibit calcification in a time-dependent manner.

    PubMed

    Xu, Jinsheng; Bai, Yaling; Jin, Jingjing; Zhang, Junxia; Zhang, Shenglei; Cui, Liwen; Zhang, Huiran

    2015-03-01

    Vascular calcification, a common complication in patients with chronic kidney disease, involves a variety of mechanisms associated with the regulation of calcification-associated factors. Previous clinical studies have indicated that magnesium is involved in the reduction of vascular calcification; however, the mechanism underlying this process remains unknown. The aim of the present study was to investigate the effects of magnesium on β-glycerophosphate (β-GP)-induced calcification and the underlying mechanisms. Primary rat vascular smooth muscle cells (VSMCs) were exposed to 10 mM β-GP in medium with or without the addition of 3 mM magnesium or 2-aminoethoxy-diphenylborate (2-APB; an inhibitor of magnesium transport), for a 14-day period. Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, quantification of calcium and enzyme-linked immunosorbent assay. The expression levels of core-binding factor α-1 (Cbfα1), matrix Gla protein (MGP) and osteopontin (OPN) were determined by reverse transcription-polymerase chain reaction or western blot analysis, following incubation for 0, 3, 6, 10 and 14 days with the different media. VSMC calcification and ALP activity was reduced significantly in the high-magnesium medium compared with the calcification medium, during the 14-day incubation. The magnesium-induced changes in the VSMCs included a β-GP-induced downregulation of Cbfα1 by day 3 of incubation, an effect that was gradually enhanced over the 14-day period. By contrast, magnesium produced notable increases in MGP and OPN expression levels, with an opposite pattern to that observed in the Cbfα1 expression levels. However, the addition of 2-APB appeared to inhibit the protective effect of magnesium on the VSMCs. Therefore, magnesium was able to effectively reduce β-GP-induced calcification in rat VSMCs by regulating the expression levels of calcification-associated factors in a time-dependent manner.

  13. Expression of FGF23 is correlated with serum phosphate level in isolated fibrous dysplasia.

    PubMed

    Kobayashi, Keisuke; Imanishi, Yasuo; Koshiyama, Hiroyuki; Miyauchi, Akimitsu; Wakasa, Kenichi; Kawata, Takehisa; Goto, Hitoshi; Ohashi, Hirotsugu; Koyano, Hajime M; Mochizuki, Ryuichi; Miki, Takami; Inaba, Masaaki; Nishizawa, Yoshiki

    2006-04-11

    Fibrous dysplasia (FD) patients sometimes suffer from concomitant hypophosphatemic rickets/osteomalacia, resulting from renal phosphate wasting. It was recently reported that FD tissue in the patients with McCune-Albright syndrome (MAS) expressed fibroblast growth factor-23 (FGF-23), which is now known to be as a pathogenic phosphaturic factor in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets. Since it remains controversial whether serum phosphate levels are influenced by FGF23 expressions in FD tissue, isolated FD patients without MAS syndrome were examined for the relationship between FGF23 expressions, circulating levels of FGF-23 and phosphate to negate the effects of MAS-associated endocrine abnormalities on serum phosphate. Eighteen paraffin embedded FD tissues and 2 frozen tissues were obtained for the study. Sixteen of 18 isolated FD tissues were successfully analyzed GNAS gene, which exhibited activated mutations observed in MAS. Eight of 16 FD tissues, which exhibited GNAS mutations, revealed positive staining for FGF-23. These evidence indicate that postzygotic activated mutations of GNAS is necessary for the FD tissue formation by mosaic distribution of mutated osteogenic cell lineage, but is not sufficient to elevate FGF23 expression causing generalized osteomalacia with severe renal phosphate wasting. The expression level of FGF23 in isolated FD tissue with hypophosphatemic osteomalacia determined by real-time PCR was abundant close to the levels in OOM tumors. Osteoblasts/osteocytes in woven bone were predominant source of circulating FGF-23 in FD tissues by immunohistochemistry. A negative correlation of the intensity of FGF-23 staining with serum inorganic phosphate levels indicated that the expression of FGF23 in focal FD tissues could be a prominent determinant of serum phosphate levels in isolated FD patient. These data provide novel insights into the regulatory mechanism of serum inorganic phosphate levels in

  14. TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins

    PubMed Central

    Prpar Mihevc, S.; Baralle, Marco; Buratti, Emanuele; Rogelj, Boris

    2016-01-01

    TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown. PMID:27665936

  15. Inconformity of CXCL3 Plasma Level and Placenta Expression in Preeclampsia and Its Effect on Trophoblast Viability and Invasion

    PubMed Central

    Jia, Jin; Gong, Yunhui; Gao, Linbo; Zhang, Lin; Zhou, Rong

    2014-01-01

    As a member of the chemokine family, CXCL3 was previously known to participate in many pathophysiological events. However, whether CXCL3 stimulates trophoblast invasion as a key process of preeclampsia pathogenesis remains largely unknown. Therefore, the aim of this study was to investigate this hypothesis and determine the effect of CXCL3 on the first trimester trophoblast. Seventy gravidas were included in this study. ELISA was used to detect CXCL3 plasma levels on preeclampsia and normal pregnant groups. CXCL3 protein and mRNA levels were detected via Western blot and real-time quantitative PCR analysis after immunolocalized in human placenta. Moreover, the CXCL3 function in HTR-8/Svneo was analyzed via WST-1 assay, flow cytometry and invasion test. The plasma CXCL3 level in preeclampsia was significantly higher than that in normal pregnancy. CXCL3 expression was observed in the cytoplasm of placental trophoblasts and vascular endothelium in all groups without significant difference between maternal and fetal sides. In addition, placenta CXCL3 expression in severe preeclampsia was significantly lower than those in normal and mild PE groups. Moreover, exogenous CXCL3 can promote the proliferation and invasion of HTR-8/Svneo; however, its effect on apoptosis remains unclear. In summary, a significant abnormality of plasma CXCL3 level and placental CXCL3 expression was discovered in severe preeclampsia; CXCL3 had a function in trophoblast invasion, which indicated its participation in shallow implantation. Therefore CXCL3 might be involved in severe preeclampsia pathogenesis. PMID:25485631

  16. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  17. Higher-order JWKB expressions for the energy levels and the wavefunction at the origin

    SciTech Connect

    Pasupathy, J.; Singh, V.

    1980-09-01

    An exact quantization condition is derived for the energy levels of a particle in a radial potential assumed finite at the origin. This is used to derive corrections to the semiclassical JWKB quantization condition. The normalization integral of the wavefunction is further related to the energy derivative of wavefunction at origin and use this expression to derive the corrections to the semiclassical JWKB expressions for the wavefunction at origin. An application to upsilon leptonic decay width is also given.

  18. Plasma concentration, genetic variation, and gene expression levels of matrix metalloproteinase 9 in Iranian patients with coronary artery disease

    PubMed Central

    Mahmoodi, Khalil; Kamali, Koorosh; Karami, Elham; Soltanpour, Mohammad Soleiman

    2017-01-01

    Background: Matrix metalloproteinase 9 (MMP9) -1562C>T (rs3918242) polymorphism has been proposed as a risk factor for coronary artery disease (CAD) with conflicting results. The aim of the present study was to investigate the association of -1562C>T genetic polymorphism, gene expression and circulating levels of MMP9 with CAD risk in an Iranian subpopulation in in Zanjan City. Materials and Methods: This was a retrospective case–control study we investigated retrospectively 100 patients with angiographically verified CAD and 100 matched controls. Genotyping of -1562C>T polymorphism was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Gene expression levels and circulating levels of MMP9 was determined by real-time reverse transcription-PCR and enzyme immunoassay method, respectively. Statistical analysis was done using Student's t-test or Chi-square test by SPSS 16 software. Results: The mean circulating levels of MMP9 were significantly higher in CAD Group than control group (P = 0.002). Mean plasma levels of MMP9 were also significantly higher in triple vessel stenosis patients than double vessel or single vessel stenosis patients (P < 0.001). Moreover, mean plasma levels and gene expression levels of MMP9 were significantly higher in T allele carrier than C allele carrier of MMP9 -1562C>T polymorphism (P = 0.002, P = 0.01, respectively). However, genotype and allele frequencies of MMP9 -1562C>T polymorphism were similar between CAD patients and controls (P > 0.05). Additionally, the -1562C>T polymorphism of MMP9 gene didn't increase the risk of CAD in dominant (P = 0.537) or recessive (P = 0.249) genetic models. Conclusion: Our study demonstrated that circulating levels of MMP9 but not -1562C>T polymorphism of MMP9 gene may be a risk factor for development and severity of CAD in an Iranian subpopulation in Zanjan.

  19. An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

    PubMed

    Seo, Jeong-Woo; Hong, Won-kyung; Rairakhwada, Dina; Seo, Pil-Soo; Choi, Min Ho; Song, Ki-Bang; Rhee, Sang-Ki; Kim, Chul Ho

    2009-06-01

    The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.

  20. Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level?

    PubMed

    Qaisar, Rizwan; Renaud, Guillaume; Morine, Kevin; Barton, Elisabeth R; Sweeney, H Lee; Larsson, Lars

    2012-03-01

    Muscle force is typically proportional to muscle size, resulting in constant force normalized to muscle fiber cross-sectional area (specific force). Mice overexpressing insulin-like growth factor-1 (IGF-1) exhibit a proportional gain in muscle force and size, but not the myostatin-deficient mice. In an attempt to explore the role of the cytoplasmic volume supported by individual myonuclei [myonuclear domain (MND) size] on functional capacity of skeletal muscle, we have investigated specific force in relation to MND and the content of the molecular motor protein, myosin, at the single muscle fiber level from myostatin-knockout (Mstn(-/-)) and IGF-1-overexpressing (mIgf1(+/+)) mice. We hypothesize that the addition of extra myonuclei is a prerequisite for maintenance of specific force during muscle hypertrophy. A novel algorithm was used to measure individual MNDs in 3 dimensions along the length of single muscle fibers from the fast-twitch extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles of Mstn(-/-) mice were correlated with the decrement in specific force and myosin content in Mstn(-/-) muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.

  1. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. PMID:23093835

  2. Effects of dietary roughage levels on the expression of adipogenic transcription factors in Wagyu steers.

    PubMed

    Yamada, T; Kawakami, S-I; Nakanishi, N

    2009-12-01

    We hypothesized that dietary roughage level would alter the expression levels of adipogenic transcription factors in adipose tissue of Japanese black (Wagyu) steers. Steers were fed whole crop rice silage at three levels: (1) high-roughage feeding group, fed 8kg silage and 5kg concentrate (HR); (2) middle roughage feeding group, fed 5kg silage and 6kg concentrate (MR); and (3) low roughage feeding group, fed 2kg silage and 7kg concentrate (LR) from 22 to 30months of age. In subcutaneous adipose tissue, there were no significant differences in the expression of the adipogenic transcription factors and adipocyte size among feeding groups. In mesenteric adipose tissue, the expression of C/EBPα in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the mesenteric adipose tissue of the LR group was significantly larger than that of the HR group. In intermuscular adipose tissue, the expression of C/EBPβ-LAP in the LR group was significantly higher than that in the HR group, and the expression of C/EBPβ-LIP in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the intermuscular adipose tissue of the LR and MR groups was significantly smaller than that of the HR group. These results suggest that dietary roughage revel affects the adipose tissue depot-specific differences in C/EBP family expression pattern and adipocyte cellularity in Wagyu steers.

  3. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  4. Tissue Specific Expression Levels of Apoptosis Involved Genes Have Correlations with Codon and Amino Acid Usage

    PubMed Central

    Sadeghi, Iman; Salavaty, Abbas; Nasiri, Habib

    2016-01-01

    Different mechanisms, including transcriptional and post transcriptional processes, regulate tissue specific expression of genes. In this study, we report differences in gene/protein compositional features between apoptosis involved genes selectively expressed in human tissues. We found some correlations between codon/amino acid usage and tissue specific expression level of genes. The findings can be significant for understanding the translational selection on these features. The selection may play an important role in the differentiation of human tissues and can be considered for future studies in diagnosis of some diseases such as cancer. PMID:28154517

  5. Protein pheromone expression levels predict and respond to the formation of social dominance networks

    PubMed Central

    Nelson, Adam C.; Cunningham, Christopher B.; Ruff, James S.; Potts, Wayne K.

    2015-01-01

    Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species. PMID:25867293

  6. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    PubMed

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  7. Addition of ammonia or amino acids to a nitrogen-depleted medium affects gene expression patterns in yeast cells during alcoholic fermentation.

    PubMed

    Jiménez-Martí, Elena; del Olmo, Marcel Lí

    2008-03-01

    Yeast cells require nitrogen and are capable of selectively using good nitrogen sources in preference to poor ones by means of the regulatory mechanism known as nitrogen catabolite repression (NCR). Herein, the effect of ammonia or amino acid addition to nitrogen-depleted medium on global yeast expression patterns in yeast cells was studied using alcoholic fermentation as a system. The results indicate that there is a differential reprogramming of the gene expression depending on the nitrogen source added. Ammonia addition resulted in a higher expression of genes involved in amino acids biosynthesis while amino acid addition prepares the cells for protein biosynthesis. Therefore, a high percentage of the genes regulated by the transcription factors involved in the regulation of amino acid biosynthesis are more expressed during the first hours after ammonia addition compared with amino acid addition. The opposite occurs for those genes regulated by the transcription factor Sfp1p, related to ribosome biosynthesis. Although both additions include rich nitrogen sources, most NCR-regulated genes are more expressed after adding ammonia than amino acids. One of the differentially expressed genes, YBR174W, is required for optimal growth in synthetic medium.

  8. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  9. Effects of phased joint intervention on Rho/ROCK expression levels in patients with portal hypertension

    PubMed Central

    Shi, Min; Wei, Jue; Meng, Wen-Ying; Wang, Na; Wang, Ting; Wang, Yu-Gang

    2016-01-01

    The current study investigated the effects of phased joint intervention on clinical efficacy and Rho/Rho-associated coil protein kinase (ROCK) expression in patients with portal hypertension complicated by esophageal variceal bleeding (EVB) and hypersplenism. Patients with portal hypertension (n=53) caused by liver cirrhosis complicated by EVB and hypersplenism treated with phased joint intervention were assessed, and portal hemodynamics, blood, liver function, complications, and rebleeding incidence were analyzed. Reverse transcription-quantitative polymerase chain reaction was used to measure Rho, ROCK1 and ROCK2 mRNA expression levels in peripheral blood mononuclear cells prior to and following phased joint intervention, and western blotting was employed to determine the protein expression levels of Rho, ROCK1, ROCK2, phosphorylated (p) myosin phosphatase target subunit 1 (MYPT1) and total-MYPT1. All patients underwent an emergency assessment of hemostasis with a 100% success rate. Varicose veins were alleviated, and portal hemodynamics and liver function improved following intervention. Furthermore, preoperative and postoperative expression levels of Rho, ROCK1 and ROCK2 mRNA were higher compared with the control group. Notably, the mRNA expression levels of Rho, ROCK1 and ROCK2 in the postoperative group were significantly lower when compared with the preoperative group. Protein expression levels of Rho, ROCK1, ROCK2 and pMYPT1 in the postoperative group were lower, as compared with the preoperative group. Concentration levels of transforming growth factor-β1, connective tissue growth factor and platelet-derived growth factor in peripheral blood were significantly reduced following phased joint intervention. Therefore, the present findings demonstrated that phased joint intervention is able to effectively treat EVB and hypersplenism, and improve liver function. The efficacy of phased joint intervention may be associated with its role in the regulation of the

  10. FMRP Expression Levels in Mouse Central Nervous System Neurons Determine Behavioral Phenotype

    PubMed Central

    Arsenault, Jason; Gholizadeh, Shervin; Niibori, Yosuke; Pacey, Laura K.; Halder, Sebok K.; Koxhioni, Enea; Konno, Ayumu; Hirai, Hirokazu; Hampson, David R.

    2016-01-01

    Fragile X mental retardation protein (FMRP) is absent or highly reduced in Fragile X Syndrome, a genetic disorder causing cognitive impairment and autistic behaviors. Previous proof-of-principle studies have demonstrated that restoring FMRP in the brain using viral vectors can improve pathological abnormalities in mouse models of fragile X. However, unlike small molecule drugs where the dose can readily be adjusted during treatment, viral vector–based biological therapeutic drugs present challenges in terms of achieving optimal dosing and expression levels. The objective of this study was to investigate the consequences of expressing varying levels of FMRP selectively in neurons of Fmr1 knockout and wild-type (WT) mice. A wide range of neuronal FMRP transgene levels was achieved in individual mice after intra-cerebroventricular administration of adeno-associated viral vectors coding for FMRP. In all treated knockout mice, prominent FMRP transgene expression was observed in forebrain structures, whereas lower levels were present in more caudal regions of the brain. Reduced levels of the synaptic protein PSD-95, elevated levels of the transcriptional modulator MeCP2, and abnormal motor activity, anxiety, and acoustic startle responses in Fmr1 knockout mice were fully or partially rescued after expression of FMRP at about 35–115% of WT expression, depending on the brain region examined. In the WT mouse, moderate FMRP over-expression of up to about twofold had little or no effect on PSD-95 and MeCP2 levels or on behavioral endophenotypes. In contrast, excessive over-expression in the Fmr1 knockout mouse forebrain (approximately 2.5–6-fold over WT) induced pathological motor hyperactivity and suppressed the startle response relative to WT mice. These results delineate a range of FMRP expression levels in the central nervous system that confer phenotypic improvement in fragile X mice. Collectively, these findings are pertinent to the development of long

  11. Relationship between TLR4 and MCP2 expression levels and habitual abortion.

    PubMed

    Li, X P; Song, L N; Tian, L P; Zhang, Y S

    2016-04-25

    Habitual abortion is associated with the altered expression of multiple genes. This study was carried out to investigate the relationship between expression of Toll-like receptor 4 (TLR4) and monocyte chemotactic protein 2 (MCP2 or CCL8) and habitual abortion. This was done by detecting and comparing their relative expression in peripheral blood and placental villi of patients and healthy fertile women. Based on our previous research, 85 subjects with habitual abortion (study group) and 40 healthy fertile women (control group), who were admitted to our hospital between June 2013 and December 2014, were enrolled in this study. After these subjects signed written informed consent, peripheral blood samples and villous tissues were collected, from which the total RNA was extracted. The expression of TLR4 and MCP2 was detected with quantitative reverse transcription-polymerase chain reaction, using GAPDH as a reference control. The expression of TLR4 and MCP2 in the peripheral blood and villous tissues of the study group was significantly higher than that of the control group (P < 0.05). A positive correlation was also observed between the changes in expression levels of TLR4 and MCP2. In conclusion, TLR4 and MCP2 expression correlated with the occurrence of habitual abortion. Detecting expression changes in TLR4 and MCP2 in the peripheral blood is a feasible method for predicting the occurrence of abortion in women of child-bearing age.

  12. Serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms.

    PubMed

    Liu, Jia; Chen, Guang; Meng, Xian-Ying; Liu, Zhong-Hui; Dong, Su

    2014-12-01

    Previous studies have demonstrated the expression of estrogen receptor (ER) and progesterone receptor (PR) in thyroid cancer; however, little is known regarding the levels of estrogen, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in serum and the expression of ER, PR, FSH receptor (FSHR), and LH receptor (LHR) in thyroid tissues of patients with different types of thyroid neoplasms. Serum levels of estrogen, progesterone, FSH, and LH were measured by chemiluminescence, and expression of ER, PR, FSHR, and LHR in thyroid tissue was detected by immunohistochemistry in female patients with thyroid adenoma (n = 70), nodular goiter (n = 73), thyroid papillary cancer (n = 149), poorly differentiated thyroid carcinoma (n = 12), or undifferentiated thyroid carcinoma (n = 8) and in normal controls (n = 60). The positive rates of serum estrogen level and ERα expression were significantly greater in patients with various types of thyroid neoplasms than in normal controls. The positive rates of ERβ expression were significantly less in various types of thyroid neoplasms than in normal thyroid tissues, especially in poorly differentiated carcinoma and undifferentiated carcinoma. The negative rates of serum progesterone level and positive rates of PR expression in thyroid tissue were significantly greater in patients with thyroid adenoma, nodular goiter, or thyroid papillary cancer than in normal controls. The positive rates of serum FSH and LH levels and FSHR and LHR expression were significantly greater in the thyroid adenoma group than in other groups. Our findings suggest that thyroid neoplasms might be sex hormone-dependent. The positive expression of ERα and PR often indicates thyroid papillary carcinoma, and the ERβ expression status is important for the diagnosis of poorly differentiated carcinoma and undifferentiated carcinoma. In addition, thyroid adenoma is often accompanied by an increase in serum FSH and LH levels, as well as

  13. Microfluidic continuum sorting of sub-populations of tumor cells via surface antibody expression levels.

    PubMed

    Jack, Rhonda; Hussain, Khadijah; Rodrigues, Danika; Zeinali, Mina; Azizi, Ebrahim; Wicha, Max; Simeone, Diane M; Nagrath, Sunitha

    2017-03-29

    The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

  14. Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer.

    PubMed

    Eftekhar, Ebrahim; Naghibalhossaini, Fakhraddin

    2014-01-01

    Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.

  15. High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

    PubMed

    Wu, Tzong-Yuan; Chen, Hsin-An; Li, Feng-Yin; Lin, Ching-Ting; Wu, Chi-Ming; Hsieh, Feng-Chia; Tzen, Jason Tze-Cheng; Hsieh, Sheng-Kuo; Ko, Jiunn-Liang; Jinn, Tzyy-Rong

    2013-02-01

    Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

  16. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  17. Correlation of Cytohistlogical Expression and Serum Level of Ca125 in Ovarian Neoplasm

    PubMed Central

    Das, Chhanda; Mukhopadhyay, Madhumita; Ghosh, Tarun; Saha, Ashis Kumar; Sengupta, Moumita

    2014-01-01

    Context or Background: CA125 is a biomarker that has potential utility across the spectrum: risk assessment, early detection, diagnosis, prognosis, monitoring and therapy. Aims and Objectives: This study was conducted to establish the validity and reliability of correlation of CA125 serum level with immunochemistry expression in imprint cytology and tissues for diagnostic purpose. Materials and Methods: A prospective study was done on 50 cases of clinically and radiologically diagnosed ovarian tumor. Imprint smears were made intraoperatively from fresh samples and stained with M.G.G. stain for air dried smears and Papanicoloau stain for alcohol fixed smears. Stained smear was assessed and compared with subsequent histopathology report. Preoperative blood samples were obtained from all patients and sent for the assay of serum CA125 levels. Analysis of CA125 immunochemistry expression in imprint cytology and tissue was done and correlated with preoperative serum blood CA125 levels. Results: Significant positive correlation was found between elevated serum CA125 levels and cytohistological expression of CA125. Overall sensitivity was 100%, specificity was 86%, positive predictive value was 74% and negative predictive value 100%. Diagnostic accuracy was 90% with high statistical significance (p<0.001). Conclusion: We considered 35 U/mL as the cut-off value when evaluating serum CA125 ovarian cancer. Patients with high serum levels show good cytohistological expression. PMID:24783076

  18. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

    PubMed Central

    2013-01-01

    Background In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. PMID:23497240

  19. Additive effects of low-level laser therapy with exercise on subacromial syndrome: a randomised, double-blind, controlled trial.

    PubMed

    Abrisham, Seyyed Mohammad Jalil; Kermani-Alghoraishi, Mohammad; Ghahramani, Rahil; Jabbari, Latife; Jomeh, Hossein; Zare, Maryam

    2011-10-01

    The subacromial syndrome is the most common source of shoulder pain. The mainstays of conservative treatment are non-steroidal anti-inflammatory drugs and exercise therapy. Recently, low-level laser therapy (LLLT) has been popularized in the treatment of various musculoskeletal disorders. The aim of this study is to evaluate the additive effects of LLLT with exercise in comparison with exercise therapy alone in treatment of the subacromial syndrome. We conducted a randomised clinical study of 80 patients who presented to clinic with subacromial syndrome (rotator cuff and biceps tendinitis). Patients were randomly allocated into two groups. In group I (n = 40), patients were given laser treatment (pulsed infrared laser) and exercise therapy for ten sessions during a period of 2 weeks. In group II (n = 40), placebo laser and the same exercise therapy were given for the same period. Patients were evaluated for the pain with visual analogue scale (VAS) and shoulder range of motion (ROM) in an active and passive movement of flexion, abduction and external rotation before and after treatment. In both groups, significant post-treatment improvements were achieved in all parameters (P = 0.00). In comparison between the two groups, a significant improvement was noted in all movements in group I (P = 0.00). Also, there was a substantial difference between the groups in VAS scores (P = 0.00) which showed significant pain reduction in group I. This study indicates that LLLT combined exercise is more effective than exercise therapy alone in relieving pain and in improving the shoulder ROM in patients with subacromial syndrome.

  20. Formation Of Transparent Exopolymeric Particles (TEP) In Mesocosms Under Increasing Turbulence Levels With And Without Additional Nutrients.

    NASA Astrophysics Data System (ADS)

    Beauvais, S.; Pedrotti, Ml.

    Transparent Exopolymeric Particles (TEP) are formed abiotically by spontaneous co- agulation of the colloidal fraction of dissolved organic polysaccharides released via phytoplankton and bacteria exudation. Their importance in the vertical fluxes of or- ganic matter in coastal and pelagic ecosystems is now well recognised. However, their production as a function of the environment features has yet to be investigated. Evolu- tion of TEP formation was followed during a two week mesocosm experiment under 4 several turbulence levels and with or without added nutrients. This study was per- formed in the framework of EC-ELOISE-NATP project. The results showed a rapid formation of TEP 24h after the phytoplankton bloom occurred. This suggests that TEP consist of fresh organic material, derived from biological process, such as phyto- plankton blooms. Their abundance increased with time in mesocosms with additional nutrients indicating that phytoplankton cells were actively exuding the precursors. The C/N ratio of particualte organic matter (POM) in mesocosms enriched with nutrients was highly correlated with TEP abundance, it confirms that TEP can have a strong impact on the biogeochemical fluxes in oceans, in particular on the carbon cycle. TEP were more abundant in the mesocosms with the highest turbulence intensity. It sug- gests that the effect of turbulence could promote encounter rates between particles increasing coagulation processes. Even if biological processes are of primary impor- tance in TEP production, this study also highlights the role of physical processes in their formation dynamics. The role of TEP in the microbial food web will be also discussed.

  1. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.

    PubMed

    Roney, Ian J; Rudner, Adam D; Couture, Jean-François; Kærn, Mads

    2016-06-21

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.

  2. HER-2 tissue expression correlated with serum levels in breast cancer patients.

    PubMed

    Pribylová, O; Springer, D; Vítková, I; Zima, T; Petruzelka, L

    2007-01-01

    We explored the relationship between circulating HER-2 extracellular domain and tissue HER-2 status in a group of 42 postmenopausal breast cancer patients. All patients were examined before adjuvant chemotherapy or other adjuvant treatment. Serum levels were measured by BAYER Advia Centaur System, Golden, CO (the cut-off level was in our conditions considered at 12 ng/ml). Tissue expression was assayed with the DAKO HercepTest, North America, Inc, Carpinteria, CA. Our findings that serum levels are in consonance with tissue expression could be important in metastatic breast cancer, when it is impossible to get a new tumour sample and establish the actual HER-2 status, which may be different from the primary tumour. Although we know that serum HER-2 concentration cannot be substituted for IHC or FISH, we have observed a statistically significant correlation between serum level concentration and tissue HER-2 status.

  3. Changes in microRNA expression levels correlate with clinicopathological features and prognoses in endometrial serous adenocarcinomas.

    PubMed

    Hiroki, Eri; Akahira, Jun-Ichi; Suzuki, Fumihiko; Nagase, Satoru; Ito, Kiyoshi; Suzuki, Takashi; Sasano, Hironobu; Yaegashi, Nobuo

    2010-01-01

    This study aimed to determine the expression profiles of microRNAs (miRNAs) in endometrial serous adenocarcinoma and to examine the association between miRNA expression and clinical outcomes. Twenty-one patients diagnosed with endometrial serous adenocarcinoma between January 2001 and December 2006 were enrolled. miRNA expression profiles were examined using miRNA microarray and qRT-PCR. miRNA expression levels were correlated with clinicopathological variables and survival rates. A total of 120 miRNAs were differentially expressed in endometrial serous adenocarcinoma compared to normal endometria. Of these, 54 miRNAs were down-regulated (>2-fold), including miR-101, miR-10b*, miR-152, and miR-29b, and the remainder were up-regulated (>2-fold), including miR-200a, miR-200b, and miR-205. Decreased expression of miR-10b*, miR-29b, and miR-455-5p was correlated with vascular invasion (P = 0.048, P = 0.013, and P = 0.032, respectively). Univariate analysis revealed that lower expression of miR-101, miR-10b*, miR-139-5p, miR-152, miR-29b, and miR-455-5p was significantly correlated with poor overall survival (P < 0.05), and reduced expression of miR-152, miR-29b, and miR-455-5p was significantly correlated with poor disease-free survival (P < 0.05). Multivariate analysis demonstrated that decreased expression of miR-152 (P = 0.021) was a statistically independent risk factor for overall survival, and decreased expression levels of miR-101 (P = 0.016) and miR-152 (P = 0.010) were statistically independent risk factors for disease-free survival. In addition, transfection of miR-101 or miR-152 precursors into an endometrial serous carcinoma cell line inhibited cell growth (P < 0.0001 and P = 0.01, respectively). Moreover, strong positive immunoreactivity of cyclooxygenase-2 (COX-2) was significantly correlated with down-regulation of miR-101 (P = 0.035). These findings suggest that the dysregulation of miRNAs is associated with the poor prognosis in endometrial serous

  4. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

  5. Novel Bifidobacterium promoters selected through microarray analysis lead to constitutive high-level gene expression.

    PubMed

    Wang, Yan; Kim, Jin Yong; Park, Myeong Soo; Ji, Geun Eog

    2012-08-01

    For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

  6. Exercise-induced increase in IL-6 level enhances GLUT4 expression and insulin sensitivity in mouse skeletal muscle.

    PubMed

    Ikeda, Shin-Ichi; Tamura, Yoshifumi; Kakehi, Saori; Sanada, Hiromi; Kawamori, Ryuzo; Watada, Hirotaka

    2016-05-13

    A single bout of exercise is known to increase the insulin sensitivity of skeletal muscle; however, the underlying mechanism of this phenomenon is not fully understood. Because a single bout of exercise induces a transient increase in blood interleukin-6 (IL-6) level, we hypothesized that the enhancement of insulin sensitivity after a single bout of exercise in skeletal muscle is mediated at least in part through IL-6-dependent mechanisms. To test this hypothesis, C57BL6J mice were intravenously injected with normal IgG or an IL-6 neutralizing antibody before exercise. Twenty-four hours after a single bout of exercise, the plantaris muscle was harvested to measure insulin sensitivity and glucose transporter (GLUT)-4 expression levels by ex-vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake and Western blotting, respectively. Compared with sedentary mice, mice that performed exercise showed enhanced IL-6 concentration, insulin-stimulated 2-DG uptake, and GLUT-4 expression in the plantaris muscle. The enhanced insulin sensitivity and GLUT4 expression were canceled by injection of the IL-6 neutralizing antibody before exercise. In addition, IL-6 injection increased GLUT4 expression, both in the plantaris muscle and the soleus muscle in C57BL6J mice. Furthermore, a short period of incubation with IL-6 increased GLUT4 expression in differentiated C2C12 myotubes. In summary, these results suggested that IL-6 increased GLUT4 expression in muscle and that this phenomenon may play a role in the post-exercise enhancement of insulin sensitivity in skeletal muscle.

  7. Ecdysteroids Regulate the Levels of Molt-Inhibiting Hormone (MIH) Expression in the Blue Crab, Callinectes sapidus

    PubMed Central

    Techa, Sirinart; Chung, J. Sook

    2015-01-01

    Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle. PMID:25849453

  8. Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

    PubMed

    Techa, Sirinart; Chung, J Sook

    2015-01-01

    Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

  9. Impact of probiotic-supplemented diet on the expression level of lactate dehydrogenase in the leukocytes of rabbits.

    PubMed

    Ghoneim, Magdy A E; Moselhy, Said S

    2014-04-01

    Probiotics are known as living, nonpathogenic microorganisms that colonize the intestine and provide benefit to the host. The present study aims to measure one important energy metabolism-related enzyme activity in blood of rabbits fed on probiotics of recommended concentration. In addition, it also aims for the evaluation of the expression level of lactate dehydrogenase (LDH) enzyme using reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Two groups of rabbits are used: control group receiving normal standardized diet and the other probiotic-supplemented group receiving the same diet containing probiotic, namely, Mega acidophilus (200 million cfu/kg body weight/day) for 4 weeks. The obtained results revealed that the rabbits supplemented with probiotics showed a significant decrease in the levels of serum total cholesterol (TC), triacylglycerol, high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) when compared with control group. Risk factors detected by measuring TC/HDL-c and LDL-c/HDL-c ratios showed statistically significant decrease in probiotic-supplemented rabbits when compared with control group. In addition, blood glucose and total LDH activity were elevated in probiotic-supplemented rabbits when compared with control group. RT-PCR products of LDH-M gene produced two specific amplicons. One amplicon has the expected size of 243 bp from all samples of rabbits as revealed by GelPro software. The level of LDH-M expression was found to be increased in the probiotic-supplemented group. However, unexpected amplicons are produced at 586 bp in all the samples, which may be a dimeric form of the amplified region. It was concluded that this probiotic blend is beneficiary for the metabolic reactions of lipids in the body. Moreover, LDH expression level can be considered as a biomarker for the effect of probiotic and hence monitoring the metabolic changes as reflected from its administration.

  10. Quercetin promotes neurite growth through enhancing intracellular cAMP level and GAP-43 expression.

    PubMed

    Chen, Ming-Ming; Yin, Zhi-Qi; Zhang, Lu-Yong; Liao, Hong

    2015-09-01

    The present study was designed to investigate the role of quercetin on neurite growth in N1E-115 cells and the underlying mechanisms. Quercetin was evaluated for its effects on cell numbers of neurites, neurite length, intracellular cAMP content, and Gap-43 expression in N1E-115 cells in vitro by use of microscopy, LANCE(tm) cAMP 384 kit, and Western blot analysis, respectively. Our results showed that quercetin could increase the neurite length in a concentration-dependent manner, but had no effect on the numbers of cells. Quercetin significantly increased the expression of cellular cAMP in a time- and concentration-dependent manner. The Gap-43 expression was up-regulated in a time-dependent manner. In conclusion, quercetin could promote neurite growth through increasing the intracellular cAMP level and Gap-43 expression.

  11. Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels.

    PubMed

    Cervigni, Gerardo D L; Paniego, Norma; Pessino, Silvina; Selva, Juan P; Díaz, Marina; Spangenberg, Germán; Echenique, Viviana

    2008-05-01

    The molecular nature of gene expression during the initiation and progress of diplosporous apomixis is still unknown. Moreover, the basis of the close correlation between diplospory and polyploidy is not clarified yet. A comparative expression analysis was performed based on expressed sequence tags (ESTs) sequencing and differential display in an Eragrostis curvula diplosporous tetraploid genotype (T, 4x apo), a sexual diploid derivative obtained from tissue culture (D, 2x sex) and an artificial sexual tetraploid obtained from the diploid seeds after colchicine treatment (C, 4x sex). From a total of 8,884 unigenes sequenced from inflorescence-derived libraries, 112 (1.26%) showed significant differential expression in individuals with different ploidy level and/or variable reproductive mode. Independent comparisons between plants with different reproductive mode (same ploidy) or different ploidy level (same reproductive mode) allowed the identification of genes modulated in response to diplosporous development or polyploidization, respectively. Surprisingly, a group of genes (Group 3) were differentially expressed or silenced only in the 4x sex plant, presenting similar levels of expression in the 4x apo and the 2x sex genotypes. A group of randomly selected differential genes was validated by QR-PCR. Differential display analysis showed that in general the 4x apo and 4x sex expression profiles were more related and different from the 2x sex one, but confirmed the existence of Group 3-type genes, in both inflorescences and leaves. The possible biological significance for the occurrence of this particular group of genes is discussed. In silico mapping onto the rice genome was used to identify candidates mapping to the region syntenic to the diplospory locus.

  12. N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

    PubMed

    Haug, Jeffrey S; He, Xi C; Grindley, Justin C; Wunderlich, Joshua P; Gaudenz, Karin; Ross, Jason T; Paulson, Ariel; Wagner, Kathryn P; Xie, Yucai; Zhu, Ruihong; Yin, Tong; Perry, John M; Hembree, Mark J; Redenbaugh, Erin P; Radice, Glenn L; Seidel, Christopher; Li, Linheng

    2008-04-10

    Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

  13. Elevated interleukin-6 expression levels are associated with intervertebral disc degeneration

    PubMed Central

    DENG, XIAO; ZHAO, FENG; KANG, BAOLIN; ZHANG, XIN

    2016-01-01

    The present study aimed to investigate whether serum interleukin-6 (IL-6) expression levels were associated with the onset and progression of intervertebral disc degeneration (IDD). A comprehensive meta-analysis of the scientific literature from numerous electronic databases was performed, in order to obtain published studies associated with the topic of interest. Relevant case-control studies that had previously assessed a correlation between IL-6 expression levels and IDD were identified using predetermined inclusion and exclusion criteria. The STATA version 12.0 software was used for statistical analysis of the extracted data. A total of 112 studies were initially retrieved, with eight studies meeting the inclusion criteria. These contained a total of 392 subjects, of which 263 were patients with IDD and 129 were healthy controls. A meta-analysis of the eight studies demonstrated that serum IL-6 protein expression levels may be associated with IDD, and this was irrespective of IDD subtype (bulging, protrusion, or sequestration). Notably, serum expression levels of the IL-6 protein were upregulated in intervertebral disc (IVD) protrusion tissue, as compared with normal IVD tissue; thus suggesting that IL-6 may have an important role in the pathophysiological process of IDD. PMID:27073460

  14. Examination of Anxiety Levels and Anger Expression Manners of Undergraduate Table Tennis Players

    ERIC Educational Resources Information Center

    Karademir, Tamer; Türkçapar, Ünal

    2016-01-01

    This research was done for the determination of how their anxiety levels' and anger expressions' get shaped according to some variances. For this reason there were 76 female 125 male totally 201 sportsmen, who participated to the table tennis championship between universities in 2016 and ages differ from 18 to 28, were included the research group.…

  15. High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals.

    PubMed

    Hirashima, Masaki; Imamura, Takayuki; Yano, Kentaro; Kawamura, Ryoichi; Meta, Akihiro; Tokieda, Yoshiyuki; Nakashima, Toshihiro

    2016-02-01

    Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, β and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, β and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.

  16. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  17. Is Nasal Polyposis Related to Levels of Serum Vitamin D and Vitamin D Receptor Gene Expression?

    PubMed Central

    Erdag, Omer; Turan, Mahfuz; Ucler, Rıfkı; Berkoz, Mehmet; Garca, Mehmet Fatih; Bozan, Nazım; Kıroglu, Ahmet Faruk; Cankaya, Hakan

    2016-01-01

    Background Nasal polyposis (NP) is the most frequent cause of nasal masses. Despite considerable research on the subject, its etiology has not been fully elucidated, and effective treatment methods have not been developed. Some etiological factors causing low or high expression of genes in genetically predisposed individuals may play a role in the pathogenesis of the disease. The purpose of this study was to assess the relation between levels of vitamin D receptor (VDR) gene expression and serum vitamin D with NP. Material/Methods The study included 46 subjects with NP (NP group) and 40 volunteers (control group). Nasal polyp tissue samples were taken from the NP group and nasal mucosa samples were taken from the control group. Levels of VDR gene expression in the tissue samples were assessed using the real-time polymerase chain reaction (RT-PCR) method. Results Mean serum 25(OH)D levels were 13.38±14.08 ng/ml in the NP group and 10.57±6.44 ng/ml in the control group (p=0.249). VDR gene expression was present in 17.5% of the NP group and 3.3% of the control group, and the difference between the 2 groups was statistically significant (likelihood ratio χ2=3.887; p=0.049). Conclusions This is the first study to assess levels of VDR gene expression in subjects with NP. Our results suggest that VDR gene expression may be associated with the pathogenesis or progression of NP. PMID:27895321

  18. Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

    PubMed

    Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

    2012-01-06

    The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

  19. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels.

  20. Expression of heat shock protein 70 is altered by age and diet at the level of transcription.

    PubMed Central

    Heydari, A R; Wu, B; Takahashi, R; Strong, R; Richardson, A

    1993-01-01

    factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes. Images PMID:7682654

  1. ClC-7 expression levels critically regulate bone turnover, but not gastric acid secretion.

    PubMed

    Supanchart, C; Wartosch, L; Schlack, C; Kühnisch, J; Felsenberg, D; Fuhrmann, J C; de Vernejoul, M-C; Jentsch, T J; Kornak, U

    2014-01-01

    Mutations in the 2Cl(-)/1H(+)-exchanger ClC-7 impair osteoclast function and cause different types of osteoclast-rich osteopetrosis. However, it is unknown to what extent ClC-7 function has to be reduced to become rate-limiting for bone resorption. In osteoclasts from osteopetrosis patients expression of the mutated ClC-7 protein did not correlate with disease severity and resorption impairment. Therefore, a series of transgenic mice expressing ClC-7 in osteoclasts at different levels was generated. Crossing of these mice with Clcn7(-/-) mutants rescued the osteopetrotic phenotype to variable degrees. One resulting double transgenic line mimicked human autosomal dominant osteopetrosis. The trabecular bone of these mice showed a reduction of osteoblast numbers, osteoid, and osteoblast marker gene expression indicative of reduced osteoblast function. In osteoclasts from these mutants ClC-7 expression levels were 20 to 30% of wildtype levels. These reduced levels not only impaired resorptive activity, but also increased numbers, size and nucleus numbers of osteoclasts differentiated in vitro. Although ClC-7 was expressed in the stomach and PTH levels were high in Clcn7(-/-) mutants loss of ClC-7 did not entail a relevant elevation of gastric pH. In conclusion, we show that in our model a reduction of ClC-7 function by approximately 70% is sufficient to increase bone mass, but does not necessarily enhance bone formation. ClC-7 does not appear to be crucially involved in gastric acid secretion, which explains the absence of an osteopetrorickets phenotype in CLCN7-related osteopetrosis.

  2. Vascular endothelial growth factor receptor 2 gene (KDR) polymorphisms and expression levels in depressive disorder.

    PubMed

    Gałecki, Piotr; Orzechowska, Agata; Berent, Dominika; Talarowska, Monika; Bobińska, Kinga; Gałecka, Elżbieta; Lewiński, Andrzej; Maes, Michael; Szemraj, Janusz

    2013-05-01

    Recent research findings suggest that vascular endothelial growth factor (VEGF) participates in the development of depressive disorder. VEGF is involved in neurogenesis and neuroprotection processes, mediated by vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 also plays a role in angiogenesis, a process related to neurogenesis and other biological processes. We examined VEGFR2 (KDR) gene polymorphism, mRNA expression levels, as well as VEGFR2 protein levels in 268 patients diagnosed with a recurrent depressive disorder (rDD) using the ICD-10 criteria, and in 200 healthy controls. Genotyping and gene expression level analysis was performed using polymerase chain reaction (PCR)-based methods. An Enzyme-Linked Immunosorbent Assay (ELISA) was used for measurement of KDR protein levels. Our study found that distribution of KDR polymorphism +1416T/A differs significantly in patients with rDD when compared to healthy subjects, while A allele and AA genotype are risk factors for rDD. KDR mRNA and protein expression are higher in patients with rDD. We also observed a significant association between the -271A/G variant and gene and protein levels. Our study is the first to demonstrate that the KDR gene may serve as a novel genetic marker that could participate in the etiology of rDD. This new pathway may play a role in the inflammatory pathophysiology of depression.

  3. Analysis of gene expression levels in individual bacterial cells without image segmentation

    SciTech Connect

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  4. High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

    PubMed

    Verma, Hemant Kumar; Shukla, Poonam; Alfatah, Md; Khare, Asheesh Kumar; Upadhyay, Udita; Ganesan, Kaliannan; Singh, Jagmohan

    2014-01-01

    Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.

  5. Comparison of microRNA expression levels between initial and recurrent glioblastoma specimens.

    PubMed

    Ilhan-Mutlu, Aysegül; Wöhrer, Adelheid; Berghoff, Anna Sophie; Widhalm, Georg; Marosi, Christine; Wagner, Ludwig; Preusser, Matthias

    2013-05-01

    Glioblastoma is the most frequent primary brain tumour in adults. Recent therapeutic advances increased patient's survival, but tumour recurrence inevitably occurs. The pathobiological mechanisms involved in glioblastoma recurrence are still unclear. MicroRNAs are small RNAs proposed o have important roles for cancer including proliferation, aggressiveness and metastases development. There exist only few data on the involvement of microRNAs in glioblastoma recurrence. We selected the following 7 microRNAs with potential relevance for glioblastoma pathobiology by means of a comprehensive literature search: microRNA-10b, microRNA-21, microRNA-181b, microRNA-181c, microRNA-195, microRNA-221 and microRNA-222. We further selected 15 primary glioblastoma patients, of whom formalin fixed and paraffin embedded tissue (FFPE) of the initial and recurrence surgery were available. All patients had received first line treatment consisting of postoperative combined radiochemotherapy with temozolomide (n = 15). Non-neoplastic brain tissue samples from 3 patients with temporal lobe epilepsy served as control. The expression of the microRNAs were analysed by RT-qPCR. These were correlated with each other and with clinical parameters. All microRNAs showed detectable levels of expressions in glioblastoma group, whereas microRNA-10b was not detectable in epilepsy patients. MicroRNAs except microRNA-21 showed significantly higher levels in epilepsy patients when compared to the levels of first resection of glioblastoma. Comparison of microRNA levels between first and second resections revealed no significant change. Cox regression analyses showed no significant association of microRNA expression levels in the tumor tissue with progression free survival times. Expression levels of microRNA-10b, microRNA-21, microRNA-181b, microRNA-181c, microRNA-195, microRNA-221 and microRNA-222 do not differ significantly between initial and recurrent glioblastoma.

  6. Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation.

    PubMed

    Connolly, Erin L; Fett, Janette P; Guerinot, Mary Lou

    2002-06-01

    Iron, an essential nutrient, is not readily available to plants because of its low solubility. In addition, iron is toxic in excess, catalyzing the formation of hydroxyl radicals that can damage cellular constituents. Consequently, plants must carefully regulate iron uptake so that iron homeostasis is maintained. The Arabidopsis IRT1 gene is the major transporter responsible for high-affinity iron uptake from the soil. Here, we show that the steady state level of IRT1 mRNA was induced within 24 h after transfer of plants to iron-deficient conditions, with protein levels peaking 72 h after transfer. IRT1 mRNA and protein were undetectable 12 h after plants were shifted back to iron-sufficient conditions. Overexpression of IRT1 did not confer dominant gain-of-function enhancement of metal uptake. Analysis of 35S-IRT1 transgenic plants revealed that although IRT1 mRNA was expressed constitutively in these plants, IRT1 protein was present only in the roots when iron is limiting. Under these conditions, plants that overexpressed IRT1 accumulated higher levels of cadmium and zinc than wild-type plants, indicating that IRT1 is responsible for the uptake of these metals and that IRT1 protein levels are indeed increased in these plants. Our results suggest that the expression of IRT1 is controlled by two distinct mechanisms that provide an effective means of regulating metal transport in response to changing environmental conditions.

  7. Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth, respiration and carbon metabolism.

    PubMed

    Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean

    2017-03-01

    Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O2 uptake, flux of carbon between sucrose and CO2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways.

  8. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    PubMed Central

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  9. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    PubMed

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

  10. Androgen Receptors in a Cichlid Fish, Astatotilapia burtoni: Structure, Localization, and Expression Levels

    PubMed Central

    HARBOTT, LENE K.; BURMEISTER, SABRINA S.; WHITE, RICHARD B.; VAGELL, MIKE; FERNALD, RUSSELL D.

    2009-01-01

    Androgens are an important output of the hypothalamic-pituitary-gonadal (HPG) axis that controls reproduction in all vertebrates. In male teleosts two androgens, testosterone and 11-ketotestosterone, control sexual differentiation and development in juveniles and reproductive behavior in adults. Androgenic signals provide feedback at many levels of the HPG axis, including the hypothalamic neurons that synthesize and release gonadotropin-releasing hormone 1 (GnRH1), but the precise cellular site of androgen action in the brain is not known. Here we describe two androgen receptor subtypes, ARα and ARβ, in the cichlid Astatotilapia burtoni and show that these subtypes are differentially located throughout the adult brain in nuclei known to function in the control of reproduction. ARα was expressed in the ventral part of the ventral telencephalon, the preoptic area (POA) of the hypothalamus and the ventral hypothalamus, whereas ARβ was more widely expressed in the dorsal and ventral telencephalon, the POA, and the ventral and dorsal hypothalamus. We provide the first evidence in any vertebrate that the GnRH1-releasing neurons, which serve as the central control point of the HPG axis, express both subtypes of AR. Using quantitative real-time PCR, we show that A. burtoni AR subtypes have different expression levels in adult tissue, with ARα showing significantly higher expression than ARβ in the pituitary, and ARβ expressed at a higher level than ARα in the anterior and middle brain. These data provide important insight into the role of androgens in regulating the vertebrate reproductive axis. PMID:17614300

  11. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato.

    PubMed

    Haroldsen, Victor M; Chi-Ham, Cecilia L; Kulkarni, Shashank; Lorence, Argelia; Bennett, Alan B

    2011-10-01

    Vitamin C (L-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 μmol m(-2) s(-1)). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato.

  12. Experimental Hyperthyroidism Decreases Gene Expression and Serum Levels of Adipokines in Obesity

    PubMed Central

    Luvizotto, Renata de Azevedo Melo; do Nascimento, André Ferreira; de Síbio, Maria Teresa; Olímpio, Regiane Marques Castro; Conde, Sandro José; Lima-Leopoldo, Ana Paula; Leopoldo, André Soares; Cicogna, Antonio Carlos; Nogueira, Célia Regina

    2012-01-01

    Aims. To analyze the influence of hyperthyroidism on the gene expression and serum concentration of leptin, resistin, and adiponectin in obese animals. Main Methods. Male Wistar rats were randomly divided into two groups: control (C)—fed with commercial chow ad libitum—and obese (OB)—fed with a hypercaloric diet. After group characterization, the OB rats continued receiving a hypercaloric diet and were randomized into two groups: obese animals (OB) and obese with 25 μg triiodothyronine (T3)/100 BW (OT). The T3 dose was administered every day for the last 2 weeks of the study. After 30 weeks the animals were euthanized. Samples of blood and adipose tissue were collected for biochemical and hormonal analyses as well as gene expression of leptin, resistin, and adiponectin. Results. T3 treatment was effective, increasing fT3 levels and decreasing fT4 and TSH serum concentration. Administration of T3 promotes weight loss, decreases all fat deposits, and diminishes serum levels of leptin, resistin, and adiponectin by reducing their gene expression. Conclusions. Our results suggest that T3 modulate serum and gene expression levels of leptin, resistin, and adiponectin in experimental model of obesity, providing new insights regarding the relationship between T3 and adipokines in obesity. PMID:22645452

  13. The human BDNF gene: peripheral gene expression and protein levels as biomarkers for psychiatric disorders

    PubMed Central

    Cattaneo, A; Cattane, N; Begni, V; Pariante, C M; Riva, M A

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates the survival and growth of neurons, and influences synaptic efficiency and plasticity. The human BDNF gene consists of 11 exons, and distinct BDNF transcripts are produced through the use of alternative promoters and splicing events. The majority of the BDNF transcripts can be detected not only in the brain but also in the blood cells, although no study has yet investigated the differential expression of BDNF transcripts at the peripheral level. This review provides a description of the human BDNF gene structure as well as a summary of clinical and preclinical evidence supporting the role of BDNF in the pathogenesis of psychiatric disorders. We will discuss several mechanisms as possibly underlying BDNF modulation, including epigenetic mechanisms. We will also discuss the potential use of peripheral BDNF as a biomarker for psychiatric disorders, focusing on the factors that can influence BDNF gene expression and protein levels. Within this context, we have also characterized, for we believe the first time, the expression of BDNF transcripts in the blood, with the aim to provide novel insights into the molecular mechanisms and signaling that may regulate peripheral BDNF gene expression levels. PMID:27874848

  14. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato

    PubMed Central

    Haroldsen, Victor M.; Chi-Ham, Cecilia L.; Kulkarni, Shashank; Lorence, Argelia; Bennett, Alan B.

    2012-01-01

    Vitamin C (l-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 µmol m−2 s−1). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato. PMID:21875809

  15. Increased SNAIL expression and low syndecan levels are associated with high Gleason grade in prostate cancer

    PubMed Central

    POBLETE, CRISTIAN E.; FULLA, JUAN; GALLARDO, MARCELA; MUÑOZ, VALENTINA; CASTELLÓN, ENRIQUE A.; GALLEGOS, IVAN; CONTRERAS, HECTOR R.

    2014-01-01

    Prostate cancer (PC) is a leading male oncologic malignancy wideworld. During malignant transformation, normal epithelial cells undergo genetic and morphological changes known as epithelial-mesenchymal transition (EMT). Several regulatory genes and specific marker proteins are involved in PC EMT. Recently, syndecans have been associated with malignancy grade and Gleason score in PC. Considering that SNAIL is mainly a gene repressor increased in PC and that syndecan promoters have putative binding sites for this repressor, we propose that SNAIL might regulate syndecan expression during PC EMT. The aim of this study was to analyze immunochemically the expression of SNAIL, syndecans 1 and 2 and other EMT markers in a tissue microarray (TMA) of PC samples and PC cell lines. The TMAs included PC samples of different Gleason grade and benign prostatic hyperplasia (BPH) samples, as non-malignant controls. PC3 and LNCaP cell lines were used as models of PC representing different tumorigenic capacities. Semi-quantitative immunohistochemistry was performed on TMAs and fluorescence immunocytochemistry and western blot analysis were conducted on cell cultures. Results show that SNAIL exhibits increased expression in high Gleason specimens compared to low histological grade and BPH samples. Accordingly, PC3 cells show higher SNAIL expression levels compared to LNCaP cells. Conversely, syndecan 1, similarly to E-cadherin (a known marker of EMT), shows a decreased expression in high Gleason grades samples and PC3 cells. Interestingly, syndecan 2 shows no changes associated to histological grade. It is concluded that increased SNAIL levels in advanced PC are associated with low expression of syndecan 1. The mechanism by which SNAIL regulates the expression of syndecan 1 remains to be investigated. PMID:24424718

  16. Lentiviral-Transduced Human Mesenchymal Stem Cells Persistently Express Therapeutic Levels of Enzyme in a Xenotransplantation Model of Human Disease

    PubMed Central

    Meyerrose, Todd E.; Roberts, Marie; Ohlemiller, Kevin K.; Vogler, Carole A.; Wirthlin, Louisa; Nolta, Jan A.; Sands, Mark S.

    2009-01-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease. PMID:18436861

  17. dParFit: A computer program for fitting diatomic molecule spectral data to parameterized level energy expressions

    NASA Astrophysics Data System (ADS)

    Le Roy, Robert J.

    2017-01-01

    This paper describes FORTRAN program dParFit, which performs least-squares fits of diatomic molecule spectroscopic data involving one or more electronic states and one or more isotopologues, to parameterized expressions for the level energies. The data may consist of any combination of microwave, infrared or electronic vibrotational bands, fluorescence series or binding energies (from photo-association spectroscopy). The level energies for each electronic state may be described by one of: (i) band constants {Gv ,Bv ,Dv , … } for each vibrational level, (ii) generalized Dunham expansions, (iii) pure near-dissociation expansions (NDEs), (iv) mixed Dunham/NDE expressions, or (v) individual term values for each distinct level of each isotopologue. Different representations may be used for different electronic states and/or for different types of constants in a given fit (e.g., Gv and Bv may be represented one way and centrifugal distortion constants another). The effect of Λ-doubling or 2Σ splittings may be represented either by band constants (qvB or γvB, qvD or γvD, etc.) for each vibrational level of each isotopologue, or by using power series expansions in (v + 1/2) to represent those constants. Fits to Dunham or NDE expressions automatically incorporate normal first-order semiclassical mass scaling to allow combined analyses of multi-isotopologue data. In addition, dParFit may fit to determine atomic-mass-dependent terms required to account for breakdown of the Born-Oppenheimer and first-order semiclassical approximations. In any of these types of fits, one or more subsets of these parameters for one or more of the electronic states may be held fixed, while a limited parameter set is varied. The program can also use a set of read-in constants to make predictions and calculate deviations [ycalc -yobs ] for any chosen input data set, or to generate predictions of arbitrary data sets.

  18. Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

    SciTech Connect

    Hufbauer, M.; Lazic, D.; Akguel, B.; Brandsma, J.L.; Pfister, H.; Weissenborn, S.J.

    2010-08-01

    Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

  19. Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris

    PubMed Central

    2013-01-01

    Background Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation. Results DNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters PG1 and PG6. Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A PG1 clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a PGAP clone with identical gene copy number, while PG6 only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of PG1 and PG6 respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L-1 HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for PG1 and with porcine carboxypeptidase B for PG6. Moreover, the molecular function of the gene under the control of PG1 was determined to encode a high-affinity glucose transporter and named GTH1. Conclusions A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using

  20. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

    SciTech Connect

    Eisenstein, R.S.; Rosen, J.M.

    1988-08-01

    The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNA was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.

  1. Biphasic Dependence of Glioma Survival and Cell Migration on CD44 Expression Level.

    PubMed

    Klank, Rebecca L; Decker Grunke, Stacy A; Bangasser, Benjamin L; Forster, Colleen L; Price, Matthew A; Odde, Thomas J; SantaCruz, Karen S; Rosenfeld, Steven S; Canoll, Peter; Turley, Eva A; McCarthy, James B; Ohlfest, John R; Odde, David J

    2017-01-03

    While several studies link the cell-surface marker CD44 to cancer progression, conflicting results show both positive and negative correlations with increased CD44 levels. Here, we demonstrate that the survival outcomes of genetically induced glioma-bearing mice and of high-grade human glioma patients are biphasically correlated with CD44 level, with the poorest outcomes occurring at intermediate levels. Furthermore, the high-CD44-expressing mesenchymal subtype exhibited a positive trend of survival with increased CD44 level. Mouse cell migration rates in ex vivo brain slice cultures were also biphasically associated with CD44 level, with maximal migration corresponding to minimal survival. Cell simulations suggest that cell-substrate adhesiveness is sufficient to explain this biphasic migration. More generally, these results highlight the potential importance of non-monotonic relationships between survival and biomarkers associated with cancer progression.

  2. Associations between joint effusion in the knee and gene expression levels in the circulation: a meta-analysis

    PubMed Central

    Peters, Marjolein J.; Ramos, Yolande F.M.; den Hollander, Wouter; Schiphof, Dieuwke; Hofman, Albert; Uitterlinden, André G.; Oei, Edwin H.G.; Slagboom, P. Eline; Kloppenburg, Margreet; Bloem, Johan L.; Bierma-Zeinstra, Sita M.A.; Meulenbelt, Ingrid; van Meurs, Joyce B.J.

    2016-01-01

    Objective: To identify molecular biomarkers for early knee osteoarthritis (OA), we examined whether joint effusion in the knee associated with different gene expression levels in the circulation. Materials and Methods: Joint effusion grades measured with magnetic resonance (MR) imaging and gene expression levels in blood were determined in women of the Rotterdam Study (N=135) and GARP (N=98). Associations were examined using linear regression analyses, adjusted for age, fasting status, RNA quality, technical batch effects, blood cell counts, and BMI. To investigate enriched pathways and protein-protein interactions, we used the DAVID and STRING webtools. Results: In a meta-analysis, we identified 257 probes mapping to 189 unique genes in blood that were nominally significantly associated with joint effusion grades in the knee. Several compelling genes were identified such as C1orf38 and NFATC1. Significantly enriched biological pathways were: response to stress, gene expression, negative regulation of intracellular signal transduction, and antigen processing and presentation of exogenous pathways. Conclusion: Meta-analyses and subsequent enriched biological pathways resulted in interesting candidate genes associated with joint effusion that require further characterization. Associations were not transcriptome-wide significant most likely due to limited power. Additional studies are required to replicate our findings in more samples, which will greatly help in understanding the pathophysiology of OA and its relation to inflammation, and may result in biomarkers urgently needed to diagnose OA at an early stage. PMID:27134727

  3. MicroRNA analysis suggests an additional level of feedback regulation in the NF-κB signaling cascade.

    PubMed

    Mechtler, Peter; Singhal, Ruchi; Kichina, Julia V; Bard, Jonathan E; Buck, Michael J; Kandel, Eugene S

    2015-07-10

    It is increasingly clear that the biological functions of a transcription factor cannot be fully understood solely on the basis of protein-coding genes that fall under its control. Many transcription factors regulate expression of miRNAs, which affect the cell by modulating translation and stability of mRNAs. The identities and the roles of NF-κB-regulated miRNAs have been attracting research interest for a long time. We revisited this issue in a system with controlled expression of one of the key regulators of NF-κB, RIPK1. Several regulated miRNAs were identified, including miR-146a, miR-215 and miR-497. The miRNAs were also inducible by IL-1β, but not when NF-κB activity was repressed by mutant IκBα. The presence of a miR-497 site was predicted in the 3'-UTR of IKBKB gene, which encodes IKKβ. Using appropriately engineered reporters, we confirmed that this site can be a target of suppressive action of miR-497. Our findings suggest that NF-κB controls expression of a miRNA, which may reduce production of IKKβ. Considering the role of IKKβ in the canonical pathway of NF-κB activation, our observations may indicate a new mechanism that modulates the magnitude of such activation, as well as the propensity of a cell to engage canonical vs. non-canonical pathways.

  4. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  5. Microarray Analysis of Gene Expression in Saccharomyces cerevisiae kap108Δ Mutants upon Addition of Oxidative Stress.

    PubMed

    Belanger, Kenneth D; Larson, Nathaniel; Kahn, Jonathan; Tkachev, Dmitry; Ay, Ahmet

    2016-04-07

    Protein transport between the nucleus and cytoplasm of eukaryotic cells is tightly regulated, providing a mechanism for controlling intracellular localization of proteins, and regulating gene expression. In this study, we have investigated the importance of nucleocytoplasmic transport mediated by the karyopherin Kap108 in regulating cellular responses to oxidative stress in Saccharomyces cerevisiae We carried out microarray analyses on wild-type and kap108 mutant cells grown under normal conditions, shortly after introduction of oxidative stress, after 1 hr of oxidative stress, and 1 hr after oxidative stress was removed. We observe more than 500 genes that undergo a 40% or greater change in differential expression between wild-type and kap108Δ cells under at least one of these conditions. Genes undergoing changes in expression can be categorized in two general groups: 1) those that are differentially expressed between wild-type and kap108Δ cells, no matter the oxidative stress conditions; and 2) those that have patterns of response dependent upon both the absence of Kap108, and introduction or removal of oxidative stress. Gene ontology analysis reveals that, among the genes whose expression is reduced in the absence of Kap108 are those involved in stress response and intracellular transport, while those overexpressed are largely involved in mating and pheromone response. We also identified 25 clusters of genes that undergo similar patterns of change in gene expression when oxidative stresses are added and subsequently removed, including genes involved in stress response, oxidation-reduction processing, iron homeostasis, ascospore wall assembly, transmembrane transport, and cell fusion during mating. These data suggest that Kap108 is important for regulating expression of genes involved in a variety of specific cell functions.

  6. The abundance of processed pseudogenes derived from glycolytic genes is correlated with their expression level.

    PubMed

    McDonell, Laura; Drouin, Guy

    2012-02-01

    The abundance of processed pseudogenes in different vertebrate species is known to be proportional to the length of their oogenesis. However, this hypothesis cannot explain why, in a given species, certain genes produce more processed pseudogenes than others. In particular, one would expect that all genes of the glycolytic pathway would generate roughly the same number of processed pseudogenes. However, some glycolitic genes generate more processed pseudogenes than others. Here, we show that there is a positive correlation between the abundance of processed pseudogene generated from glycolytic genes and their level of expression. The variation in expression level of different glycolytic genes likely reflects the fact that some of them, such a GAPDH, have functions other than those they play in glycolysis. Furthermore, the age distribution of GAPDH-processed pseudogenes corresponds to the age distribution of LINE1 elements, which are the source of the reverse transcriptase that generates processed pseudogenes. These results support the hypothesis that gene expression levels affect the level of processed pseudogene production.

  7. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels

    PubMed Central

    Steinacher, Arno; Bates, Declan G.; Akman, Ozgur E.; Soyer, Orkun S.

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  8. Neuropeptide Y mRNA expression levels following chronic olanzapine, clozapine and haloperidol administration in rats.

    PubMed

    Huang, X-F; Deng, Chao; Zavitsanou, Katerina

    2006-06-01

    Using quantitative in situ hybridization, this study examined regional changes in rat brain mRNA levels encoding neuropeptide Y (NPY) following olanzapine, clozapine and haloperidol administration (1.2, 1.5 and 2.0 mg/kg, oral) for 36 days. The NPY mRNA expression levels and patterns were examined after the last drug administration at both time points enabling the measurement of immediate effect at 2h and the effects after 48 h of drug administration. It was found that all these drugs had an immediate effect on NPY mRNA expression, while virtually all these changes normalized 48 h after the drug treatments. A similarity in altered NPY mRNA expression patterns was seen between the olanzapine and clozapine groups; however, haloperidol was very different. Olanzapine and clozapine administration decreased NPY mRNA levels in the nucleus accumbens, striatum and anterior cingulate cortex (from -60% to -77%, p<0.05). Haloperidol decreased NPY mRNA expression in the amygdala and hippocampus (-69%, -64%, p<0.05). In the lateral septal nucleus, NPY mRNA levels significantly decreased in the olanzapine group (-66%, p<0.05), a trend toward a decrease was observed in the clozapine group, and no change was found in the haloperidol treated group. These results suggest that the different effects of atypical and typical antipsychotics on NPY systems may reflect the neural chemical mechanisms responsible for the differences between these drugs in their effects in treating positive and negative symptoms of schizophrenia. The immediate decrease of NPY mRNA levels suggests an immediate reduction of NPY biosynthesis in response to these drugs.

  9. Global Gene Expression Profiling of a Population Exposed to a Range of Benzene Levels

    PubMed Central

    McHale, Cliona M.; Zhang, Luoping; Lan, Qing; Vermeulen, Roel; Li, Guilan; Hubbard, Alan E.; Porter, Kristin E.; Thomas, Reuben; Portier, Christopher J.; Shen, Min; Rappaport, Stephen M.; Yin, Songnian; Smith, Martyn T.; Rothman, Nathaniel

    2011-01-01

    Background Benzene, an established cause of acute myeloid leukemia (AML), may also cause one or more lymphoid malignancies in humans. Previously, we identified genes and pathways associated with exposure to high (> 10 ppm) levels of benzene through transcriptomic analyses of blood cells from a small number of occupationally exposed workers. Objectives The goals of this study were to identify potential biomarkers of benzene exposure and/or early effects and to elucidate mechanisms relevant to risk of hematotoxicity, leukemia, and lymphoid malignancy in occupationally exposed individuals, many of whom were exposed to benzene levels < 1 ppm, the current U.S. occupational standard. Methods We analyzed global gene expression in the peripheral blood mononuclear cells of 125 workers exposed to benzene levels ranging from < 1 ppm to > 10 ppm. Study design and analysis with a mixed-effects model minimized potential confounding and experimental variability. Results We observed highly significant widespread perturbation of gene expression at all exposure levels. The AML pathway was among the pathways most significantly associated with benzene exposure. Immune response pathways were associated with most exposure levels, potentially providing biological plausibility for an association between lymphoma and benzene exposure. We identified a 16-gene expression signature associated with all levels of benzene exposure. Conclusions Our findings suggest that chronic benzene exposure, even at levels below the current U.S. occupational standard, perturbs many genes, biological processes, and pathways. These findings expand our understanding of the mechanisms by which benzene may induce hematotoxicity, leukemia, and lymphoma and reveal relevant potential biomarkers associated with a range of exposures. PMID:21147609

  10. AEG-1 expression correlates with CD133 and PPP6c levels in human glioma tissues

    PubMed Central

    Guo, Jia; Chen, Xin; Xi, Ruxing; Chang, Yuwei; Zhang, Xuanwei; Zhang, Xiaozhi

    2014-01-01

    Abstract Astrocyte elevated gene-1 (AEG-1) is associated with tumor genesis and progression in a variety of human cancers. This study aimed to explore the significance of AEG-1 in glioma and investigate whether it correlated with radioresistance of glioma cells. Immunohistochemical staining showed that the intensity of AEG-1, CD133 and PPP6c protein expression in glioma tissues increased significantly, mainly in the cytoplasm. The expression rate of AEG-1, CD133 and PPP6c were 85.9% (67/78), 60.3% (47/78) and 65.8% (51/78), respectively. AEG-1 expression was correlated with age (r = 0.227, P = 0.045), clinical stage (r = 0.491, P<0.001) and clinical grade (r = 0.450, P<0.001). No correlation was found between AEG-1 expression and other clinicopathologic parameters (P>0.05). The expression of AEG-1 was positively correlated with the expression of CD133 (r = 0.240, P  =  0.035) and PPP6c (r =  0.250, P  =  0.027). In addition, retrieved data on TCGA implied co-occurrence of genomic alterations of AEG-1 and PPP6c in glioblastoma. Our findings indicate that AEG-1 is positively correlated with CD133 and AEG-1 expression. It may play an important role in the progression of glioma and may serve as potential novel marker of chemoresistance and radioresistance. PMID:25332711

  11. Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size

    PubMed Central

    Toonen, Ruud F. G.; Wierda, Keimpe; Sons, Michèle S.; de Wit, Heidi; Cornelisse, L. Niels; Brussaard, Arjen; Plomp, Jaap J.; Verhage, Matthijs

    2006-01-01

    Prompt recovery after intense activity is an essential feature of most mammalian synapses. Here we show that synapses with reduced expression of the presynaptic gene munc18-1 suffer from increased depression during intense stimulation at glutamatergic, GABAergic, and neuromuscular synapses. Conversely, munc18-1 overexpression makes these synapses recover faster. Concomitant changes in the readily releasable vesicle pool and its refill kinetics were found. The number of vesicles docked at the active zone and the total number of vesicles per terminal correlated with both munc18-1 expression levels and the size of the releasable vesicle pool. These data show that varying expression of a single gene controls synaptic recovery by modulating the number of docked, release-ready vesicles and thereby replenishment of the secretion capacity. PMID:17110441

  12. High-Level Transient Expression of ER-Targeted Human Interleukin 6 in Nicotiana benthamiana

    PubMed Central

    Nausch, Henrik; Mikschofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T0 transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T2 plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein. PMID:23152824

  13. Comprehensive expression analysis of FSHD candidate genes at the mRNA and protein level.

    PubMed

    Klooster, Rinse; Straasheijm, Kirsten; Shah, Bharati; Sowden, Janet; Frants, Rune; Thornton, Charles; Tawil, Rabi; van der Maarel, Silvère

    2009-12-01

    In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

  14. Identification of Gene Expression Signatures in the Chicken Intestinal Intraepithelial Lymphocytes in Response to Herb Additive Supplementations

    PubMed Central

    Won, Kyeong-Hye; Song, Ki-Duk; Park, Jong-Eun; Kim, Duk-Kyung; Na, Chong-Sam

    2016-01-01

    Anethole and garlic have an immune modulatory effects on avian coccidiosis, and these effects are correlated with gene expression changes in intestinal epithelial lymphocytes (IELs). In this study, we integrated gene expression datasets from two independent experiments and investigated gene expression profile changes by anethole and garlic respectively, and identified gene expression signatures, which are common targets of these herbs as they might be used for the evaluation of the effect of plant herbs on immunity toward avian coccidiosis. We identified 4,382 and 371 genes, which were differentially expressed in IELs of chickens supplemented with garlic and anethole respectively. The gene ontology (GO) term of differentially expressed genes (DEGs) from garlic treatment resulted in the biological processes (BPs) related to proteolysis, e.g., “modification-dependent protein catabolic process”, “proteolysis involved in cellular protein catabolic process”, “cellular protein catabolic process”, “protein catabolic process”, and “ubiquitin-dependent protein catabolic process”. In GO analysis, one BP term, “Proteolysis”, was obtained. Among DEGs, 300 genes were differentially regulated in response to both garlic and anethole, and 234 and 59 genes were either up- or down-regulated in supplementation with both herbs. Pathway analysis resulted in enrichment of the pathways related to digestion such as “Starch and sucrose metabolism” and “Insulin signaling pathway”. Taken together, the results obtained in the present study could contribute to the effective development of evaluation system of plant herbs based on molecular signatures related with their immunological functions in chicken IELs. PMID:26954117

  15. Correlations among PPARγ, DNMT1, and DNMT3B Expression Levels and Pancreatic Cancer.

    PubMed

    Pazienza, Valerio; Tavano, Francesca; Benegiamo, Giorgia; Vinciguerra, Manlio; Burbaci, Francesca Paola; Copetti, Massimiliano; di Mola, Fabio Francesco; Andriulli, Angelo; di Sebastiano, Pierluigi

    2012-01-01

    Emerging evidence indicates that peroxisome proliferator-activated receptor γ (PPARγ) and DNA methyltransferases (DNMTs) play a role in carcinogenesis. In this study we aimed to evaluate the expression of PPARγ, DNMT1, and DNMT3B and their correlation with clinical-pathological features in patients with pancreatic cancer (PC), and to define the effect of PPARγ activation on DNMTs expression in PC cell lines. qRT-PCR analysis showed that DNMT3B expression was downregulated in tumors compared to normal tissues (P = 0.03), whereas PPARγ and DNMT1 levels did not show significant alterations in PC patients. Expression levels between PPARγ and DNMT1 and between DNMT1 and DNMT3B were highly correlated (P = 0.008 and P = 0.05 resp.). DNMT3B overexpression in tumor tissue was positively correlated with both lymph nodes spreading (P = 0.046) and resection margin status (P = 0.04), and a borderline association with perineural invasion (P = 0.06) was found. Furthermore, high levels of DNMT3B expression were significantly associated with a lower mortality in the whole population (HR = 0.485; 95%CI = 0.262-0.895, P = 0.02) and in the subgroup of patients without perineural invasion (HR = 0.314; 95%CI = 0.130-0.758; P = 0.01), while such association was not observed in patients with tumor invasion into perineural structures (P = 0.70). In conclusion, in vitro and in vivo PPARγ and DNMTs appear interrelated in PC, and this interaction might influence cell phenotype and disease behavior.

  16. Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin II expression.

    PubMed

    Tsai, Wen-Chung; Hsu, Chih-Chin; Pang, Jong-Hwei S; Lin, Miao-Sui; Chen, Ying-Hsun; Liang, Fang-Chen

    2012-01-01

    Low-level laser therapy (LLLT) is commonly used to treat sports-related tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of laser on tenocyte migration. Furthermore, the correlation between this effect and expression of dynamin 2, a positive regulator of cell motility, was also investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm with energy density at 1.0, 1.5, and 2.0 J/cm(2)). Tenocyte migration was evaluated by an in vitro wound healing model and by transwell filter migration assay. The messenger RNA (mRNA) and protein expressions of dynamin 2 were determined by reverse transcription/real-time polymerase chain reaction (real-time PCR) and Western blot analysis respectively. Immunofluorescence staining was used to evaluate the dynamin 2 expression in tenocytes. Tenocytes with or without laser irradiation was treated with dynasore, a dynamin competitor and then underwent transwell filter migration assay. In vitro wound model revealed that more tenocytes with laser irradiation migrated across the wound border to the cell-free zone. Transwell filter migration assay confirmed that tenocyte migration was enhanced dose-dependently by laser. Real-time PCR and Western-blot analysis demonstrated that mRNA and protein expressions of dynamin 2 were up-regulated by laser irradiation dose-dependently. Confocal microscopy showed that laser enhanced the expression of dynamin 2 in cytoplasm of tenocytes. The stimulation effect of laser on tenocytes migration was suppressed by dynasore. In conclusion, low-level laser irradiation stimulates tenocyte migration in a process that is mediated by up-regulation of dynamin 2, which can be suppressed by dynasore.

  17. Zinc Fortification Decreases ZIP1 Gene Expression of Some Adolescent Females with Appropriate Plasma Zinc Levels

    PubMed Central

    Méndez, Rosa O.; Santiago, Alejandra; Yepiz-Plascencia, Gloria; Peregrino-Uriarte, Alma B.; de la Barca, Ana M. Calderón; García, Hugo S.

    2014-01-01

    Zinc homeostasis is achieved after intake variation by changes in the expression levels of zinc transporters. The aim of this study was to evaluate dietary intake (by 24-h recall), absorption, plasma zinc (by absorption spectrophotometry) and the expression levels (by quantitative PCR), of the transporters ZIP1 (zinc importer) and ZnT1 (zinc exporter) in peripheral white blood cells from 24 adolescent girls before and after drinking zinc-fortified milk for 27 day. Zinc intake increased (p < 0.001) from 10.5 ± 3.9 mg/day to 17.6 ± 4.4 mg/day, and its estimated absorption from 3.1 ± 1.2 to 5.3 ± 1.3 mg/day. Mean plasma zinc concentration remained unchanged (p > 0.05) near 150 µg/dL, but increased by 31 µg/dL (p < 0.05) for 6/24 adolescents (group A) and decreased by 25 µg/dL (p < 0.05) for other 6/24 adolescents (group B). Expression of ZIP1 in blood leukocytes was reduced 1.4-fold (p < 0.006) in group A, while for the expression of ZnT1 there was no difference after intervention (p = 0.39). An increase of dietary zinc after 27-days consumption of fortified-milk did not increase (p > 0.05) the plasma level of adolescent girls but for 6/24 participants from group A in spite of the formerly appropriation, which cellular zinc uptake decreased as assessed by reduction of the expression of ZIP1. PMID:24922175

  18. Effects of a phytogenic feed additive on susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to investigate the effect of a phytogenic feed additive (Digestarom® P.E.P. MGE) on growth performance and disease susceptibility to Edwardsiella ictaluri. Two hundred and fifty juvenile channel catfish (7.2 ± 0.1 g) were allotted into the following treatments: Control (float...

  19. The Effects of Computer-Assisted Instruction on Student Achievement in Addition and Subtraction at First Grade Level.

    ERIC Educational Resources Information Center

    Spivey, Patsy M.

    This study was conducted to determine whether the traditional classroom approach to instruction involving the addition and subtraction of number facts (digits 0-6) is more or less effective than the traditional classroom approach plus a commercially-prepared computer game. A pretest-posttest control group design was used with two groups of first…

  20. MicroRNA analysis suggests an additional level of feedback regulation in the NF-κB signaling cascade

    PubMed Central

    Kichina, Julia V.; Bard, Jonathan E.; Buck, Michael J.; Kandel, Eugene S.

    2015-01-01

    It is increasingly clear that the biological functions of a transcription factor cannot be fully understood solely on the basis of protein-coding genes that fall under its control. Many transcription factors regulate expression of miRNAs, which affect the cell by modulating translation and stability of mRNAs. The identities and the roles of NF-κB-regulated miRNAs have been attracting research interest for a long time. We revisited this issue in a system with controlled expression of one of the key regulators of NF-κB, RIPK1. Several regulated miRNAs were identified, including miR-146a, miR-215 and miR-497. The miRNAs were also inducible by IL-1β, but not when NF-κB activity was repressed by mutant IκBα. The presence of a miR-497 site was predicted in the 3′-UTR of IKBKB gene, which encodes IKKβ. Using appropriately engineered reporters, we confirmed that this site can be a target of suppressive action of miR-497. Our findings suggest that NF-κB controls expression of a miRNA, which may reduce production of IKKβ. Considering the role of IKKβ in the canonical pathway of NF-κB activation, our observations may indicate a new mechanism that modulates the magnitude of such activation, as well as the propensity of a cell to engage canonical vs. non-canonical pathways. PMID:26020802

  1. Effect of heat stress and feeding phosphorus levels on pig electron transport chain gene expression.

    PubMed

    Weller, M M D C A; Alebrante, L; Campos, P H R F; Saraiva, A; Silva, B A N; Donzele, J L; Oliveira, R F M; Silva, F F; Gasparino, E; Lopes, P S; Guimarães, S E F

    2013-12-01

    The purpose of this study was to evaluate the effect of temperature and different levels of available phosphorus (aP) on the expression of nine genes encoding electron transport chain proteins in the Longissimus dorsi (LD) muscle of pigs. Two trials were carried out using 48 high-lean growth pigs from two different growth phases: from 15 to 30 kg (phase 1) and from 30 to 60 kg (phase 2). Pigs from growth phase 1 were fed with three different levels of dietary aP (0.107%, 0.321% or 0.535%) and submitted either to a thermoneutral (24°C and RH at 76%) or to a heat stress (34°C and RH at 70%) environment. Pigs from growth phase 2 were fed with three different levels of dietary aP (0.116%, 0.306% or 0.496%) and submitted either to a thermoneutral (22ºC and RH at 77%) or to a heat stress (32ºC and RH at 73%) environment. Heat stress decreased (P<0.001) average daily feed intake at both growth phases. At 24°C, pigs in phase 1 fed the 0.321% aP diet had greater average daily gain and feed conversion (P<0.05) than those fed the 0.107% or 0.535% while, at 34°C pigs fed the 0.535% aP had the best performance (P<0.05). Pigs from phase 2 fed the 0.306% aP had best performance in both thermal environments. Gene expression profile was analyzed by quantitative real-time polymerase chain reaction. Irrespective of growing phase, the expression of six genes was lower (P<0.05) at high temperature than at thermoneutrality. The lower expression of these genes under high temperatures evidences the effects of heat stress by decreasing oxidative metabolism, through adaptive physiological mechanisms in order to reduce heat production. In pigs from phase 1, six genes were differentially expressed across aP levels (P<0.05) in the thermoneutral and one gene in the heat stress. In pigs from phase 2, two genes were differentially expressed across aP levels (P<0.05) in both thermal environments. These data revealed strong evidence that phosphorus and thermal environments are key factors to

  2. Caveolin-1 Expression Level in Cancer Associated Fibroblasts Predicts Outcome in Gastric Cancer

    PubMed Central

    Gao, Jun; Fan, Lifang; Li, Zonghuan; Yang, Guifang; Chen, Honglei

    2013-01-01

    Aims Altered expression of epithelial or stromal caveolin-1 (Cav-1) is observed in various types of human cancers. However, the clinical significance of Cav-1 expression in gastric cancer (GC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of both tumor cells and cancer associated fibroblasts (CAFs) Cav-1 in GC. Methods and Results Quantum dots immunofluorescence histochemistry was performed to examine the expression of Cav-1 in 20 cases of gastritis without intestinal metaplasia (IM), 20 cases of gastritis with IM and 286 cases of GC. Positive rates of epithelial Cav-1 in gastritis without IM, gastritis with IM and GC showed a decreasing trend (P = 0.012). Low expression of Cav-1 in CAFs but not in tumor cells was an independent predictor of poor prognosis in GC patients (P = 0.034 and 0.005 respectively in disease free survival and overall survival). Cav-1 level in tumor cells and CAFs showed no significant correlation with classic clinicopathological features. Conclusions Loss of epithelial Cav-1 may promote malignant progression and low CAFs Cav-1 level herald worse outcome of GC patient, suggesting CAFs Cav-1 may be a candidate therapeutic target and a useful prognostic marker of GC. PMID:23527097

  3. Potato steroidal glycoalkaloid levels and the expression of key isoprenoid metabolic genes.

    PubMed

    Krits, Pinchas; Fogelman, Edna; Ginzberg, Idit

    2007-12-01

    The potato steroidal glycoalkaloids (SGA) are toxic secondary metabolites, and their total content in tubers should not exceed 20 mg/100 g fresh weight. The two major SGA in cultivated potato (Solanum tuberosum) are alpha-chaconine and alpha-solanine. SGA biosynthetic genes and the genetic factors that control their expression have not yet been determined. In the present study, potato genotypes exhibiting different levels of SGA content showed an association between high SGA levels in their leaves and tubers and high expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (hmg1) and squalene synthase 1 (pss1), genes of the mevalonic/isoprenoid pathway. Transcripts of other key enzymes of branches of the isoprenoid pathway, vetispiradiene/sesquiterpene synthase (pvs1) and sterol C24-methyltransferase type1 (smt1), were undetectable or exhibited stable expression regardless of SGA content, respectively, suggesting facilitated precursor flow to the SGA biosynthetic branch. The transcript ratio of solanidine glucosyltransferase (sgt2) to solanidine galactosyltransferase (sgt1) was correlated to the documented chaconine-to-solanine ratio in the tested genotypes. Significantly higher expression of hmg1, pss1, smt1, sgt1 and sgt2 was monitored in the tuber phelloderm than in the parenchyma of the tuber's flesh, targeting the former as the main SGA-producing tissue in the tuber, in agreement with the known high SGA content in the layers directly under the tuber skin.

  4. The hESC line Envy expresses high levels of GFP in all differentiated progeny.

    PubMed

    Costa, Magdaline; Dottori, Mirella; Ng, Elizabeth; Hawes, Susan M; Sourris, Koula; Jamshidi, Pegah; Pera, Martin F; Elefanty, Andrew G; Stanley, Edouard G

    2005-04-01

    Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.

  5. Gene expression suggests conserved aspects of Hox gene regulation in arthropods and provides additional support for monophyletic Myriapoda.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2010-07-05

    Antisense transcripts of Ultrabithorax (aUbx) in the millipede Glomeris and the centipede Lithobius are expressed in patterns complementary to that of the Ubx sense transcripts. A similar complementary expression pattern has been described for non-coding RNAs (ncRNAs) of the bithoraxoid (bxd) locus in Drosophila, in which the transcription of bxd ncRNAs represses Ubx via transcriptional interference. We discuss our findings in the context of possibly conserved mechanisms of Ubx regulation in myriapods and the fly.Bicistronic transcription of Ubx and Antennapedia (Antp) has been reported previously for a myriapod and a number of crustaceans. In this paper, we show that Ubx/Antp bicistronic transcripts also occur in Glomeris and an onychophoran, suggesting further conserved mechanisms of Hox gene regulation in arthropods.Myriapod monophyly is supported by the expression of aUbx in all investigated myriapods, whereas in other arthropod classes, including the Onychophora, aUbx is not expressed. Of the two splice variants of Ubx/Antp only one could be isolated from myriapods, representing a possible further synapomorphy of the Myriapoda.

  6. Endogenous Oxytocin Levels Are Associated with the Perception of Emotion in Dynamic Body Expressions in Schizophrenia

    PubMed Central

    Strauss, Gregory P.; Keller, William R.; Koenig, James I.; Sullivan, Sara K.; Gold, James M.; Buchanan, Robert W.

    2015-01-01

    Lower endogenous oxytocin levels have been associated with impaired social cognition in schizophrenia, particularly facial affect identification. Little is known about the relationship between oxytocin and other forms of emotion perception. In the current study, 41 individuals with schizophrenia (SZ) and 22 demographically matched healthy controls (CN) completed a forced-choice affective body expression classification task. Stimuli included dynamic videos of male and female actors portraying 4 discrete emotions: happiness, sadness, anger, and neutral. Plasma oxytocin levels were determined via radioimmunoassay. Results indicated that SZ had significantly higher plasma oxytocin concentrations than CN. SZ were also less accurate at identifying expressions of happiness and sadness; however, there were no group differences for anger or neutral stimuli. A group x sex interaction was also present, such that female CN were more accurate than male CN, whereas male SZ were more accurate than female SZ. Higher endogenous oxytocin levels were associated with better total recognition in both SZ and CN; this association was specific to females in SZ. Findings indicate that sex plays an important role in identifying emotional expressions in body gestures in SZ, and that individual differences in endogenous oxytocin predict emotion perception accuracy. PMID:25620121

  7. Deimination level and peptidyl arginine deiminase 2 expression are elevated in astrocytes with increased incubation temperature.

    PubMed

    Enriquez-Algeciras, Mabel; Bhattacharya, Sanjoy K; Serra, Horacio M

    2015-09-01

    Astrocytes respond to environmental cues, including changes in temperatures. Increased deimination, observed in many progressive neurological diseases, is thought to be contributed by astrocytes. We determined the level of deimination and expression of peptidyl arginine deiminase 2 (PAD2) in isolated primary astrocytes in response to changes on either side (31°C and 41°C) of the optimal temperature (37°C). We investigated changes in the astrocytes by using a number of established markers and accounted for cell death with the CellTiter-Blue assay. We found increased expression of glial fibrillary acidic protein, ALDH1L1, and J1-31, resulting from increased incubation temperature and increased expression of TSP1, S100β, and AQP4, resulting from decreased incubation temperature vs. optimal temperature, suggesting activation of different biochemical pathways in astrocytes associated with different incubation temperatures. Mass spectrometric analyses support such trends. The PAD2 level was increased only as a result of increased incubation temperature with a commensurate increased level of deimination. Actin cytoskeleton and iso[4]LGE, a lipid peroxidase modification, also showed an increase with higher incubation temperature. Altogether, these results suggest that temperature, as an environmental cue, activates astrocytes in a different manner on either side of the optimal temperature and that increase in deimination is associated only with the higher temperature side of the spectrum.

  8. Comprehensive data base of high-level nuclear waste glasses: September 1987 status report: Volume 2, Additional appendices

    SciTech Connect

    Kindle, C.H.; Kreiter, M.R.

    1987-12-01

    The Materials Characterization Center (MCC) is assembling a comprehensive data base (CDB) of experimental data collected for high-level nuclear waste package components. The status of the CDB is summarized in Volume I of this report. Volume II contains appendices that present data from the data base and an evaluation of glass durability models applied to the data base.

  9. Relationship between expression levels and atherogenesis in scavenger receptor Class B, Type I Transgenics

    SciTech Connect

    Ueda, Yukihiko; Gong, Elaine; Royer, Lori; Cooper, Philip N.; Francone, Omar L; Rubin, Edward M.

    2000-03-15

    Both in vitro and in vivo studies of SR-BI have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate SR-BI's effect on atherogenesis we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) in SR-BI expression in an inbred mouse background hemizygous for a human apo B transgene. Unlike non-HDL cholesterol levels which minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol while the high expression SR-BI transgene was associated with two-fold decreases in HDL as well as dramatic alterations in HDL composition and size including the near absence of a-migrating particles as determined by 2-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a two-fold decrease in the development of diet induced fatty streak lesions compared t o the apo B transgenics (4448{+-}1908 {mu}m2/aorta to 10133 {+-} 4035 {mu}m2/aorta; p<0.001), while the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692{+-}7238 {mu}m2/aorta) but three-fold greater than the low SR-BI/apo B mice (p<0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences illustrating the complexity of the relationship between SR-BI and atherogenesis.

  10. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared towards the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically-expressed enzymes. Here we show that the addit...

  11. Additive effects of ulinastatin and docetaxel on growth of breast cancer xenograft in nude mice and expression of PGE2, IL-10, and IL-2 in primary breast cancer cells.

    PubMed

    Zhong, Biao; Shen, Hongyan; Sun, Xin; Wang, Hong; Zhang, Yonghua; Sun, Zhijun

    2012-05-01

    Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

  12. Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

    PubMed Central

    McSpadden, Luke C.; Kirkton, Robert D.; Bursac, Nenad

    2009-01-01

    Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle α-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from −71.6 ± 4.9 mV in controls to −65.0 ± 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue. PMID:19494239

  13. Expression Level of Genes Coding for Cell Adhesion Molecules of Cadherin Group in Colorectal Cancer Patients

    PubMed Central

    Lorenc, Zbigniew; Opiłka, Mieszko Norbert; Kruszniewska-Rajs, Celina; Rajs, Antoni; Waniczek, Dariusz; Starzewska, Małgorzata; Lorenc, Justyna; Mazurek, Urszula

    2015-01-01

    Background Colorectal Cancer (CRC) is one of the most frequently diagnosed neoplasms and also one of the main death causes. Cell adhesion molecules are taking part in specific junctions, contributing to tissue integrality. Lower expression of the cadherins may be correlated with poorer differentiation of the CRC, and its more aggressive phenotype. The aim of the study is to designate the cadherin genes potentially useful for the diagnostics, prognostics, and the treatment of CRC. Material/Method Specimens were collected from 28 persons (14 female and 14 male), who were operated for CRC. The molecular analysis was performed using oligonucleotide microarrays, mRNA used was collected from adenocarcinoma, and macroscopically healthy tissue. The results were validated using qRT-PCR technique. Results Agglomerative hierarchical clustering of normalized mRNA levels has shown 4 groups with statistically different gene expression. The control group was divided into 2 groups, the one was appropriate control (C1), the second (C2) had the genetic properties of the CRC, without pathological changes histologically and macroscopically. The other 2 groups were: LSC (Low stage cancer) and HSC (High stage cancer). Consolidated results of the fluorescency of all of the differential genes, designated two coding E-cadherin (CDH1) with the lower expression, and P-cadherin (CDH3) with higher expression in CRC tissue. Conclusions The levels of genes expression are different for several groups of cadherins, and are related with the stage of CRC, therefore could be potentially the useful marker of the stage of the disease, also applicable in treatment and diagnostics of CRC. PMID:26167814

  14. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    PubMed Central

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

  15. Effect of sodium intake on gene expression and plasma levels of ANF in rats

    SciTech Connect

    Lattion, A.L.; Aubert, J.F.; Flueckiger, J.P.; Nussberger, J.; Waeber, B.; Brunner, H.R. )

    1988-08-01

    The effect of short- and long-term sodium loading and sodium restriction on the gene expression as well as on circulating plasma levels of atrial natriuretic factor (ANF) was evaluated in normotensive Wistar rats. These rats were fed either a low-, a regular-, or a high-sodium diet (regular diet and 1% saline as drinking fluid) and studied after 1 and 3 wk. The ANF mRNA was determined in pooled atria and ventricles of the different groups of rats, using the dot-blot technique. Plasma ANF levels were measured with a radioimmunoassay. After 1 wk on the high-sodium diet, ANF mRNA was increased in right atria and ventricles together with circulating ANF levels when compared with animals maintained for the same period on a low-sodium diet. After 3 wk on the various diets, the differences in cardiac ANF mRNA and in plasma ANF levels had disappeared. Gene expression of ANF was also looked for in different areas of the brain, lung, thyroid, adrenals, and the kidney; no hybridization was detected in any of these organs. These data suggest that in rats, the transcription of the ANF gene and peptide release is enhanced only during short-term adaptation to dietary sodium loading.

  16. Tubular B7-1 expression parallels proteinuria levels, but not clinical outcomes in adult minimal change disease patients

    PubMed Central

    Lee, Sung Woo; Baek, Seon Ha; Paik, Jin Ho; Kim, Sejoong; Na, Ki Young; Chae, Dong-Wan; Chin, Ho Jun

    2017-01-01

    B7-1 is thought to play a pathogenic role in minimal-change disease (MCD). Recently, however, doubts have arisen regarding the role of B7-1 expression in MCD. Therefore, we aimed to identify the presence and clinical significance of B7-1 expression in MCD patients. The study participants included 28 adult MCD patients for whom kidney specimens were available. The intensity of B7-1 expression was assessed by two independent specialists. We analysed the association between the intensity of B7-1 expression and clinicopathological variables. No B7-1 expression in the glomeruli was observed in any of the 28 patients. Unexpectedly, however, 75.0% of the patients exhibited tubular B7-1 expression, with 35.7% demonstrating weak positive expressions and 39.3% demonstrating strong positive expressions. The level of proteinuria significantly increased as the intensity of tubular B7-1 expression increased. We also found trends of increasing blood urea nitrogen and serum creatinine levels with increased intensity of tubular B7-1 expression. However, we could not observe definite differences in long- and short-term clinical outcomes depending on the intensity of tubular B7-1 expression. In conclusion, B7-1 was expressed in renal tubular cells but not in glomeruli in adult MCD patients. The intensity of tubular B7-1 expression paralleled proteinuria levels, but not clinical outcomes. PMID:28150736

  17. EFFECTS OF QUARTZ PARTICLE SIZE AND SUCROSE ADDITION ON MELTING BEHAVIOR OF A MELTER FEED FOR HIGH-LEVEL GLASS

    SciTech Connect

    MARCIAL J; KRUGER AA; HRMA PR; SCHWEIGER MJ; SWEARINGEN KJ; TEGROTENHUIS WE; HENAGER SH

    2010-07-28

    The behavior of melter feed (a mixture of nuclear waste and glass-forming additives) during waste-glass processing has a significant impact on the rate of the vitrification process. We studied the effects of silica particle size and sucrose addition on the volumetric expansion (foaming) of a high-alumina feed and the rate of dissolution of silica particles in feed samples heated at 5 C/min up to 1200 C. The initial size of quartz particles in feed ranged from 5 to 195 {micro}m. The fraction of the sucrose added ranged from 0 to 0.20 g per g glass. Extensive foaming occurred only in feeds with 5-{micro}m quartz particles; particles {ge}150 {micro}m formed clusters. Particles of 5 {micro}m completely dissolved by 900 C whereas particles {ge}150 {micro}m did not fully dissolve even when the temperature reached 1200 C. Sucrose addition had virtually zero impact on both foaming and the dissolution of silica particles. Over 100 sites in the United States are currently tasked with the storage of nuclear waste. The largest is the Hanford Site located in southeastern Washington State with 177 subterranean tanks containing over fifty-million gallons of nuclear waste from plutonium production from 1944 through 1987. This waste will be vitrified at the Hanford Tank Waste Treatment and Immobilization Plant. In the vitrification process, feed is charged into a melter and converted into glass to be ultimately stored in a permanent repository. The duration of waste-site cleanups by the vitrification process depends on the rate of melting, i.e., on the rate of the feed-to-glass conversion. Foaming associated with the melting process and the rate of dissolution of quartz particles (silica being the major glass-forming additive) are assumed to be important factors that influence the rate of melting. Previous studies on foaming of high-alumina feed demonstrated that varying the makeup of a melter feed has a significant impact on foaming. The volume of feeds that contained 5-{micro

  18. High level expression of human enteropeptidase light chain in Pichia pastoris.

    PubMed

    Pepeliaev, Stanislav; Krahulec, Ján; Černý, Zbyněk; Jílková, Jana; Tlustá, Marcela; Dostálová, Jana

    2011-10-20

    Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.

  19. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

    PubMed

    Guzman, L M; Belin, D; Carson, M J; Beckwith, J

    1995-07-01

    We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

  20. Claudin-1, -2, -4, and -5: comparison of expression levels and distribution in equine tissues

    PubMed Central

    Lee, Bonn; Kang, Hee Young; Lee, Dong Oh; Ahn, Changhwan

    2016-01-01

    Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenly observed in equine tissues, claudin-4 was abundantly detected in the liver, kidney and uterus, and claudin-5 was strongly expressed in the lung, duodenum, ovary, and uterus, as determined by Western blotting method. The localization of equine claudins was observed by immunohistochemistry methods. These findings provide knowledge regarding the expression patterns and localization of equine claudins, as well as valuable information to understand tight junction-related diseases according to tissue specificity and function of claudins in horses. PMID:27030194

  1. Identification of a gene, FMP21, whose expression levels are involved in thermotolerance in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Elucidation of the mechanism of high temperature tolerance in yeasts is important for the molecular breeding of high temperature-tolerant yeasts that can be used in bioethanol production. We identified genes whose expression is correlated with the degree of thermotolerance in Saccharomyces cerevisiae by DNA microarray analysis. Gene expression profiles of three S. cerevisiae strains showing different levels of thermotolerance were compared, and we chose three of them as candidate genes. Among these genes, FMP21 was investigated as a thermotolerance-related gene in S. cerevisiae by comparing the growth at high temperature with the gene expression in eight strains. The expression ratio of FMP21 at 37°C was correlated with the doubling time ratio at a coefficient of determination of 0.787. The potential involvement of the Fmp21 in the thermotolerance of yeasts was evaluated. The FMP21 deletion variant showed a decreased respiratory growth rate and increased thermosensitivity. Furthermore, the overexpression of FMP21 improved thermotolerance in yeasts. In conclusion, the function of Fmp21 is important for thermotolerance in yeasts. PMID:25177541

  2. Effects of zinc deficiency and zinc supplementation on homocysteine levels and related enzyme expression in rats.

    PubMed

    Jing, Mingyan; Rech, Leslie; Wu, Yinghong; Goltz, Douglas; Taylor, Carla G; House, James D

    2015-04-01

    Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n=8/group) for 3 weeks: Zn deficient (ZD; <1mg Zn/kg); Zn control (ZC; 30mg Zn/kg); Zn supplemented (ZS; 300mg Zn/kg); pair fed (PF; 30mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P<0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P<0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P<0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P<0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.

  3. Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression.

    PubMed

    Gómez-Sánchez, Rubén; Gegg, Matthew E; Bravo-San Pedro, José M; Niso-Santano, Mireia; Alvarez-Erviti, Lydia; Pizarro-Estrella, Elisa; Gutiérrez-Martín, Yolanda; Alvarez-Barrientos, Alberto; Fuentes, José M; González-Polo, Rosa Ana; Schapira, Anthony H V

    2014-02-01

    Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

  4. Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

    PubMed Central

    Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

    2015-01-01

    Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

  5. Divergent prion strain evolution driven by PrPC expression level in transgenic mice

    PubMed Central

    Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

    2017-01-01

    Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

  6. Diazepam affects the nuclear thyroid hormone receptor density and their expression levels in adult rat brain.

    PubMed

    Constantinou, Caterina; Bolaris, Stamatis; Valcana, Theony; Margarity, Marigoula

    2005-07-01

    Thyroid hormones (THs) are involved in the occurrence of anxiety and affective disorders; however, the effects following an anxiolytic benzodiazepine treatment, such as diazepam administration, on the mechanism of action of thyroid hormones has not yet been investigated. The effect of diazepam on the in vitro nuclear T3 binding, on the relative expression of the TH receptors (TRs) and on the synaptosomal TH availability were examined in adult rat cerebral hemispheres 24 h after a single intraperitoneal dose (5 mg/kg BW) of this tranquillizer. Although, diazepam did not affect the availability of TH either in blood circulation or in the synaptosomal fraction, it decreased (33%) the nuclear T3 maximal binding density (B(max)). No differences were observed in the equilibrium dissociation constant (K(d)). The TRalpha2 variant (non-T3-binding) mRNA levels were increased by 33%, whereas no changes in the relative expression of the T3-binding isoforms of TRs (TRalpha1, TRbeta1) were observed. This study shows that a single intraperitoneal injection of diazepam affects within 24 h, the density of the nuclear TRs and their expression pattern. The latest effect occurs in an isoform-specific manner involving specifically the TRalpha2 mRNA levels in adult rat brain.

  7. Exposure to caregiver maltreatment alters expression levels of epigenetic regulators in the medial prefrontal cortex

    PubMed Central

    Blaze, Jennifer; Roth, Tania L

    2013-01-01

    Quality of maternal care experienced during infancy is a key factor that can confer vulnerability or resilience to psychiatric disorders later in life. Research continues to indicate that early-life experiences can affect developmental trajectories through epigenetic alterations capable of affecting gene regulation and neural plasticity. Previously, our lab has shown that experiences within an adverse caregiving environment (i.e. maltreatment) produce aberrant DNA methylation patterns at various gene loci in the medial prefrontal cortex (mPFC) of developing and adult rats. This study aimed to determine whether caregiver maltreatment likewise affects expression levels of several genes important in regulating DNA methylation patterns (Dnmt1, Dnmt3a, MeCP2, Gadd45b, and Hdac1). While we observed minimal changes in gene expression within the mPFC of developing rats, we observed expression changes for all genes in adult animals. Specifically, exposure to maltreatment produced a significant decrease in mRNA levels of all epigenetic regulators in adult males and a significant decrease in Gadd45b in adult females. Our results here provide further empirical support for the long-term and sex-specific epigenetic consequences of caregiver maltreatment on the mPFC. PMID:24120634

  8. Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level

    PubMed Central

    Dolatabadi, Soheila; Candia, Julián; Akrap, Nina; Vannas, Christoffer; Tesan Tomic, Tajana; Losert, Wolfgang; Landberg, Göran; Åman, Pierre; Ståhlberg, Anders

    2017-01-01

    Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (G0/G1 – S – G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics. PMID:28179914

  9. Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells

    PubMed Central

    Meier, Elizabeth M.; Wu, Bin; Siddiqui, Aamir; Tepper, Donna G.; Longaker, Michael T.

    2016-01-01

    Background: Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells. Methods: Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied. Results: Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers. Conclusion: Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated. PMID:27757329

  10. Endotoxemia enhances expression of the signaling receptor (GP130) on protein and molecular level.

    PubMed

    Marsik, Claudia; Halama, Thomas; Cardona, Francesco; Schlifke, Irene; Mittermayer, Friedrich; Jilma, Bernd

    2005-03-01

    Interleukin 6 (IL-6) performs a prominent role during sepsis. To examine the molecular regulation of IL-6, IL-6 receptor, and signaling receptor gp130 during endotoxemia, nine healthy young volunteers received a bolus injection of lipopolysaccharide (LPS) on day 1 and saline on day 2 in a double blind, randomized, placebo-controlled trial. LPS enhanced IL-6 release 300-fold. IL-6 mRNA expression was not significantly altered in blood samples at any time after LPS infusion in vivo, while incubation of whole blood with 50 pg/ml LPS up-regulated IL-6 mRNA levels 8000- to 50,000-fold in vitro. LPS infusion increased synthesis of gp130 mRNA 5.5-fold compared to baseline at 4 h (P < 0.05), while no significant change was observed in the placebo period (P = 0.001 between groups). LPS increased the percentage of gp130 positive neutrophils gp130 700% over baseline at 8 h (P < 0.01 versus baseline and placebo). IL-6 receptor levels were not significantly altered by low-grade endotoxemia. In conclusion, endotoxemia up-regulates gp130 expression in vivo and in vitro. Quantification of IL-6 mRNA expression in circulating leukocytes is unlikely a suitable marker for monitoring of endotoxemia.

  11. Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

    PubMed

    Bröker, M; Bäuml, O; Göttig, A; Ochs, J; Bodenbenner, M; Amann, E

    1991-03-01

    The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

  12. Interleukin-10 neutralizing antibody for detection of intestinal luminal levels and as a dietary additive in Eimeria challenged broiler chicks.

    PubMed

    Arendt, Maria K; Sand, Jordan M; Marcone, Taylor M; Cook, Mark E

    2016-02-01

    Interleukin-10 (IL-10) mRNA levels are increased within intestinal mucosa after Eimeria infection. IL-10 apical receptor presence on enterocytes suggests IL-10 is secreted into the intestinal lumen. Increased IL-10 has been shown to be central to the pathogenesis of numerous intracellular pathogens; we hypothesize luminal secretion of IL-10 enables Eimeria spp. infection in chickens. This study examines intestine luminal IL-10 levels and performance in broilers challenged with Eimeria when fed an anti-IL-10 antibody. Chicks were fed a diet (1 to 21 d) with control or anti-IL-10 antibody (0.34 g egg yolk antibody powder/Kg diet) with a saline or 10× dose of Advent coccidiosis vaccine on d 3. One chick per pen was euthanized on days 2, 4, 7, 10, 13, 16, and 19 post-challenge, bled, and intestines were collected for luminal fluid IL-10 concentrations. Body weight and feed intake were measured on d 21, and oocyst shedding was assessed on d 7 post-challenge. A significant Eimeria × antibody interaction on d 21 body weight (P < 0.05) showed chicks fed control antibody, but not anti-IL-10, had significant reductions in body weight when challenged with Eimeria spp. Oocyst shedding was increased with Eimeria challenge, but dietary antibody had no effect. Plasma carotenoid levels were reduced in Eimeria challenged chicks 4, 7, 10, and 16 days post-challenge compared to unchallenged chicks. Lack of an Eimeria × antibody interaction showed anti-IL-10 was not protective against Eimeria-induced decreases in plasma carotenoids. Eimeria challenge increased intestine luminal IL-10 on days 4 and 7 post-challenge in the cecum and jejunum, respectively, compared to unchallenged. Dietary anti-IL-10 decreased luminal IL-10 in the ileum on day 2 post-challenge when compared to control antibody fed chicks. No interaction between Eimeria challenge and antibody was observed on intestine luminal contents of IL-10, suggesting anti-IL-10 was ineffective at preventing increased Eimeria

  13. Recurrent selection for transgene expression levels in maize results in proxy selection for a native gene with the same promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High expression levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High expression levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurre...

  14. Electromagnetic Properties of (Gd, Y)Ba2Cu3Ox Superconducting Tapes With High Levels of Zr Addition

    SciTech Connect

    Liu, Y; Yao, Y; Chen, Y; Khatri, ND; Liu, J; Galtsyan, E; Lei, C; Selvamanickam, V

    2013-06-01

    The dependence of the critical current density (J(c)) on the orientation of applied magnetic fields was studied in Zr-doped (Gd, Y)Ba2Cu3Ox tapes fabricated by metal organic chemical vapor deposition. The in-field performance of J(c) of (Gd, Y)Ba2Cu3Ox tapes with Zr-doping levels of 7.5-30 at.% was investigated up to 5 T over a temperature range of 40-77 K. The highest critical currents (I-c) at H parallel to c and the highest values of minimum Ic in angular dependence measurements were achieved in the tapes with 20% Zr doping over a broad range of temperature and magnetic field conditions measured. The electromagnetic properties have been related to the changes in BaZrO3 content and microstructure.

  15. Ascorbic acid inhibits PMP22 expression by reducing cAMP levels.

    PubMed

    Kaya, Ferdinand; Belin, Sophie; Bourgeois, Patrice; Micaleff, Joelle; Blin, Olivier; Fontés, Michel

    2007-03-01

    Charcot-Marie-Tooth [CMT] syndrome is the most common hereditary peripheral neuropathy. CMT1A, which accounts for 50% of all CMT cases, usually results from triploidy of the PMP22 gene. Preclinical trials using an animal model show that disabled mice force-fed with high doses of ascorbic acid partially recover muscular strength after a few months of treatment, and suggest that high doses of ascorbic acid repress PMP22 expression. In this study, we demonstrated that ascorbic acid represses PMP22 gene expression by acting on intracellular cAMP levels and adenylate cyclase activity. This action is dose dependent and specific to ascorbic acid, since repression is not observed after treatment with other antioxidants. The new properties of ascorbic acid are discussed, along with the implications of these findings for CMT disease treatment.

  16. Level of CYP4G19 Expression Is Associated with Pyrethroid Resistance in Blattella germanica.

    PubMed

    Guo, Guang-Zhou; Geng, Yi-Jie; Huang, Da-Na; Xue, Cai-Fang; Zhang, Ren-Li

    2010-01-01

    German cockroaches have become a large problem in the Shenzhen area because of their pesticide resistance, especially to pyrethroid. A pyrethroid called "Jia Chong Qing" to prevent pests for a long time were found to be resistant to "Jia Chong Qing" with resistance index of 3.88 measured using RT-PCR and immunohistochemistry analysis showed that both CYP4G19 mRNA and CYP4G19 protein expression levels in the wild strain were substantially higher than that of a sensitive strain. dsRNA segments derived from the target gene CYP4G19 were prepared using in vitro transcription and were microinjected into abdomens of the wild strain. Two to eight days after injection, the result showed that CYP4G19 mRNA expressions were significantly reduced in the groups injected with dsRNAs.

  17. Effects of pulp and paper mill effluent extractives on aromatase CYP19a gene expression and sex steroid levels in juvenile triploid rainbow trout.

    PubMed

    Orrego, Rodrigo; McMaster, Mark; Van Der Kraak, Glen; Holdway, Douglas

    2010-05-10

    We evaluated plasma testosterone (T) and 17beta-estradiol (E2) levels and ovarian aromatase CYP19a gene expression following a single intraperitoneal injection of Chilean pulp and paper mill effluent extracts into juvenile triploid rainbow trout. Fish injected with untreated effluent extracts had increased plasma T after 4 days, while plasma E2 concentration was increased in fish injected with both primary and secondary treated effluent extracts at the same sampling period. Ovarian CYP19a gene expression as measured by qRT-PCR was significantly induced in fish injected with the untreated, primary and secondary treated pulp and paper mill effluent extracts. Similar induction of CYP19a expression was found in fish injected with the androgens androstenedione (ADD) and T. A Principal Component Analysis (PCA) was conducted in order to identify structure in relationships between all measured variables and identifying which factors were most responsible for the variance observed within the plasma steroid levels, upregulation of ovarian CYP19a gene expression and the final estrogenic effect of increased plasma VTG levels. This analysis indicated a cluster correlation between plasma T levels and CYP19a gene expression (Factor 1, explaining 27.2% of total variance), a cluster including condition factor and liver somatic index (Factor 2, explaining 17.3%) and an additional cluster including plasma E2 and vitellogenin levels (Factor 3, explaining an additional 15.8%). The present results indicate that Chilean pulp and paper mill effluent extracts cause estrogenic effects in triploid rainbow trout. These effects could be related to the compounds present in the effluent that act as estrogen receptor agonists, or that induce changes leading to increased amounts of endogenous estrogens, reflected by increased E2 levels and induced aromatase expression/activity.

  18. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma

    PubMed Central

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)–transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease. PMID:26147378

  19. Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

    PubMed

    Roselló, Paula L; Vigliocco, Ana E; Andrade, Andrea M; Riera, Natalí V; Calafat, Mario; Molas, María L; Alemano, Sergio G

    2016-05-01

    Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself.

  20. The levels of RAC3 expression are up regulated by TNF in the inflammatory response.

    PubMed

    Alvarado, Cecilia Viviana; Rubio, María Fernanda; Fernández Larrosa, Pablo Nicolas; Panelo, Laura Carolina; Azurmendi, Pablo Javier; Ruiz Grecco, Marina; Martínez-Nöel, Giselle Astrid; Costas, Mónica Alejandra

    2014-01-01

    RAC3 is a coactivator of glucocorticoid receptor and nuclear factor-κB (NF-κB) that is usually over-expressed in tumors and which also has important functions in the immune system. We investigated the role of the inflammatory response in the control of RAC3 expression levels in vivo and in vitro. We found that inflammation regulates RAC3 levels. In mice, sub-lethal doses of lipopolysaccharide induce the increase of RAC3 in spleen and the administration of the synthetic anti-inflammatory glucocorticoid dexamethasone has a similar effect. However, the simultaneous treatment with both stimuli is mutually antagonistic. In vitro stimulation of the HEK293 cell line with tumor necrosis factor (TNF), one of the cytokines induced by lipopolysaccharide, also increases the levels of RAC3 mRNA and protein, which correlates with an enhanced transcription dependent on the RAC3 gene promoter. We found that binding of the transcription factor NF-κB to the RAC3 gene promoter could be responsible for these effects. Our results suggest that increase of RAC3 during the inflammatory response could be a molecular mechanism involved in the control of sensitivity to both pro- and anti-inflammatory stimuli in order to maintain the normal healthy course of the immune response.

  1. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.

  2. Increases in c-Yes expression level and activity promote motility but not proliferation of human colorectal carcinoma cells.

    PubMed

    Barraclough, Jane; Hodgkinson, Cassandra; Hogg, Alison; Dive, Caroline; Welman, Arkadiusz

    2007-09-01

    Increases in the levels and/or activity of nonreceptor tyrosine kinases c-Src and c-Yes are often associated with colorectal carcinogenesis. The physiological consequences of increased c-Yes activity during the early and late stages of tumorigenesis, in addition to the degree of redundancy between c-Yes and c-Src in colorectal cancer cells, remain elusive. To study the consequences of increases in c-Yes levels and activity in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Yes levels and activity can be inducibly increased by a tightly controlled expression of wild-type c-Yes or by constitutively active mutants of c-Yes, c-YesY537F, and c-Yes Delta t6aa. c-Yes induction resulted in increased cell motility but did not promote proliferation either in vitro or in vivo. These results suggest that in later stages of colorectal carcinogenesis, elevations in c-Yes levels/activity may promote cancer spread and metastasis rather than tumor growth.

  3. Improving Polycyclic Aromatic Hydrocarbon Biodegradation in Contaminated Soil Through Low-Level Surfactant Addition After Conventional Bioremediation.

    PubMed

    Adrion, Alden C; Singleton, David R; Nakamura, Jun; Shea, Damian; Aitken, Michael D

    2016-09-01

    Efficacy of bioremediation for soil contaminated with polycyclic aromatic hydrocarbons (PAHs) may be limited by the fractions of soil-bound PAHs that are less accessible to PAH-degrading microorganisms. In previous test-tube-scale work, submicellar doses of nonionic surfactants were screened for their ability to enhance the desorption and biodegradation of residual PAHs in soil after conventional bioremediation in a laboratory-scale, slurry-phase bioreactor. Polyoxyethylene sorbitol hexaoleate (POESH) was the optimum surfactant for enhancing PAH removal, especially the high-molecular weight PAHs. This work extends that concept by treating the effluent from the slurry-phase bioreactor in a second-stage batch reactor, to which POESH was added, for an additional 7 or 12 days. Surfactant amendment removed substantial amounts of the PAHs and oxy-PAHs remaining after conventional slurry-phase bioremediation, including more than 80% of residual 4-ring PAHs. Surfactant-amended treatment decreased soil cytotoxicity, but often increased the genotoxicity of the soil as measured using the DT-40 chicken lymphocyte DNA damage response assay. Potential ecotoxicity, measured using a seed germination assay, was reduced by bioreactor treatment and was reduced further after second-stage treatment with POESH. Of bacteria previously implicated as potential PAH degraders under POESH-amended conditions in a prior study, members of the Terrimonas genus were associated with differences in high-molecular weight PAH removal in the current study. Research using submicellar doses of surfactant as a second-stage treatment step is limited and these findings can inform the design of bioremediation systems at field sites treating soil contaminated with PAHs and other hydrophobic contaminants that have low bioaccessibility.

  4. GENE EXPRESSION PROFILES IN ARSENIC-TREATED MCF-7 BREAST CANCER CELLS EXPRESSING DIFFERENT LEVELS OF HSP70

    EPA Science Inventory

    Gene expression profiles in arsenic-treated MCF-7 breast cancer cells expressing different levels of HSP70

    Gail Nelson, Susan Hester, Ernest Winkfield, Jill Barnes, James Allen
    Environmental Carcinogenesis Division, NHEERL, ORD, US Environmental Protection Agency, Rese...

  5. Strategies for achieving high-level expression of genes in Escherichia coli.

    PubMed Central

    Makrides, S C

    1996-01-01

    Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

  6. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae

    PubMed Central

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

    Abstract Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon‐optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half‐lives of 40 and 5 min, respectively. The commercial substrate Nano‐Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat‐shock promoter (PCYC1–HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  7. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd.

  8. Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure.

    PubMed

    Thomas, Reuben; Hubbard, Alan E; McHale, Cliona M; Zhang, Luoping; Rappaport, Stephen M; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z; Jinot, Jennifer; Sonawane, Babasaheb R; Smith, Martyn T

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings.

  9. Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure

    PubMed Central

    Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

  10. Non-additive hepatic gene expression elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) co-treatment in C57BL/6 mice

    SciTech Connect

    Kopec, Anna K.; D'Souza, Michelle L.; Mets, Bryan D.; Burgoon, Lyle D.; Reese, Sarah E.; Archer, Kellie J.; Potter, Dave; Tashiro, Colleen; Sharratt, Bonnie; Harkema, Jack R.; Zacharewski, Timothy R.

    2011-10-15

    Interactions between environmental contaminants can lead to non-additive effects that may affect the toxicity and risk assessment of a mixture. Comprehensive time course and dose-response studies with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), non-dioxin-like 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) and their mixture were performed in immature, ovariectomized C57BL/6 mice. Mice were gavaged once with 30 {mu}g/kg TCDD, 300 mg/kg PCB153, a mixture of 30 {mu}g/kg TCDD with 300 mg/kg PCB153 (MIX) or sesame oil vehicle for 4,12, 24,72 or 168 h. In the 24 h dose-response study, animals were gavaged with TCDD (0.3,1, 3, 6, 10, 15, 30, 45 {mu}g/kg), PCB153 (3,10, 30, 60, 100, 150, 300, 450 mg/kg), MIX (0.3 + 3, 1 + 10, 3 + 30, 6 + 60, 10 + 100, 15 + 150, 30 + 300, 45 {mu}g/kg TCDD + 450 mg/kg PCB153, respectively) or vehicle. All three treatments significantly increased relative liver weights (RLW), with MIX eliciting significantly greater increases compared to TCDD and PCB153 alone. Histologically, MIX induced hepatocellular hypertrophy, vacuolization, inflammation, hyperplasia and necrosis, a combination of TCDD and PCB153 responses. Complementary lipid analyses identified significant increases in hepatic triglycerides in MIX and TCDD samples, while PCB153 had no effect on lipids. Hepatic PCB153 levels were also significantly increased with TCDD co-treatment. Microarray analysis identified 167 TCDD, 185 PCB153 and 388 MIX unique differentially expressed genes. Statistical modeling of quantitative real-time PCR analysis of Pla2g12a, Serpinb6a, Nqo1, Srxn1, and Dysf verified non-additive expression following MIX treatment compared to TCDD and PCB153 alone. In summary, TCDD and PCB153 co-treatment elicited specific non-additive gene expression effects that are consistent with RLW increases, histopathology, and hepatic lipid accumulation. - Graphical abstract: Display Omitted Highlights: > MIX (TCDD:PCB153 at 1:10,000 ratio) exposure leads to non-additive gene expression

  11. Identification of a sugar beet BvM14-MADS box gene through differential gene expression analysis of monosomic addition line M14.

    PubMed

    Ma, Chunquan; Wang, Yuguang; Wang, Yuting; Wang, Lifa; Chen, Sixue; Li, Haiying

    2011-11-01

    Monosomic addition line M14 carrying an additional chromosome 9 from Beta corolliflora Zosimovic ex Buttler was obtained through hybridization between the wild species B. corolliflora and a cultivated species Beta vulgaris L. var Saccharifera Alef. The M14 line showed diplosporic reproduction and stress tolerance. To identify differentially expressed genes in M14, a subtractive cDNA library was prepared by suppression subtractive hybridization (SSH) between M14 (2n=18+1) and B. vulgaris (2n=18). A total of 190 unique sequences were identified in the library and their putative functions were analyzed using Gene Ontology (GO). One of the genes, designated as BvM14-MADS box, encodes a MADS box transcription factor. It was cloned from M14 and over-expressed in transgenic tobacco plants. Interestingly, this gene was located on chromosome 2 of B. vulgaris, not on the additional chromosome 9. Overexpression of BvM14-MADS box led to significant phenotypic changes in tobacco. The differential expression of BvM14-MADS box gene in M14 may be caused by the interaction between the additional chromosome 9 from B. corolliflora and the B. vulgaris chromosomes in M14.

  12. Regulation of Connexins Expression Levels by MicroRNAs, an Update

    PubMed Central

    Calderón, Juan F.; Retamal, Mauricio A.

    2016-01-01

    Control of cell-cell coordination and communication is regulated by several factors, including paracrine and autocrine release of biomolecules, and direct exchange of soluble factors between cells through gap junction channels. Additionally, hemichannels also participate in cell-cell coordination through the release of signaling molecules, such as ATP and glutamate. A family of transmembrane proteins named connexins forms both gap junction channels and hemichannels. Because of their importance in cell and tissue coordination, connexins are controlled both by post-translational and post-transcriptional modifications. In recent years, non-coding RNAs have garnered research interest due to their ability to exert post-transcriptional regulation of gene expression. One of the most recent, well-documented control mechanisms of protein synthesis is found through the action of small, single-stranded RNA, called micro RNAs (miRNAs or miRs). Put simply, miRNAs are negative regulators of the expression of a myriad proteins involved in many physiological and pathological processes. This mini review will briefly summarize what is currently known about the action of miRNAs over Cxs expression/function in different organs under some relevant physiological and pathological conditions. PMID:27932990

  13. Regulation of Connexins Expression Levels by MicroRNAs, an Update.

    PubMed

    Calderón, Juan F; Retamal, Mauricio A

    2016-01-01

    Control of cell-cell coordination and communication is regulated by several factors, including paracrine and autocrine release of biomolecules, and direct exchange of soluble factors between cells through gap junction channels. Additionally, hemichannels also participate in cell-cell coordination through the release of signaling molecules, such as ATP and glutamate. A family of transmembrane proteins named connexins forms both gap junction channels and hemichannels. Because of their importance in cell and tissue coordination, connexins are controlled both by post-translational and post-transcriptional modifications. In recent years, non-coding RNAs have garnered research interest due to their ability to exert post-transcriptional regulation of gene expression. One of the most recent, well-documented control mechanisms of protein synthesis is found through the action of small, single-stranded RNA, called micro RNAs (miRNAs or miRs). Put simply, miRNAs are negative regulators of the expression of a myriad proteins involved in many physiological and pathological processes. This mini review will briefly summarize what is currently known about the action of miRNAs over Cxs expression/function in different organs under some relevant physiological and pathological conditions.

  14. Effect of Feed Melting, Temperature History and Minor Component Addition on Spinel Crystallization in High-Level Waste Glass

    SciTech Connect

    Izak, Pavel; Hrma, Pavel R.; Arey, Bruce W.; Plaisted, Trevor J.

    2001-08-01

    This study was undertaken to help design mathematical models for high-level waste (HLW) glass melter that simulate spinel behavior in molten glass. Spinel, (Fe,Ni,Mn) (Fe,Cr)2O4, is the primary solid phase that precipitates from HLW glasses containing Fe and Ni in sufficient concentrations. Spinel crystallization affects the anticipated cost and risk of HLW vitrification. To study melting reactions, we used simulated HLW feed, prepared with co-precipitated Fe, Ni, Cr, and Mn hydroxides. Feed samples were heated up at a temperature-increase rate (4C/min) close to that which the feed experiences in the HLW glass melter. The decomposition, melting, and dissolution of feed components (such as nitrates, carbonates, and silica) and the formation of intermediate crystalline phases (spinel, sodalite [Na8(AlSiO4)6(NO2)2], and Zr-containing minerals) were characterized using evolved gas analysis, volume-expansion measurement, optical microscope, scanning electron microscope, thermogravimetric analysis, differential scanning calorimetry, and X-ray diffraction. Nitrates and quartz, the major feed components, converted to a glass-forming melt by 880C. A chromium-free spinel formed in the nitrate melt starting from 520C and Sodalite, a transient product of corundum dissolution, appeared above 600C and eventually dissolved in glass. To investigate the effects of temperature history and minor components (Ru,Ag, and Cu) on the dissolution and growth of spinel crystals, samples were heated up to temperatures above liquidus temperature (TL), then subjected to different temperature histories, and analyzed. The results show that spinel mass fraction, crystals composition, and crystal size depend on the chemical and physical makeup of the feed and temperature history.

  15. Activation of AMPKα mediates additive effects of solamargine and metformin on suppressing MUC1 expression in castration-resistant prostate cancer cells

    PubMed Central

    Xiang, SongTao; Zhang, QiuHong; Tang, Qing; Zheng, Fang; Wu, JingJing; Yang, LiJun; Hann, Swei Sunny

    2016-01-01

    Prostate cancer is the second most common cause of cancer-related deaths worldwide. The mucin 1 (MUC1) oncoprotein is highly expressed in human prostate cancers with aggressive features. However, the role for MUC1 in occurrence and progression of castration-resistant prostate cancer (CRPC) remained elusive. In this study, we showed that solamargine, a major steroidal alkaloid glycoside, inhibited the growth of CRPC cells, which was enhanced in the presence of metformin. Furthermore, we found that solamargine increased phosphorylation of AMPKα, whereas reducing the protein expression and promoter activity of MUC1. A greater effect was observed in the presence of metformin. In addition, solamargine reduced NF-κB subunit p65 protein expression. Exogenously expressed p65 resisted solamargine-reduced MUC1 protein and promoter activity. Interestingly, exogenously expressed MUC1 attenuated solamargine-stimulated phosphorylation of AMPKα and, more importantly reversed solamargine-inhibited cell growth. Finally, solamargine increased phosphorylation of AMPKα, while inhibiting MUC1, p65 and tumor growth were observed in vivo. Overall, our results show that solamargine inhibits the growth of CRPC cells through AMPKα-mediated inhibition of p65, followed by reduction of MUC1 expression in vitro and in vivo. More importantly, metformin facilitates the antitumor effect of solamargine on CRPC cells. PMID:27830724

  16. Regulation of Ubx expression by epigenetic enhancer silencing in response to Ubx levels and genetic variation.

    PubMed

    Crickmore, Michael A; Ranade, Vikram; Mann, Richard S

    2009-09-01

    For gene products that must be present in cells at defined concentrations, expression levels must be tightly controlled to ensure robustness against environmental, genetic, and developmental noise. By studying the regulation of the concentration-sensitive Drosophila melanogaster Hox gene Ultrabithorax (Ubx), we found that Ubx enhancer activities respond to both increases in Ubx levels and genetic background. Large, transient increases in Ubx levels are capable of silencing all enhancer input into Ubx transcription, resulting in the complete silencing of this gene. Small increases in Ubx levels, brought about by duplications of the Ubx locus, cause sporadic silencing of subsets of Ubx enhancers. Ubx enhancer silencing can also be induced by outcrossing laboratory stocks to D. melanogaster strains established from wild flies from around the world. These results suggest that enhancer activities are not rigidly determined, but instead are sensitive to genetic background. Together, these findings suggest that enhancer silencing may be used to maintain gene product levels within the correct range in response to natural genetic variation.

  17. Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

    PubMed Central

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

    2013-01-01

    Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

  18. Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging.

    PubMed

    Zhang, Fenni; Wang, Shaopeng; Yin, Linliang; Yang, Yunze; Guan, Yan; Wang, Wei; Xu, Han; Tao, Nongjian

    2015-10-06

    Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 10(5) M(-1) s(-1) and (1.4 ± 0.5) × 10(-4) s(-1), respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm(2) with an estimation of 5 × 10(5) EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

  19. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

    PubMed Central

    Kanobe, Milly N.; Rodermel, Steven R.; Bailey, Theodore; Scott, M. Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls. PMID:23785377

  20. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene.

    PubMed

    Kanobe, Milly N; Rodermel, Steven R; Bailey, Theodore; Scott, M Paul

    2013-01-01

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337) on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium, and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

  1. Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

    PubMed

    Solomon, R G; Appels, R

    1999-06-01

    The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

  2. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    PubMed

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  3. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression

    PubMed Central

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels (χKruskal2-Wallis, df(3) = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = −7.133, P = 0.002) and Non-PSD group (FBonferroni = −5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081–1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656–0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression. PMID:28082897

  4. The Role of Neuropeptide Y mRNA Expression Level in Distinguishing Different Types of Depression.

    PubMed

    Yue, Yingying; Jiang, Haitang; Yin, Yingying; Zhang, Yuqun; Liang, Jinfeng; Li, Shenghua; Wang, Jun; Lu, Jianxin; Geng, Deqin; Wu, Aiqin; Yuan, Yonggui

    2016-01-01

    Previous studies demonstrate that the protein of neuropeptide Y (NPY) is abnormal in depression patients, but the changes of NPY in different types of depression are unclear. This study was aimed to examine protein and mRNA expression levels of NPY in 159 cases with four groups including post-stroke depression (PSD) group, stroke without depression (Non-PSD) group, major depressive disorder (MDD) group and normal control (NC) group. The protein and gene expression analysis were performed by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction-based methods. One way analysis of variance (ANOVA), chi-square tests and nonparametric test were used to evaluate general characteristics, clinical and biological materials. In order to explore the role of NPY in different types of depression, the partial correlations, binary logistic regression analysis and receiver operating characteristic (ROC) curve were calculated for PSD and MDD groups. There are significant differences of NPY protein (Fdf(3) = 5.167, P = 0.002) and mRNA expression levels ([Formula: see text] = 20.541, P < 0.001) among four groups. Bonferroni multiple comparisons found that the NPY protein was significantly decreased in PSD (FBonferroni = -7.133, P = 0.002) and Non-PSD group (FBonferroni = -5.612, P = 0.018) compared with NC group. However, contrasted with MDD group, the mRNA expression was increased in PSD and Non-PSD group by nonparametric test (all P < 0.05). In binary logistic analyses, NPY mRNA expression was independent predictors of PSD (odds ratio: 1.452, 95% CI, 1.081-1.951, P = 0.013). The ROC curve showed NPY mRNA had a general prognostic accuracy (area under the curve: 0.766, 95% CI, 0.656-0.876, P < 0.001). This is the first study to explore the distinguishing function of NPY in different types of depression. It will provide help in the identification of different subtypes of depression.

  5. High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

    PubMed

    Chen, Yu-Chia; Chang, Jan-Gowth; Jong, Yuh-Jyh; Liu, Ting-Yuan; Yuo, Chung-Yee

    2015-01-01

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn-/- SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.

  6. Direct effect of acaricides on pathogen loads and gene expression levels in honey bees Apis mellifera.

    PubMed

    Boncristiani, Humberto; Underwood, Robyn; Schwarz, Ryan; Evans, Jay D; Pettis, Jeffery; vanEngelsdorp, Dennis

    2012-05-01

    The effect of using acaricides to control varroa mites has long been a concern to the beekeeping industry due to unintended negative impacts on honey bee health. Irregular ontogenesis, suppression of immune defenses, and impairment of normal behavior have been linked to pesticide use. External stressors, including parasites and the pathogens they vector, can confound studies on the effects of pesticides on the metabolism of honey bees. This is the case of Varroa destructor, a mite that negatively affects honey bee health on many levels, from direct parasitism, which diminishes honey bee productivity, to vectoring and/or activating other pathogens, including many viruses. Here we present a gene expression profile comprising genes acting on diverse metabolic levels (detoxification, immunity, and development) in a honey bee population that lacks the influence of varroa mites. We present data for hives treated with five different acaricides; Apiguard (thymol), Apistan (tau-fluvalinate), Checkmite (coumaphos), Miteaway (formic acid) and ApiVar (amitraz). The results indicate that thymol, coumaphos and formic acid are able to alter some metabolic responses. These include detoxification gene expression pathways, components of the immune system responsible for cellular response and the c-Jun amino-terminal kinase (JNK) pathway, and developmental genes. These could potentially interfere with the health of individual honey bees and entire colonies.

  7. Utilizing the Carba NP test as an indicator of expression level of carbapenemase genes in Enterobacteriaceae.

    PubMed

    Segawa, Takaya; Matsui, Mari; Suzuki, Masato; Tsutsui, Atsuko; Kuroda, Makoto; Shibayama, Keigo; Suzuki, Satowa

    2017-02-01

    The Carba NP test was developed to detect carbapenemase-producing Enterobacteriaceae, and uses imipenem as the reaction substrate. In Japan, IMP-6 metallo-β-lactamase (MBL) producers, which are usually resistant to meropenem but susceptible to imipenem, and IMP-1 MBL producers, which are usually resistant to both carbapenems are prevalent. We performed the Carba NP test with IMP-6 and IMP-1 MBL producers, and both types were detected by the Carba NP test with high sensitivity. All IMP-1 MBL producers were detected by the Carba NP test, but the minimum inhibitory concentrations (MICs) of imipenem varied from 0.25 to >32μg/mL, and the time to positivity varied from 0 to 30min. Time to positivity was significantly correlated with expression levels of blaIMP-1, but not with MICs of imipenem. These results suggested that the Carba NP test can be used as a screening assay for carbapenemase gene expression levels among producers of the same type of carbapenemase. Using this approach, it is possible to determine whether the carbapenem resistance of each carbapenemase-producing Enterobacteriaceae isolate is primarily due to carbapenemase production, or to another mechanism such as outer membrane impermeability.

  8. Expression of DMP-1 in the human pulp tissue using low level laser therapy

    NASA Astrophysics Data System (ADS)

    Lourenço Neto, Natalino; Teixeira Marques, Nádia Carolina; Fernandes, Ana Paula; Oliveira Rodini, Camila; Cruvinel Silva, Thiago; Moreira Machado, Maria Aparecida Andrade; Marchini Oliveira, Thais

    2015-09-01

    This study aimed to evaluate the effects of low-level laser therapy (LLLT) on DMP-1 expression in pulp tissue repair of human primary teeth. Twenty mandibular primary molars were randomly assigned into the following groups: Group I—Buckley’s Formocresol (FC); Group II—Calcium Hydroxide (CH); Group III—LLLT + CH and Group IV—LLLT + Zinc oxide/Eugenol. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Descriptive analysis was performed on the dentin pulp complex. Histopathological assessment showed internal resorption in group FC. Groups CH and LLLT + CH provided better pulpal repair due to the absence of inflammation and the formation of hard tissue barrier. These two groups presented odontoblastic layer expressing DMP-1. According to this study, low level laser therapy preceding the use of calcium hydroxide exhibited satisfactory bio-inductive activity on pulp tissue repair of human primary teeth. However, other histological and cellular studies are needed to confirm the laser tissue action and efficacy.

  9. Estradiol replacement enhances fear memory formation, impairs extinction and reduces COMT expression levels in the hippocampus of ovariectomized female mice.

    PubMed

    McDermott, Carmel M; Liu, Dan; Ade, Catherine; Schrader, Laura A

    2015-02-01

    Females experience depression, posttraumatic stress disorder (PTSD), and anxiety disorders at approximately twice the rate of males, but the mechanisms underlying this difference remain undefined. The effect of sex hormones on neural substrates presents a possible mechanism. We investigated the effect of ovariectomy at two ages, before puberty and in adulthood, and 17β-estradiol (E2) replacement administered chronically in drinking water on anxiety level, fear memory formation, and extinction. Based on previous studies, we hypothesized that estradiol replacement would impair fear memory formation and enhance extinction rate. Females, age 4 weeks and 10 weeks, were divided randomly into 4 groups; sham surgery, OVX, OVX+low E2 (200nM), and OVX+high E2 (1000nM). Chronic treatment with high levels of E2 significantly increased anxiety levels measured in the elevated plus maze. In both age groups, high levels of E2 significantly increased contextual fear memory but had no effect on cued fear memory. In addition, high E2 decreased the rate of extinction in both ages. Finally, catechol-O-methyltransferase (COMT) is important for regulation of catecholamine levels, which play a role in fear memory formation and extinction. COMT expression in the hippocampus was significantly reduced by high E2 replacement, implying increased catecholamine levels in the hippocampus of high E2 mice. These results suggest that estradiol enhanced fear memory formation, and inhibited fear memory extinction, possibly stabilizing the fear memory in female mice. This study has implications for a neurobiological mechanism for PTSD and anxiety disorders.

  10. The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma.

    PubMed

    Hoebeeck, Jasmien; Vandesompele, Jo; Nilsson, Helén; De Preter, Katleen; Van Roy, Nadine; De Smet, Els; Yigit, Nurten; De Paepe, Anne; Laureys, Geneviève; Påhlman, Sven; Speleman, Frank

    2006-08-01

    Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.

  11. Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

    PubMed

    Barrachina, Marta; Castaño, Esther; Dalfó, Esther; Maes, Tamara; Buesa, Carlos; Ferrer, Isidro

    2006-05-01

    Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

  12. In situ detection of specific gene expression during and immediately after transcription at electron microscopic level.

    PubMed

    Kitazawa, Sohei; Kitazawa, Riko

    2006-01-01

    In situ hybridization (ISH) is a widely applied technique used for visualizing specific nucleic acid sequences at chromosomal, cytologic, and histologic levels. It sometimes fails, however, to demonstrate precise cell identity, early stages of gene expression and variants of alternative splicing because of its limited resolution. To overcome this shortcoming, we have developed an improved ISH technique at the electron microscopic (EM) level by conducting en bloc hybridization before embedding (pre-embedding) and immuno-EM detection after ultra-thin sectioning (post-embedding). We applied this technique to demonstrate both the dynamic expression of interleukin (IL)-6 mRNA immediately after lipopolysaccharide (LPS) treatment, and the static expression of osteonectin mRNA in a differentiating osteoblastic cell linage. Tissue samples were diced into 1mm cubes, fixed with 4% paraformaldehyde, and then successively hybridized en bloc with the digoxigenin (DIG)-labeled single-stranded probe measuring 200-300 bp with the aid of microwave treatment. After washing, for EM observation, the cubes were embedded in epon for ultra-thin sectioning, and a gold-colloid-labeled anti-DIG antibody was used for post-embedding immuno-EM; some of the cubes was directly incubated with anti-DIG antibody and developed en bloc for stereoscopic and light microscopic observation. IL-6 mRNA during and immediately after transcription was demonstrated in the nuclei of the alveolar macrophages and in neutrophils of mouse lung tissue as early as 15 min after LPS treatment, which was of better sensitivity than that by Northern blot or nuclear run-on techniques. Moreover, in mouse calvaria tissue, osteonectin mRNA both in the nucleus and the cytoplasm was observed in a differentiating osteoblastic cell linage in a differentiation-specific manner. This technique is useful in identifying specific cell types during and immediately after transcribing specific mRNA based on ultrastructural morphology.

  13. Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC

    PubMed Central

    2013-01-01

    Background Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2‾), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs. Method Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2‾ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement. Results We showed that the O2‾ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high

  14. Endogenous galectin-3 expression levels modulate immune responses in galectin-3 transgenic mice.

    PubMed

    Chaudhari, Aparna D; Gude, Rajiv P; Kalraiya, Rajiv D; Chiplunkar, Shubhada V

    2015-12-01

    Galectin-3 (Gal-3), a β-galactoside-binding mammalian lectin, is involved in cancer progression and metastasis. However, there is an unmet need to identify the underlying mechanisms of cancer metastasis mediated by endogenous host galectin-3. Galectin-3 is also known to be an important regulator of immune responses. The present study was aimed at analysing how expression of endogenous galectin-3 regulates host immunity and lung metastasis in B16F10 murine melanoma model. Transgenic Gal-3(+/-) (hemizygous) and Gal-3(-/-) (null) mice exhibited decreased levels of Natural Killer (NK) cells and lower NK mediated cytotoxicity against YAC-1 tumor targets, compared to Gal-3(+/+) (wild-type) mice. On stimulation, Gal-3(+/-) and Gal-3(-/-) mice splenocytes showed increased T cell proliferation than Gal-3(+/+) mice. Intracellular calcium flux was found to be lower in activated T cells of Gal-3(-/-) mice as compared to T cells from Gal-3(+/+) and Gal-3(+/-) mice. In Gal-3(-/-) mice, serum Th1, Th2 and Th17 cytokine levels were found to be lowest, exhibiting dysregulation of pro-inflammatory and anti-inflammatory cytokines balance. Marked decrease in serum IFN-γ levels and splenic IFN-γR1 (IFN-γ Receptor 1) expressing T and NK cell percentages were observed in Gal-3(-/-) mice. On recombinant IFN-γ treatment of splenocytes in vitro, Suppressor of Cytokine Signaling (SOCS) 1 and SOCS3 protein expression was higher in Gal-3(-/-) mice compared to that in Gal-3(+/+) and Gal-3(+/-) mice; suggesting possible attenuation of Signal Transducer and Activator of Transcription (STAT) 1 mediated IFN-γ signaling in Gal-3(-/-) mice. The ability of B16F10 melanoma cells to form metastatic colonies in the lungs of Gal-3(+/+) and Gal-3(-/-) mice remained comparable, whereas it was found to be reduced in Gal-3(+/-) mice. Our data indicates that complete absence of endogenous host galectin-3 facilitates lung metastasis of B16F10 cells in mice, which may be contributed by dysregulated immune

  15. Development of ileal cytokine and immunoglobulin expression levels in response to early feeding in broilers and layers.

    PubMed

    Simon, K; de Vries Reilingh, G; Kemp, B; Lammers, A

    2014-12-01

    Provision of feed in the immediate posthatch period may influence interaction between intestinal microbiota and immune system, and consequently immunological development of the chick. This study addressed ileal immune development in response to early feeding in 2 chicken breeds selected for different production traits: broilers and layers. Chicks of both breeds either received feed and water immediately posthatch or were subjected to a 72-h feed and water delay. Ileal cytokine and immunoglobulin mRNA expression levels were determined at different time points. Effects of early feeding were limited, but breeds differed strikingly regarding cytokine and immunoglobulin expression levels. Cytokine expression levels in broilers were low compared with layers and showed a transient drop in the second to third week of life. In contrast, broilers showed considerably higher expression levels of IgA, IgM, and IgY. These findings indicate that the 2 breeds use different immune strategies, at least on the ileal level.

  16. Glutaminase expression is a poor prognostic factor in node-positive triple-negative breast cancer patients with a high level of tumor-infiltrating lymphocytes.

    PubMed

    Kim, Joo Young; Heo, Sun-Hee; Choi, Seul Ki; Song, In Hye; Park, In Ah; Kim, Young-Ae; Park, Hye Seon; Park, Suk Young; Bang, Won Seon; Gong, Gyungyub; Lee, Hee Jin

    2017-04-01

    Glutamine metabolism is emerging as one aspect of dysregulated metabolism of tumors. Triple-negative breast cancer (TNBC) cells are glutamine dependent, whereas luminal-type cells tend to be glutamine independent. Therefore, TNBC patients might benefit from therapies targeting glutamine metabolism. To investigate the clinical significance of glutamine metabolism, we examined expression and prognostic significance of glutaminase in tumor cells and tumor-infiltrating lymphocytes (TILs) in TNBC. We retrieved 658 surgically resected TNBCs and analyzed glutaminase expression in tumor cells and TILs by immunohistochemical staining. Glutaminase expression was observed in 237 cases (36.0%) in tumor cells and 104 cases (15.5%) in TILs. Although glutaminase expression in tumor cells was significantly associated with a low level of TILs (p = 0.018), glutaminase expression in TILs was significantly higher in cases with a high level of TILs (p = 0.031). Glutaminase expression in tumor cells was significantly associated with poor disease-free survival in patients with lymph node metastasis and high levels of TILs (p = 0.020). In addition, it was an independent poor prognostic factor (hazard ratio = 10.643, 95% confidence interval = 1.999-56.668; p = 0.006). Glutaminase expression in tumor cells was observed in a subset of TNBC patients. It was significantly associated with a low level of TILs and poor disease-free survival in TNBCs presenting with lymph node metastasis and high levels of TILs.

  17. Relationship between Rgs2 gene expression level and anxiety and depression-like behaviour in a mutant mouse model: serotonergic involvement.

    PubMed

    Lifschytz, Tzuri; Broner, Esther Channah; Zozulinsky, Polina; Slonimsky, Alexandra; Eitan, Renana; Greenbaum, Lior; Lerer, Bernard

    2012-10-01

    RGS2 is a member of a family of proteins that negatively modulate G-protein coupled receptor transmission. Variations in the RGS2 gene were found to be associated in humans with anxious and depressive phenotypes. We sought to study the relationship of Rgs2 expression level to depression and anxiety-like behavioural features, sociability and brain 5-HT1A and 5-HT1B receptor expression. We studied male mice carrying a mutation that causes lower Rgs2 gene expression, employing mice heterozygous (Het) or homozygous (Hom) for this mutation, or wild-type (WT). Mice were subjected to behavioural tests reflecting depressive-like behaviour [forced swim test (FST), novelty suppressed feeding test (NSFT)], elevated plus maze (EPM) for evaluation of anxiety levels and the three-chamber sociability test. The possible involvement of raphe nucleus 5-HT1A receptors in these behavioural features was examined by 8-OH-DPAT-induced hypothermia. Expression levels of 5-HT1A and 5-HT1B receptors in the cortex, raphe nucleus and hypothalamus were compared among mice of the different Rgs2 genotype groups. NSFT results demonstrated that Hom mice showed more depressive-like features than Rgs2 Het and WT mice. A trend for such a relationship was also suggested by the FST results. EPM and sociability test results showed Hom and Het mice to be more anxious and less sociable than WT mice. In addition Hom and Het mice were characterized by lower basal body temperature and demonstrated less 8-OH-DPAT-induced hypothermia than WT mice. Finally, Hom and Het mice had significantly lower 5-HT1A and 5-HT1B receptor expression levels in the raphe than WT mice. Our findings demonstrate a relationship between Rgs2 gene expression level and a propensity for anxious and depressive-like behaviour and reduced social interaction that may involve changes in serotonergic receptor expression.

  18. Neuroblastoma patient outcomes, tumor differentiation, and ERK activation are correlated with expression levels of the ubiquitin ligase UBE4B

    PubMed Central

    Woodfield, Sarah E.; Guo, Rong Jun; Liu, Yin; Major, Angela M.; Hollingsworth, Emporia Faith; Indiviglio, Sandra; Whittle, Sarah B.; Mo, Qianxing; Bean, Andrew J.; Ittmann, Michael; Lopez-Terrada, Dolores; Zage, Peter E.

    2016-01-01

    Background UBE4B is an E3/E4 ubiquitin ligase whose gene is located in chromosome 1p36.22. We analyzed the associations of UBE4B gene and protein expression with neuroblastoma patient outcomes and with tumor prognostic features and histology. Methods We evaluated the association of UBE4B gene expression with neuroblastoma patient outcomes using the R2 Platform. We screened neuroblastoma tumor samples for UBE4B protein expression using immunohistochemistry. FISH for UBE4B and 1p36 deletion was performed on tumor samples. We then evaluated UBE4B expression for associations with prognostic factors and with levels of phosphorylated ERK in neuroblastoma tumors and cell lines. Results Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma and with worse outcomes in all patient subgroups. UBE4B protein expression was associated with neuroblastoma tumor differentiation, and decreased UBE4B protein levels were associated with high-risk features. UBE4B protein levels were also associated with levels of phosphorylated ERK. Conclusions We have demonstrated associations between UBE4B gene expression and neuroblastoma patient outcomes and prognostic features. Reduced UBE4B protein expression in neuroblastoma tumors was associated with high-risk features, a lack of differentiation, and with ERK activation. These results suggest UBE4B may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions and that UBE4B expression may mediate neuroblastoma differentiation. PMID:27014418

  19. Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

    PubMed

    Lindberg, Karolina; Ström, Anders; Lock, John G; Gustafsson, Jan-Ake; Haldosén, Lars-Arne; Helguero, Luisa A

    2010-01-01

    Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

  20. Identification of genes differentially expressed during early interactions between the stem rot fungus (Sclerotium rolfsii) and peanut (Arachis hypogaea) cultivars with increasing disease resistance levels.

    PubMed

    Jogi, Ansuya; Kerry, John W; Brenneman, Timothy B; Leebens-Mack, James H; Gold, Scott E

    2016-03-01

    Sclerotium rolfsii, a destructive soil-borne fungal pathogen causes stem rot of the cultivated peanut, Arachis hypogaea. This study aimed to identify differentially expressed genes associated with peanut resistance and fungal virulence. Four peanut cultivars (A100-32, Georgia Green, GA-07W and York) with increasing resistance levels were inoculated with a virulent S. rolfsii strain to study the early plant-pathogen interaction. 454 sequencing was performed on RNAs from infected tissue collected at 4 days post inoculation, generating 225,793 high-quality reads. Normalized read counts and fold changes were calculated and statistical analysis used to identify differentially expressed genes. Several genes identified as differential in the RNA-seq experiment were selected based on functions of interest and real-time PCR employed to corroborate their differential expression. Expanding the analysis to include all four cultivars revealed a small but interesting set of genes showing colinearity between cultivar resistance and expression levels. This study identified a set of genes possibly related to pathogen response that may be useful marker assisted selection or transgenic disease control strategies. Additionally, a set of differentially expressed genes that have not been functionally characterized in peanut or other plants and warrant additional investigation were identified.

  1. IL-15 up-regulates the MMP-9 expression levels and induces inflammatory infiltration of macrophages in polymyositis through regulating the NF-kB pathway.

    PubMed

    Yan, Wang; Fan, Weinv; Chen, Caijing; Wu, Yunqin; Fan, Zhenyi; Chen, Jiaqi; Chen, Zhaoying; Chen, Huimin

    2016-10-10

    This study was aimed to research the effects of IL-15 on inducing inflammatory infiltration of macrophages in polymyositis (PM) through the NF-kB pathway, and whether IL-15 was able to further regulate MMP-9 expression levels. Prepared PM cells, collected from the patients suffering from PM, were administered to SD rats. Also, a group of healthy SD rats was undergoing the same treatment as the control group. The test animals were treated with either anti-IL-15, IL-15, MMP-9 siRNA or ERK1/2 inhibitor. The blood toxicological parameters creatine kinase (CK) and CD163 were tested by using ELISA and immunohistochemistry assay. In addition, NF-kB expression in macrophages was measured by immunocytochemical assay. To measure the degree of cell infiltration the Transwell assay was performed. Lastly, western blot and zymography were carried out to compare MMP-9 and ERK expression levels between the two groups, both in vivo and in vitro. The results showed that S-CK, IL-15 and IL-15Rα levels increased rapidly after the conventional treatment was introduced to the PM infected SD rats. The PM model establishment and IL-15 treatment significantly increased the expressions of IL-15Rα, MMP-9, p-ERK and p-IKBα. However, the same effect can be suppressed by using anti-IL-15, MMP-9 siRNA or ERK1/2 inhibitor (P < 0.05). In addition, IL-15 is proved to increase cell migration and nucleus expression of NF-kB in the macrophages. IL-15 is able to significantly regulate the inflammatory infiltration of macrophages in PM patients through affecting the NF-kB pathway and MMP-9 expression levels.

  2. Perception of Emotional Facial Expressions in Amyotrophic Lateral Sclerosis (ALS) at Behavioural and Brain Metabolic Level

    PubMed Central

    Aho-Özhan, Helena E. A.; Keller, Jürgen; Heimrath, Johanna; Uttner, Ingo; Kassubek, Jan; Birbaumer, Niels; Ludolph, Albert C.; Lulé, Dorothée

    2016-01-01

    Introduction Amyotrophic lateral sclerosis (ALS) primarily impairs motor abilities but also affects cognition and emotional processing. We hypothesise that subjective ratings of emotional stimuli depicting social interactions and facial expressions is changed in ALS. It was found that recognition of negative emotions and ability to mentalize other’s intentions is reduced. Methods Processing of emotions in faces was investigated. A behavioural test of Ekman faces expressing six basic emotions was presented to 30 ALS patients and 29 age-, gender and education matched healthy controls. Additionally, a subgroup of 15 ALS patients that were able to lie supine in the scanner and 14 matched healthy controls viewed the Ekman faces during functional magnetic resonance imaging (fMRI). Affective state and a number of daily social contacts were measured. Results ALS patients recognized disgust and fear less accurately than healthy controls. In fMRI, reduced brain activity was seen in areas involved in processing of negative emotions replicating our previous results. During processing of sad faces, increased brain activity was seen in areas associated with social emotions in right inferior frontal gyrus and reduced activity in hippocampus bilaterally. No differences in brain activity were seen for any of the other emotional expressions. Inferior frontal gyrus activity for sad faces was associated with increased amount of social contacts of ALS patients. Conclusion ALS patients showed decreased brain and behavioural responses in processing of disgust and fear and an altered brain response pattern for sadness. The negative consequences of neurodegenerative processes in the course of ALS might be counteracted by positive emotional activity and positive social interactions. PMID:27741285

  3. Chloral hydrate-dependent reduction in the peptidoglycan-induced inflammatory macrophage response is associated with lower expression levels of toll-like receptor 2.

    PubMed

    Pan, Qingjun; Liu, Yuan; Zhu, Xuezhi; Liu, Huafeng

    2014-05-01

    The aim of this study was to investigate the effect and mechanism of action of chloral hydrate on the peptidoglycan (PGN)-induced inflammatory macrophage response. The effect of chloral hydrate on the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) by murine peritoneal macrophages with PGN-stimulation was investigated. In addition, RAW264.7 cells transfected with a nuclear factor-κB (NF-κB) luciferase reporter plasmid stimulated by PGN were used to study the effect of chloral hydrate on the levels NF-κB activity. Flow cytometry and western blotting were performed to investigate the expression levels of toll-like receptor 2 (TLR2) in the treated RAW264.7 cells. It was identified that chloral hydrate reduced the levels of IL-6 and TNF-α produced by the peritoneal macrophages stimulated with PGN. The levels of NF-κB activity of the RAW264.7 cells stimulated by PGN decreased following treatment with chloral hydrate, which was associated with a reduction in the expression levels of TLR2 and reduced levels of TLR2 signal transduction. These data demonstrate that chloral hydrate reduced the magnitude of the PGN-induced inflammatory macrophage response associated with lower expression levels of TLR2.

  4. The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato.

    PubMed

    Hong, Ya-Fang; Liu, Chang-Yeu; Cheng, Kuo-Joan; Hour, Ai-Ling; Chan, Min-Tsair; Tseng, Tung-Hai; Chen, Kai-Yi; Shaw, Jei-Fu; Yu, Su-May

    2008-07-01

    The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.

  5. The influence of genetic polymorphisms in TLR4 and TIRAP, and their expression levels in peripheral blood, on susceptibility to sepsis

    PubMed Central

    ZHANG, JIANPING; YANG, JINGPING; XU, XIYUAN; LIANG, LIANGSHEN; SUN, HAIXIA; LIU, GUOHUA; ZHANG, LIHONG; SU, YUN

    2016-01-01

    The present study aimed to investigate whether genetic polymorphisms in the Toll-like receptor (TLR)-4 and Toll/interleukin-1 receptor (TIR)-associated protein (TIRAP) genes, and/or their expression levels, influence the susceptibility of a patient to sepsis. A total of 106 patients with sepsis were divided into two groups on the basis of their acute physiology and chronic health evaluation (APACHE) II scores: i) Sepsis group A (APACHE II <20) and ii) Sepsis group B (APACHE II >20). In addition, 100 healthy volunteers were enrolled into the control group. Polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the following genetic polymorphisms: The Ser180Leu allele of the TIRAP gene and the Asp299Gly and Thr399I1e alleles of the TLR4 gene. Furthermore, the protein expression levels of TLR4 and TIRAP were analyzed using an enzyme-linked immunosorbent assay. Genetic polymorphisms were not detected for the TLR4 and TIRAP genes; however, the protein expression levels of TLR4 and TIRAP differed significantly between the control, sepsis A and sepsis B groups (P<0.01). An APACHE II score of 20 was used as a baseline in order to differentiate sepsis severity. Pearson analysis demonstrated that TLR4 and TIRAP protein expression levels were positively correlated with sepsis severity (r=0.931 and 0.972; P<0.05), and TLR4 protein expression levels were positively correlated with those of TIRAP (r=0.936; P<0.05). The results of the present study suggested that the protein expression levels of, but not genetic polymorphisms in, TLR4 and TIRAP were associated with the severity of sepsis. PMID:26889229

  6. Cholecalciferol Additively Reduces Serum Parathyroid Hormone and Increases Vitamin D and Cathelicidin Levels in Paricalcitol-Treated Secondary Hyperparathyroid Hemodialysis Patients

    PubMed Central

    Zheng, Jing-Quan; Hou, Yi-Chou; Zheng, Cai-Mei; Lu, Chien-Lin; Liu, Wen-Chih; Wu, Chia-Chao; Huang, Ming-Te; Lin, Yuh-Feng; Lu, Kuo-Cheng

    2016-01-01

    Background: Active Vitamin D analogues are used clinically for prevention and treatment of secondary hyperparathyroidism (SHPT) in hemodialysis (HD) patients. Nutritional vitamin D supplementation is used for additional local parathyroid (PTH) suppression, with lower incidence of hypercalcemia and hyperphosphatemia. This study evaluates the possible beneficial effects of combined vitamin D treatment (paricalcitol and cholecalciferol). Methods: Sixty HD patients with serum parathyroid hormone (iPTH) >300 pg/mL were enrolled. All patients administered 2 mcg/day of paricalcitol and were randomly allocated into control group (placebo) or study group (cholecalciferol) for 16 weeks. Serum 25(OH)D3, iPTH and human cathelicidin (hCAP-18) were measured at baseline and during follow-up. Results: iPTH levels decreased in the study group appropriately and were more significantly decreased at 16 weeks. Study group had significantly increased 25(OH)D3 levels. In addition, the study group had significantly increased serum hCAP-18 levels compared with control group. Correlation analysis showed a significant correlation between the percentage increase in serum hCAP-18 and 25(OH)D3 levels. Conclusions: Cholecalciferol, in combination with paricalcitol, additively lowers the iPTH levels in a significant number of patients after 16 weeks of supplementation. A dose of 5000 IU/week of cholecalciferol could maintain serum 25(OH)D3 levels above 30 ng/dL as early as 8 weeks after beginning supplementation. Doubling of serum cathelicidin levels were noted after 16 weeks of cholecalciferol supplementation in 40% of study patients. PMID:27827962

  7. Meta-analysis on Methane Mitigating Properties of Saponin-rich Sources in the Rumen: Influence of Addition Levels and Plant Sources

    PubMed Central

    Jayanegara, Anuraga; Wina, Elizabeth; Takahashi, Junichi

    2014-01-01

    Saponins have been considered as promising natural substances for mitigating methane emissions from ruminants. However, studies reported that addition of saponin-rich sources often arrived at contrasting results, i.e. either it decreased methane or it did not. The aim of the present study was to assess ruminal methane emissions through a meta-analytical approach of integrating related studies from published papers which described various levels of different saponin-rich sources being added to ruminant feed. A database was constructed from published literature reporting the addition of saponin-rich sources at various levels and then monitoring ruminal methane emissions in vitro. Accordingly, levels of saponin-rich source additions as well as different saponin sources were specified in the database. Apart from methane, other related rumen fermentation parameters were also included in the database, i.e. organic matter digestibility, gas production, pH, ammonia concentration, short-chain fatty acid profiles and protozoal count. A total of 23 studies comprised of 89 data points met the inclusion criteria. The data obtained were subsequently subjected to a statistical meta-analysis based on mixed model methodology. Accordingly, different studies were treated as random effects whereas levels of saponin-rich source additions or different saponin sources were considered as fixed effects. Model statistics used were p-value and root mean square error. Results showed that an addition of increasing levels of a saponin-rich source decreased methane emission per unit of substrate incubated as well as per unit of total gas produced (p<0.05). There was a decrease in acetate proportion (linear pattern; p<0.001) and an increase in propionate proportion (linear pattern; p<0.001) with increasing levels of saponin. Log protozoal count decreased (p<0.05) at higher saponin levels. Comparing between different saponin-rich sources, all saponin sources, i.e. quillaja, tea and yucca saponins

  8. Rapid N2O fluxes at high level of nitrate nitrogen addition during freeze-thaw events in boreal peatlands of Northeast China

    NASA Astrophysics Data System (ADS)

    Cui, Qian; Song, Changchun; Wang, Xianwei; Shi, Fuxi; Wang, Lili; Guo, Yuedong

    2016-06-01

    Freeze-thaw (FT) events and increasing nitrogen (N) availability may alter N turnover and nitrous oxide (N2O) emissions in permafrost peatlands. However, the responses of N2O emissions to different N levels and additions during FT events are far from clear. We conducted an incubation study to investigate the impacts of different N addition levels (LN: 0.07 mg N g-1, HN: 0.14 mg N g-1) and N addition forms (AC: ammonium chloride, NS: sodium nitrate) on the emissions of N2O under FT and non-freeze-thaw (NFT) conditions in boreal peatlands of Northeast China. Results indicated that the FT condition significantly increased N2O emissions compared with the NFT condition and peaks occurred during thawing. Compared with AC treatments, NS treatments significantly elevated the accumulation of N2O emissions under the FT condition, exhibiting significant differences in different NS levels. N2O emissions were also positively dependent on soil NO3- concentrations to supply nitrate for denitrification. Nitrate-N addition was mainly responsible for the burst of N2O with denitrification as the main process during FT events. Therefore, these results suggest that N2O emissions potentially increase during FT events with increasing nitrate-N deposition in permafrost peatlands, which would contribute to global climate warming.

  9. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-04-21

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.

  10. Vitamin D Levels Are Associated with Expression of SLE, but Not Flare Frequency

    PubMed Central

    Squance, Marline L.; Reeves, Glenn E. M.; Tran, Huy A.

    2014-01-01

    This study explores links between vitamin D deficiency (25(OH)D = 50 nmol/L) and serological autoimmunity (ANA > 1 : 80) and frequency of self-reported flares (SRF) in participants with clinical autoimmunity (SLE). 25(OH)D levels of 121 females were quantified and compared. The cohort consisted of 80 ACR defined SLE patients and 41 age and sex matched controls. Association analysis of log2 (25(OH)D) levels and ANA 80 positivity was undertaken via two-sample t-tests and regression models. Significant differences were found for 25(OH)D levels (mean: control 74 nmol/L (29.5 ng/ml); SLE 58 nmol/L (23.1 ng/ml), P = 0.04), 25(OH)D deficiency (P = 0.02). Regression models indicate that, for a twofold rise in 25(OH)D level, the odds ratio (OR) for ANA-positivity drops to 36% of the baseline OR. No link was found between SRF-days and 25(OH)D levels. Our results support links between vitamin D deficiency and expression of serological autoimmunity and clinical autoimmunity (SLE). However, no demonstrable association between 25(OH)D and SRF was confirmed, suggesting independent influences of other flare-inducing factors. Results indicate that SLE patients have high risk of 25(OH)D deficiency and therefore supplementation with regular monitoring should be considered as part of patient management. PMID:25506363

  11. Intake of red wine in different meals modulates oxidized LDL level, oxidative and inflammatory gene expression in healthy people: a randomized crossover trial.

    PubMed

    Di Renzo, Laura; Carraro, Alberto; Valente, Roberto; Iacopino, Leonardo; Colica, Carmen; De Lorenzo, Antonino

    2014-01-01

    Several studies have found that adherence to the Mediterranean Diet, including consumption of red wine, is associated with beneficial effects on oxidative and inflammatory conditions. We evaluate the outcome of consumption of a McDonald's Meal (McD) and a Mediterranean Meal (MM), with and without the additive effect of red wine, in order to ascertain whether the addition of the latter has a positive impact on oxidized (ox-) LDL and on expression of oxidative and inflammatory genes. A total of 24 subjects were analyzed for ox-LDL, CAT, GPX1, SOD2, SIRT2, and CCL5 gene expression levels, before and after consumption of the 4 different meal combinations with washout intervals between each meal. When red wine is associated with McD or MM, values of ox-LDL are lowered (P < 0.05) and expression of antioxidant genes is increased, while CCL5 expression is decreased (P < 0.05). SIRT2 expression after MM and fasting with red wine is significantly correlated with downregulation of CCL5 and upregulation of CAT (P < 0.001). GPX1 increased significantly in the comparison between baseline and all conditions with red wine. We highlighted for the first time the positive effect of red wine intake combined with different but widely consumed meal types on ox-LDL and gene expression. Trial Registration. This trial is registered with ClinicalTrials.gov NCT01890070.

  12. Intake of Red Wine in Different Meals Modulates Oxidized LDL Level, Oxidative and Inflammatory Gene Expression in Healthy People: A Randomized Crossover Trial

    PubMed Central

    Di Renzo, Laura; Valente, Roberto; Colica, Carmen

    2014-01-01

    Several studies have found that adherence to the Mediterranean Diet, including consumption of red wine, is associated with beneficial effects on oxidative and inflammatory conditions. We evaluate the outcome of consumption of a McDonald's Meal (McD) and a Mediterranean Meal (MM), with and without the additive effect of red wine, in order to ascertain whether the addition of the latter has a positive impact on oxidized (ox-) LDL and on expression of oxidative and inflammatory genes. A total of 24 subjects were analyzed for ox-LDL, CAT, GPX1, SOD2, SIRT2, and CCL5 gene expression levels, before and after consumption of the 4 different meal combinations with washout intervals between each meal. When red wine is associated with McD or MM, values of ox-LDL are lowered (P < 0.05) and expression of antioxidant genes is increased, while CCL5 expression is decreased (P < 0.05). SIRT2 expression after MM and fasting with red wine is significantly correlated with downregulation of CCL5 and upregulation of CAT (P < 0.001). GPX1 increased significantly in the comparison between baseline and all conditions with red wine. We highlighted for the first time the positive effect of red wine intake combined with different but widely consumed meal types on ox-LDL and gene expression. Trial Registration. This trial is registered with ClinicalTrials.gov NCT01890070. PMID:24876915

  13. Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.).

    PubMed

    González-Agüero, Mauricio; Troncoso, Sebastián; Gudenschwager, Orianne; Campos-Vargas, Reinaldo; Moya-León, María A; Defilippi, Bruno G

    2009-05-01

    Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adhEU395433; Pa-pdcEU395434; and Pa-loxEU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.

  14. BCL6 mRNA Expression Level in Invasive Duct Carcinoma not otherwise Specified

    PubMed Central

    Badr, Eman; Masoud, Eman; Eldien, Marwa Serag

    2016-01-01

    Introduction B-Cell Lymphoma 6 (BCL6) has an oncogenic role in tumourigenesis of various malignancies. It represses genes involved in terminal differentiation and plays complementary role with Signal Transducer and Activator of Transcription 3 (STAT3) in triple-negative breast cancer cellular function. Aim To evaluate the expression of BCL6 in cancer breast and determine its correlation with the clinico-pathological features including the molecular subtype of breast carcinoma. Materials and Methods This prospective case control study was carried out on 150 patients, divided into 100 cases of invasive duct carcinoma not otherwise specified and 50 benign breast lesions including fibroadenoma and fibrocystic disease. Fresh tissues were excised, which were then subjected to RNA extraction. The BCL6 mRNA level was assessed using real-time reverse transcription Polymerase Chain Reaction (PCR). Results There was a significant higher levels of BCL6 mRNA in malignant cases compared to benign ones (p<0.001). The level of BCL6 mRNA was higher in cases showing advanced tumor stage (p<0.04), triple negative subtype and associated in situ component (p<0.001) compared to cases with an early stage, luminal or Her 2-neu positive subtypes and those lacking in situ component. Conclusion BCL6 is up-regulated in breast cancer and is associated with poor prognostic features such as advanced stage and triple negative molecular subtype. BCL6 inhibitors might be considered as targeted therapy for breast cancer. PMID:28208987

  15. Chronic stress alters the expression levels of longevity-related genes in the rat hippocampus.

    PubMed

    Sánchez-Hidalgo, Ana C; Muñoz, Mario F; Herrera, Antonio J; Espinosa-Oliva, Ana M; Stowell, Rianne; Ayala, Antonio; Machado, Alberto; Venero, José L; de Pablos, Rocío M

    2016-07-01

    The molecular mechanisms underlying the negative effects of psychological stress on cellular stress during aging and neurodegenerative diseases are poorly understood. The main objective of this study was to test the effect of chronic psychological stress, and the consequent increase of circulating glucocorticoids, on several hippocampal genes involved in longevity. Sirtuin-1, p53, thioredoxin-interacting protein, and heat shock protein 70 were studied at the mRNA and protein levels in stressed and non-stressed animals. Stress treatment for 10 days decreased sirtuin-1 and heat shock protein 70 levels, but increased levels of p53, thioredoxin-interacting protein and the NADPH oxidase enzyme. Examination of protein expression following two months of stress treatment indicated that sirtuin-1 remained depressed. In contrast, an increase was observed for thioredoxin-interacting protein, heat shock protein 70, p53 and the NADPH oxidase enzyme. The effect of stress was reversed by mifepristone, a glucocorticoid receptor antagonist. These data suggest that chronic stress could contribute to aging in the hippocampus.

  16. Expression of catalytically active recombinant Helicobacter pylori urease at wild-type levels in Escherichia coli.

    PubMed Central

    Hu, L T; Mobley, H L

    1993-01-01

    The genes encoding Helicobacter pylori urease, a nickel metalloenzyme, have been cloned and expressed in Escherichia coli. Enzymatic activity, however, has been very weak compared with that in clinical isolates of H. pylori. Conditions under which near wild-type urease activity was achieved were developed. E. coli. SE5000 containing recombinant H. pylori urease genes was grown in minimal medium containing no amino acids, NiCl2 was added to 0.75 microM, and structural genes ureA and ureB (pHP902) were overexpressed in trans to the complete urease gene cluster (pHP808). Under these conditions, E. coli SE5000 pHP808/pHP902) expressed a urease activity up to 87 mumol of urea per min per mg of protein (87 U/mg of protein), a level approaching that of wild-type H. pylori UMAB41 (100 U/mg of protein), from which the genes were cloned. Poor catalytic activity of recombinant clones grown in Luria broth or M9 medium containing 0.5% Casamino Acids was due to chelation of nickel ions by medium components, particularly histidine and cysteine. In cultures containing these amino acids, 63Ni2+ was prevented from being transported into cells and was not incorporated into urease protein. As a consequence, M9 minimal medium cultures containing histidine or cysteine produced only 0.05 and 0.9%, respectively, of active urease produced by control cultures containing no amino acids. We conclude that recombinant H. pylori urease is optimally expressed when Ni2+ transport is not inhibited and when sufficient synthesis of urease subunits UreA and UreB is provided. Images PMID:8500893

  17. Stress levels of glucocorticoids inhibit LHβ-subunit gene expression in gonadotrope cells.

    PubMed

    Breen, Kellie M; Thackray, Varykina G; Hsu, Tracy; Mak-McCully, Rachel A; Coss, Djurdjica; Mellon, Pamela L

    2012-10-01

    Increased glucocorticoid secretion is a common response to stress and has been implicated as a mediator of reproductive suppression upon the pituitary gland. We utilized complementary in vitro and in vivo approaches in the mouse to investigate the role of glucocorticoids as a stress-induced intermediate capable of gonadotrope suppression. Repeated daily restraint stress lengthened the ovulatory cycle of female mice and acutely reduced GnRH-induced LH secretion and synthesis of LH β-subunit (LHβ) mRNA, coincident with increased circulating glucocorticoids. Administration of a stress level of glucocorticoid, in the absence of stress, blunted LH secretion in ovariectomized female mice, demonstrating direct impairment of reproductive function by glucocorticoids. Supporting a pituitary action, glucocorticoid receptor (GR) is expressed in mouse gonadotropes and treatment with glucocorticoids reduces GnRH-induced LHβ expression in immortalized mouse gonadotrope cells. Analyses revealed that glucocorticoid repression localizes to a region of the LHβ proximal promoter, which contains early growth response factor 1 (Egr1) and steroidogenic factor 1 sites critical for GnRH induction. GR is recruited to this promoter region in the presence of GnRH, but not by dexamethasone alone, confirming the necessity of the GnRH response for GR repression. In lieu of GnRH, Egr1 induction is sufficient for glucocorticoid repression of LHβ expression, which occurs via GR acting in a DNA- and dimerization-independent manner. Collectively, these results expose the gonadotrope as an important neuroendocrine site impaired during stress, by revealing a molecular mechanism involving Egr1 as a critical integrator of complex formation on the LHβ promoter during GnRH induction and GR repression.

  18. Molecular Characterization of the NLRC4 Expression in Relation to Interleukin-18 Levels

    PubMed Central

    Zeller, Tanja; Haase, Tina; Müller, Christian; Riess, Helene; Lau, Denise; Zeller, Simon; Krause, Jasmin; Baumert, Jens; Pless, Ole; Dupuis, Josée; Wild, Philipp S.; Eleftheriadis, Medea; Waldenberger, Melanie; Zeilinger, Sonja; Ziegler, Andreas; Peters, Annette; Tiret, Laurence; Proust, Carole; Marzi, Carola; Munzel, Thomas; Strauch, Konstantin; Prokisch, Holger; Lackner, Karl J.; Herder, Christian; Thorand, Barbara; Benjamin, Emilia J.; Blankenberg, Stefan; Koenig, Wolfgang; Schnabel, Renate B.

    2015-01-01

    Background Interleukin-18 (IL-18) is a pleiotropic cytokine centrally involved in the cytokine cascade with complex immunomodulatory functions in innate and acquired immunity. Circulating IL-18 concentrations are associated with type 2 diabetes, cardiovascular events and diverse inflammatory and autoimmune disorders. Methods and Results To identify causal variants affecting circulating IL-18 concentrations, we applied various omics and molecular biology approaches. By GWAS, we confirmed association of IL-18 levels with a SNP in the untranslated exon 2 of the inflammasome component NLRC4 (NLR family, CARD domain containing 4) gene on chromosome 2 (rs385076, P=2.4×10−45). Subsequent molecular analyses by gene expression analysis and reporter gene assays indicated an effect of rs385076 on NLRC4 expression and differential isoform usage by modulating binding of the transcription factor PU.1. Conclusions Our study provides evidence for the functional causality of SNP rs385076 within the NLRC4 gene in relation to IL-18 activation. PMID:26362438

  19. High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector.

    PubMed

    Choi, J Y; Woo, S D; Lee, H K; Hong, H K; Je, Y H; Park, J H; Song, J Y; An, S H; Kang, S K

    2000-01-01

    For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 x 10(3) HA units/ml from infected Bm5 cells, 2.1x 10(5) HA units/larvae from infected larval fat body, and 1.6x 10(6) HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines.

  20. A cloud-based workflow to quantify transcript-expression levels in public cancer compendia

    PubMed Central

    Tatlow, PJ; Piccolo, Stephen R.

    2016-01-01

    Public compendia of sequencing data are now measured in petabytes. Accordingly, it is infeasible for researchers to transfer these data to local computers. Recently, the National Cancer Institute began exploring opportunities to work with molecular data in cloud-computing environments. With this approach, it becomes possible for scientists to take their tools to the data and thereby avoid large data transfers. It also becomes feasible to scale computing resources to the needs of a given analysis. We quantified transcript-expression levels for 12,307 RNA-Sequencing samples from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas. We used two cloud-based configurations and examined the performance and cost profiles of each configuration. Using preemptible virtual machines, we processed the samples for as little as $0.09 (USD) per sample. As the samples were processed, we collected performance metrics, which helped us track the duration of each processing step and quantified computational resources used at different stages of sample processing. Although the computational demands of reference alignment and expression quantification have decreased considerably, there remains a critical need for researchers to optimize preprocessing steps. We have stored the software, scripts, and processed data in a publicly accessible repository (https://osf.io/gqrz9). PMID:27982081

  1. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

    PubMed

    Núñez-Acuña, Gustavo; Gallardo-Escárate, Cristian

    2014-02-15

    Myostatin (MSTN) is a protein of the Transforming Growth Factor-β (TGF-β) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

  2. High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production.

    PubMed

    Kang, Mi-Suk; Han, Sang-Soo; Kim, Mi-Young; Kim, Bu-Youn; Huh, Jong-Pil; Kim, Hak-Sung; Lee, Jin-Ho

    2014-05-01

    The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

  3. High level expression of an acid-stable phytase from Citrobacter freundii in Pichia pastoris.

    PubMed

    Zhao, Wei; Xiong, Aisheng; Fu, Xiaoyan; Gao, Feng; Tian, Yongsheng; Peng, Rihe

    2010-12-01

    To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²+-NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5-8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca²+, and Mg²+. These characteristics contribute to its potential application in feed industry.

  4. [Significant regression of glioblastoma with low level of Mgmt gene expression following radiotherapy].

    PubMed

    Matsko, M V; Luchin, E I; Ievleva, A G; Bakholdin, D V; Abysheva, S N; Zavgorodniaia, E V; Potapova, O N; Imianitov, E N; Ulitin, A Iu; Matsko, D E

    2011-01-01

    Radiochemotherapy is leading the universal research effort in fighting lethality: it is improving relapse-free survival of patients with inoperable glioblastoma, the most pernicious brain tumor in adults. Its effectiveness was found to depend on expression of Mgmt gene of tumor DNA reparation following radiochemotherapy and adequate medication based on the molecular phenotype of tumor. Our study involved a 40-year old male with a low level of Mgmt gene expression as established by stereotactic biopsy. The patient received hypofractionated three-dimensional conformational proton therapy with the benefit of temozolomide (140 mg/24 hr). Subsequently, the dose was raised to 360 mg/24 hr, on days 1-5 of the cycle. Contrast-enhanced MRI examination established significant diminishing of the size of tumors on completion of cycles 7 and 8; patients felt better, memory and blood indices improved. As of the time this paper was written, relapse-free survival was 17.5 months, as compared with the literature data on inoperable glioblastoma--5.5 months.

  5. Accurate Estimation of Expression Levels of Homologous Genes in RNA-seq Experiments

    NASA Astrophysics Data System (ADS)

    Paşaniuc, Bogdan; Zaitlen, Noah; Halperin, Eran

    Next generation high throughput sequencing (NGS) is poised to replace array based technologies as the experiment of choice for measuring RNA expression levels. Several groups have demonstrated the power of this new approach (RNA-seq), making significant and novel contributions and simultaneously proposing methodologies for the analysis of RNA-seq data. In a typical experiment, millions of short sequences (reads) are sampled from RNA extracts and mapped back to a reference genome. The number of reads mapping to each gene is used as proxy for its corresponding RNA concentration. A significant challenge in analyzing RNA expression of homologous genes is the large fraction of the reads that map to multiple locations in the reference genome. Currently, these reads are either dropped from the analysis, or a naïve algorithm is used to estimate their underlying distribution. In this work, we present a rigorous alternative for handling the reads generated in an RNA-seq experiment within a probabilistic model for RNA-seq data; we develop maximum likelihood based methods for estimating the model parameters. In contrast to previous methods, our model takes into account the fact that the DNA of the sequenced individual is not a perfect copy of the reference sequence. We show with both simulated and real RNA-seq data that our new method improves the accuracy and power of RNA-seq experiments.

  6. Accurate estimation of expression levels of homologous genes in RNA-seq experiments.

    PubMed

    Paşaniuc, Bogdan; Zaitlen, Noah; Halperin, Eran

    2011-03-01

    Abstract Next generation high-throughput sequencing (NGS) is poised to replace array-based technologies as the experiment of choice for measuring RNA expression levels. Several groups have demonstrated the power of this new approach (RNA-seq), making significant and novel contributions and simultaneously proposing methodologies for the analysis of RNA-seq data. In a typical experiment, millions of short sequences (reads) are sampled from RNA extracts and mapped back to a reference genome. The number of reads mapping to each gene is used as proxy for its corresponding RNA concentration. A significant challenge in analyzing RNA expression of homologous genes is the large fraction of the reads that map to multiple locations in the reference genome. Currently, these reads are either dropped from the analysis, or a naive algorithm is used to estimate their underlying distribution. In this work, we present a rigorous alternative for handling the reads generated in an RNA-seq experiment within a probabilistic model for RNA-seq data; we develop maximum likelihood-based methods for estimating the model parameters. In contrast to previous methods, our model takes into account the fact that the DNA of the sequenced individual is not a perfect copy of the reference sequence. We show with both simulated and real RNA-seq data that our new method improves the accuracy and power of RNA-seq experiments.

  7. Dependency of Escherichia coli cell-division size, and independency of nucleoid segregation on the mode and level of ftsZ expression.

    PubMed

    Palacios, P; Vicente, M; Sánchez, M

    1996-06-01

    Expression of ftsZ in strain VIP205 is dissociated from its natural promoters, and is under the control of an inducible tac promoter. This abolishes the oscillation in ftsZ transcription observed in the wild type, allowing different levels of ftsZ expression. We demonstrate that this construction does not affect the expression of other genes, and has no effects on replication or nucleoid segregation. A shift in IPTG from 30 microM, that supports division at wild-type sizes, to lower (6 microM) or higher (100 microM) concentrations, indicates that VIP205 cells can divide within a broad range of FtsZ concentrations. Analysis of the morphological parameters during the transition from one IPTG concentration to another suggests that the correct timing of ftsZ expression, and the correct FtsZ concentration, are required for division to occur at normal cell sizes. After a transient division delay during the transition to lower IPTG concentrations, cells in which ftsZ is expressed continuously (yielding 80% of the wild-type FtsZ levels) divide with the same division time as the wild type, but at the expense of becoming 1.5 times larger. A precise control of ftsZ expression is required for normal division, but the existence of additional regulators to maintain the correct timing during the cell cycle cannot be ruled out.

  8. Soft mean spherical approximation for dusty plasma liquids: Level of accuracy and analytic expressions

    SciTech Connect

    Tolias, P.; Ratynskaia, S.; Angelis, U. de

    2015-08-15

    The soft mean spherical approximation is employed for the study of the thermodynamics of dusty plasma liquids, the latter treated as Yukawa one-component plasmas. Within this integral theory method, the only input necessary for the calculation of the reduced excess energy stems from the solution of a single non-linear algebraic equation. Consequently, thermodynamic quantities can be routinely computed without the need to determine the pair correlation function or the structure factor. The level of accuracy of the approach is quantified after an extensive comparison with numerical simulation results. The approach is solved over a million times with input spanning the whole parameter space and reliable analytic expressions are obtained for the basic thermodynamic quantities.

  9. DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein.

    PubMed

    Ferreira, Daniela M; Miyaji, Eliane N; Oliveira, Maria Leonor S; Darrieux, Michelle; Arêas, Ana Paula M; Ho, Paulo L; Leite, Luciana C C

    2006-04-01

    Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

  10. Superoxide dismutase activity and gene expression levels in Saudi women with recurrent miscarriage

    PubMed Central

    GHNEIM, HAZEM K.; AL-SHEIKH, YAZEED A.; ALSHEBLY, MASHAEL M.; ABOUL-SOUD, MOURAD A. M.

    2016-01-01

    The antioxidant activities of superoxide dismutase 1 (SOD1) and SOD2, as well as the levels of the oxidant superoxide anion (SOA) and the micronutrients zinc (Zn), copper (Cu) and manganese (Mn), were assayed in plasma, whole blood and placental tissue of non-pregnant (NP), healthy pregnant (HP) women and recurrent miscarriage (RM) patients. The results showed that SOD1 and SOD2 activities and the levels of Zn, Cu and Mn in plasma and whole blood of HP women were slightly, but significantly lower, and even more significantly decreased in RM patients compared to those observed in NP women (P<0.05 and P<0.0001, respectively). Additionally, whereas plasma SOD1 and SOD2 activities and Zn, Cu and Mn levels were significantly lower in RM patients, those of whole blood and placental tissue were significantly lower when compared to HP women (P<0.001 and P<0.0001, respectively). Concurrently, there were consistent increases of equal magnitude and statistical significance in SOA levels in all the assayed samples as identified by a comparison between the subjects. The findings thus supported oxidative metabolism and excessive reactive oxygen species generation. The resultant oxidative stress, identified in whole blood and placental tissues of RM patients, may have been a primary cause of RM. Dietary supplementation of Zn, Cu and Mn may be beneficial to these patients pre- and post-conception. PMID:26821085

  11. Constitutive high-level SOS1 expression and absence of HKT1;1 expression in the salt-accumulating halophyte Salicornia dolichostachya.

    PubMed

    Katschnig, D; Bliek, T; Rozema, J; Schat, H

    2015-05-01

    We investigated the effects of salinity on ion accumulation and expression of candidate salt tolerance genes in the highly tolerant salt accumulating halophyte Salicornia dolichostachya and the taxonomically related glycophytic Spinacia oleracea. S. dolichostachya, in comparison with S. oleracea, constitutively expressed SOS1 at a high level, but did not detectably express HKT1;1. These findings suggest that the constitutive high level of shoot salt accumulation in S. dolichostachya is accomplished through enhancement of SOS1-mediated Na(+) xylem loading, in combination with complete suppression of HKT1;1-mediated Na(+) retrieval from the xylem. Our findings demonstrate the importance of gene expression comparisons between highly tolerant halophytes and taxonomically related glycophytes to improve the understanding of mechanisms of Na(+) movement and salt tolerance in plants.

  12. The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells.

    PubMed

    Ram, Sripad; Kim, Dongyoung; Ober, Raimund J; Ward, E Sally

    2014-01-01

    The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2.

  13. A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos.

    PubMed

    Mebatsion, T; Verstegen, S; De Vaan, L T; Römer-Oberdörfer, A; Schrier, C C

    2001-01-01

    Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

  14. Diabetic retinopathy alters light-induced clock gene expression and dopamine levels in the mouse retina

    PubMed Central

    Lahouaoui, Hasna; Coutanson, Christine; Cooper, Howard M.; Bennis, Mohamed

    2016-01-01

    Purpose Diabetic retinopathy is one of the most common consequences of diabetes that affects millions of working-age adults worldwide and leads to progressive degeneration of the retina, visual loss, and blindness. Diabetes is associated with circadian disruption of the central and peripheral circadian clocks, but the mechanisms responsible for such alterations are unknown. Using a streptozotocin (STZ)-induced model of diabetes, we investigated whether diabetes alters 1) the circadian regulation of clock genes in the retina and in the central clocks, 2) the light response of clock genes in the retina, and/or 3) light-driven retinal dopamine (DA), a major output marker of the retinal clock. Methods To quantify circadian expression of clock and clock-controlled genes, retinas and suprachiasmatic nucleus (SCN) from the same animals were collected every 4 h in circadian conditions, 12 weeks post-diabetes. Induction of Per1, Per2, and c-fos mRNAs was quantified in the retina after the administration of a pulse of monochromatic light (480 nm, 1.17×1014 photons/cm2/s, 15 min) at circadian time 16. Gene expression was assessed with real-time reverse transcription PCR (RT–PCR). Pooled retinas from the control and STZ-diabetic mice were collected 2 h after light ON and light OFF (Zeitgeber time (ZT)2 and ZT14), and DA and its metabolite were analyzed with high-performance liquid chromatography (HPLC). Results We found variable effects of diabetes on the expression of clock genes in the retina and only slight differences in phase and/or amplitude in the SCN. c-fos and Per1 induction by a 480 nm light pulse was abolished in diabetic animals at 12 weeks post-induction of diabetes in comparison with the control mice, suggesting a deficit in light-induced neuronal activation of the retinal clock. Finally, we quantified a 56% reduction in the total number of tyrosine hydroxylase (TH) immunopositive cells, associated with a decrease in DA levels during the subjective day (ZT2

  15. FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle.

    PubMed

    Varela-Rodríguez, B M; Pena-Bello, L; Juiz-Valiña, P; Vidal-Bretal, B; Cordido, F; Sangiao-Alvarellos, S

    2016-07-19

    Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different.

  16. FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle

    PubMed Central

    Varela-Rodríguez, B. M.; Pena-Bello, L.; Juiz-Valiña, P.; Vidal-Bretal, B.; Cordido, F.; Sangiao-Alvarellos, S.

    2016-01-01

    Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different. PMID:27432282

  17. Neuropeptide Receptor Transcript Expression Levels and Magnitude of Ionic Current Responses Show Cell Type-Specific Differences in a Small Motor Circuit

    PubMed Central

    Garcia, Veronica J.; Daur, Nelly; Temporal, Simone; Schulz, David J.

    2015-01-01

    We studied the relationship between neuropeptide receptor transcript expression and current responses in the stomatogastric ganglion (STG) of the crab, Cancer borealis. We identified a transcript with high sequence similarity to crustacean cardioactive peptide (CCAP) receptors in insects and mammalian neuropeptide S receptors. This transcript was expressed throughout the nervous system, consistent with the role of CCAP in a range of different behaviors. In the STG, single-cell qPCR showed expression in only a subset of neurons. This subset had previously been shown to respond to CCAP with the activation of a modulator-activated inward current (IMI), with one exception. In the one cell type that showed expression but no IMI responses, we found CCAP modulation of synaptic currents. Expression levels within STG neuron types were fairly variable, but significantly different between some neuron types. We tested the magnitude and concentration dependence of IMI responses to CCAP application in two identified neurons, the lateral pyloric (LP) and the inferior cardiac (IC) neurons. LP had several-fold higher expression and showed larger current responses. It also was more sensitive to low CCAP concentrations and showed saturation at lower concentrations, as sigmoid fits showed smaller EC50 values and steeper slopes. In addition, occlusion experiments with proctolin, a different neuropeptide converging onto IMI, showed that saturating concentrations of CCAP activated all available IMI in LP, but only approximately two-thirds in IC, the neuron with lower receptor transcript expression. The implications of these findings for comodulation are discussed. PMID:25926455

  18. Heat Stress Regulates the Expression of Genes at Transcriptional and Post-Transcriptional Levels, Revealed by RNA-seq in Brachypodium distachyon

    PubMed Central

    Chen, Shoukun; Li, Haifeng

    2017-01-01

    Heat stress greatly affects plant growth/development and influences the output of crops. With the increased occurrence of extreme high temperature, the negative influence on cereal products from heat stress becomes severer and severer. It is urgent to reveal the molecular mechanism in response to heat stress in plants. In this research, we used RNA-seq technology to identify differentially expressed genes (DEGs) in leaves of seedlings, leaves and inflorescences at heading stage of Brachypodium distachyon, one model plant of grasses. Results showed many genes in responding to heat stress. Of them, the expression level of 656 DEGs were altered in three groups of samples treated with high temperature. Gene ontology (GO) analysis showed that the highly enriched DEGs were responsible for heat stress and protein folding. According to KEGG pathway analysis, the DEGs were related mainly to photosynthesis-antenna proteins, the endoplasmic reticulum, and the spliceosome. Additionally, the expression level of 454 transcription factors belonging to 49 gene families was altered, as well as 1,973 splicing events occurred after treatment with high temperature. This research lays a foundation for characterizing the molecular mechanism of heat stress response and identifying key genes for those responses in plants. These findings also clearly show that heat stress regulates the expression of genes not only at transcriptional level, but also at post-transcriptional level. PMID:28119730

  19. Effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and expression of genes related to the reproductive axis in laying hens.

    PubMed

    Long, L; Wu, S G; Yuan, F; Zhang, H J; Wang, J; Qi, G H

    2016-09-24

    This experiment was conducted to evaluate the effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and gene expression related to reproductive axis in laying hens to confirm the reproduction-promoting function of octacosanol. In total, 360 Hy-Line Brown (67-wk-old) laying hens were randomly assigned to one of three treatments with 0, 5, and 10 mg octacosanol (extracted from rice bran, purity >92%)/kg feed. The feeding trial lasted for 10 weeks. The results showed that the dietary addition of 5 and 10 mg/kg octacosanol improved feed efficiency by 4.9% and 3.4% (P < 0.01), increased the albumen height by 20.5% and 13.3% (P < 0.01), the Haugh unit score by 12.9% and 8.7% (P < 0.01), and the eggshell strength by 39.5% and 24.5% (P < 0.01), respectively, compared with the control diet. Dietary octacosanol addition significantly affected serum triiodothyronine, estradiol, follicle-stimulating hormone levels (P < 0.05), and progesterone and luteinizing hormone level (P < 0.01). Compared with the control, dietary addition of octacosanol at 5 mg/kg promoted the follicle-stimulating hormone receptor (FSHR) mRNA expression in different-sized follicles, and significantly increased the FSHR mRNA expression of granulosa cells from the F2 and F3 follicles (P < 0.05). Dietary supplementation with both 5 and 10 mg/kg octacosanol promoted the mRNA expression of luteinizing hormone receptor and prolactin receptor in different-sized follicles, and significantly up-regulated the expression levels in F1 granulosa cells (P < 0.05). The ovarian weight was significantly increased with the dietary addition of 5 mg/kg octacosanol (P < 0.05). The numbers of small yellow follicles and large white follicles were increased with the addition of dietary 5 and 10 mg/kg octacosanol (P < 0.01). This study provides evidence that octacosanol has the capacity to improve reproductive performance, indicating that it is a potentially effective

  20. TWO-LAYER MATHEMATICAL MODELING OF GENE EXPRESSION: INCORPORATING DNA-LEVEL INFORMATION AND SYSTEM DYNAMICS*

    PubMed Central

    DRESCH, JACQUELINE M.; THOMPSON, MARC A.; ARNOSTI, DAVID N.; CHIU, CHICHIA

    2014-01-01

    High-throughput genome sequencing and transcriptome analysis have provided researchers with a quantitative basis for detailed modeling of gene expression using a wide variety of mathematical models. Two of the most commonly employed approaches used to model eukaryotic gene regulation are systems of differential equations, which describe time-dependent interactions of gene networks, and thermodynamic equilibrium approaches that can explore DNA-level transcriptional regulation. To combine the strengths of these approaches, we have constructed a new two-layer mathematical model that provides a dynamical description of gene regulatory systems, using detailed DNA-based information, as well as spatial and temporal transcription factor concentration data. We also developed a semi-implicit numerical algorithm for solving the model equations and demonstrate here the efficiency of this algorithm through stability and convergence analyses. To test the model, we used it together with the semi-implicit algorithm to simulate a Drosophila gene regulatory circuit that drives development in the dorsal-ventral axis of the blastoderm-stage embryo, involving three genes. For model validation, we have done both mathematical and statistical comparisons between the experimental data and the model’s simulated data. Where protein and cis-regulatory information is available, our two-layer model provides a method for recapitulating and predicting dynamic aspects of eukaryotic transcriptional systems that will greatly improve our understanding of gene regulation at a global level. PMID:25328249

  1. Relationship between Fluorescence Intensity of GFP and the Expression Level of Prestin in a Prestin-Expressing Chinese Hamster Ovary Cell Line

    NASA Astrophysics Data System (ADS)

    Iida, Koji; Nagaoka, Tomoyuki; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

    Outer hair cells (OHCs) in mammals can elongate and contract at frequencies up to 100kHz in response to changes in their membrane potential. The origin of this unique motility is the motor protein prestin, which is densely packed in the lateral membrane of the OHCs. In a previous work, we constructed a prestin-expressing cell line using Chinese hamster ovary (CHO) cells to obtain a stable supply of prestin. When we research prestin using constructed cells, it is necessary to estimate the expression level of prestin in the cells easily and non-invasively. As the prestin gene and a green fluorescent protein (GFP) gene were introduced into constructed cells using the same vector, the expression level of prestin and fluorescence intensity of GFP are possibly correlated. Since this correlation is not clear, however, in this study, we therefore investigated whether the expression level of prestin evaluated by patch-clamp recording and the fluorescence intensity of GFP obtained from fluorescence images are correlated or not. As a result, it was demonstrated that they were correlated. The expression level of prestin can therefore be evaluated by measuring the fluorescence intensity of GFP.

  2. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels.

    PubMed

    Hall, R E; Lee, C S; Alexander, I E; Shine, J; Clarke, C L; Sutherland, R L

    1990-12-15

    The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.

  3. Increasing CACNA1C expression in placenta containing high Cd level: an implication of Cd toxicity.

    PubMed

    Phuapittayalert, Laorrat; Saenganantakarn, Phisid; Supanpaiboon, Wisa; Cheunchoojit, Supaporn; Hipkaeo, Wiphawi; Sakulsak, Natthiya

    2016-12-01

    Cadmium (Cd) has known to produce many adverse effects on organs including placenta. Many essential transporters are involved in Cd transport pathways such as DMT-1, ZIP as well as L-VDCC. Fourteen pregnant women participated and were divided into two groups: high and low Cd-exposed (H-Cd, L-Cd) groups on the basis of their residential areas, Cd concentrations in the blood (B-Cd), urine (U-Cd), and placenta (P-Cd). The results showed that the B-Cd and U-Cd were significantly increased in H-Cd group (p < 0.05). Interestingly, the P-Cd in H-Cd group was elevated (p < 0.05) and positively related to their B-Cd and U-Cd values (p < 0.05). However, the mean cord blood Cd (C-Cd) concentration in H-Cd group was not significantly increased about 2.5-fold when comparing to L-Cd group. To determine the Cd accumulation in placental tissues, metallothionein-1A (MT-1A) and metallothionein-2A (MT-2A) expressions were used as biomarkers. The results revealed that mean MT-1A and MT-2A mRNAs and MT-1/2 proteins were up-regulated in H-Cd group (p < 0.05). In addition, the Ca channel alpha 1C (CACNA1C) mRNA and protein expressions were noticeably elevated in H-Cd group (p < 0.05). From these findings, we suggested that CACNA1C might be implicated in Cd transport in human placenta.

  4. High levels of acetoacetate and glucose increase expression of cytokines in bovine hepatocytes, through activation of the NF-κB signalling pathway.

    PubMed

    Li, Yu; Ding, Hongyan; Wang, Xichun; Liu, Lei; Huang, Dan; Zhang, Renhe; Guo, Lihui; Wang, Zhe; Li, Xiaobing; Liu, Guowen; Wu, Jinjie; Li, Xinwei

    2016-02-01

    Elevated levels of blood interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) increase insulin resistance and result in inflammation. It is not clear whether elevated blood level of acetoacetate (ACAC) and decreased blood level of glucose, which are the predominant characteristics of clinical biochemistry in ketotic dairy cows, increase proinflammatory cytokines and subsequent inflammation. The objective of this study was to test the hypothesis that ACAC and glucose activate the NF-κB signalling pathway to regulate cytokines expression in bovine hepatocytes. Bovine hepatocytes were cultured with ACAC (0-4.8 mm) and glucose (0-5.55 mm) with or without NF-κB inhibitor PDTC for 24 h. The secretion and mRNA levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The NF-κB signalling pathway activation was evaluated by western blotting. Results showed that the secretion and expression of IL-1β, IL-6 and TNF-α increased in an ACAC dose-dependent manner. Additionally, there was an increase in the secretion and mRNA expression of these three cytokines in glucose treatment group, which increased significantly when the glucose concentrations exceed 3.33 mm. Furthermore, both ACAC and glucose upregulated NF-κB p65 protein expression and IκBα phosphorylation levels. However, these effects were reduced by PDTC. These results demonstrate that elevated levels of ACAC and glucose increase the synthesis and expression of proinflammatory factors by activating NF-κB signalling pathway in hepatocytes, which may contribute to inflammation injury in ketotic dairy cows.

  5. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats

    PubMed Central

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects. PMID:27652270

  6. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

  7. Epigallocatechin gallate induces a hepatospecific decrease in the CYP3A expression level by altering intestinal flora.

    PubMed

    Ikarashi, Nobutomo; Ogawa, Sosuke; Hirobe, Ryuta; Kon, Risako; Kusunoki, Yoshiki; Yamashita, Marin; Mizukami, Nanaho; Kaneko, Miho; Wakui, Nobuyuki; Machida, Yoshiaki; Sugiyama, Kiyoshi

    2017-03-30

    In previous studies, we showed that a high-dose intake of green tea polyphenol (GP) induced a hepatospecific decrease in the expression and activity of the drug-metabolizing enzyme cytochrome P450 3A (CYP3A). In this study, we examined whether this decrease in CYP3A expression is induced by epigallocatechin gallate (EGCG), which is the main component of GP. After a diet containing 1.5% EGCG was given to mice, the hepatic CYP3A expression was measured. The level of intestinal bacteria of Clostridium spp., the concentration of lithocholic acid (LCA) in the feces, and the level of the translocation of pregnane X receptor (PXR) to the nucleus in the liver were examined. A decrease in the CYP3A expression level was observed beginning on the second day of the treatment with EGCG. The level of translocation of PXR to the nucleus was significantly lower in the EGCG group. The fecal level of LCA was clearly decreased by the EGCG treatment. The level of intestinal bacteria of Clostridium spp. was also decreased by the EGCG treatment. It is clear that the hepatospecific decrease in the CYP3A expression level observed after a high-dose intake of GP was caused by EGCG. Because EGCG, which is not absorbed from the intestine, causes a decrease in the level of LCA-producing bacteria in the colon, the level of LCA in the liver decreases, resulting in a decrease in the nuclear translocation of PXR, which in turn leads to the observed decrease in the expression level of CYP3A.

  8. Does addition of low-level laser therapy (LLLT) in conservative care of knee arthritis successfully postpone the need for joint replacement?

    PubMed

    Ip, David

    2015-12-01

    The current study evaluates whether the addition of low-level laser therapy into standard conventional physical therapy in elderly with bilateral symptomatic tri-compartmental knee arthritis can successfully postpone the need for joint replacement surgery. A prospective randomized cohort study of 100 consecutive unselected elderly patients with bilateral symptomatic knee arthritis with each knee randomized to receive either treatment protocol A consisting of conventional physical therapy or protocol B which is the same as protocol A with added low-level laser therapy. The mean follow-up was 6 years. Treatment failure was defined as breakthrough pain which necessitated joint replacement surgery. After a follow-up of 6 years, patients clearly benefited from treatment with protocol B as only one knee needed joint replacement surgery, while nine patients treated with protocol A needed surgery (p < 0.05). We conclude low-level laser therapy should be incorporated into standard conservative treatment protocol for symptomatic knee arthritis.

  9. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  10. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

  11. Protection against UVA-induced photooxidative damage in mammalian cell lines expressing increased levels of metallothionein

    SciTech Connect

    Dudek, E.J. Illinois Inst. of Tech., Chicago, IL . Dept. of Biology); Peak, J.G.; Peak, M.J. ); Roth, R.M. . Dept. of Biology)

    1990-01-01

    Metallothionein (MT) is an endogenous low molecular weight protein that is inducible in a variety of eukaryotic cells and has the ability to selectivity bind heavy metal ions such as zinc and the cadmium. Although the exact physiological role of MT is still not understood, there is strong evidence that MT is involved in providing cellular resistance against the damaging effects of heavy metals and in the regulation of intracellular zinc and copper. Recently, it has been demonstrated that MT can scavenge radiation-induced reactive oxygen intermediates in vitro, specifically hydroxyl and superoxide radicals, and because of these observations it has been suggested that MT may provide protection against radiation-induced oxidative stress in vivo. Cell lines expressing increased levels of MT have demonstrated resistance to ionizing radiation, to ultraviolet radiation, and also to various DNA damaging agents including melphalan and cis-diaminedichloroplatinum. It is therefore important to gain some insight into the relationship between cellular MT content and cellular resistance to radiation and other DNA damaging agents. In this study we investigated the role of MT in providing protection against monochromatic 365-nm UVA radiation, which is known to generate intracellular reactive oxygen species that are involved in both DNA damage and cell killing. For this purpose, we used zinc acetate, a potent inducer of MT, to elevate MT levels in V79 Chinese hamster fibroblasts prior to UVA exposure and determined cell survival for uninduced and induced cultures. In order to eliminate any zinc effects other than MT induction, we also isolated and characterized cadmium chloride-resistant clones of V79 cells that have increased steady-state levels of both MT mRNA and protein, and we examined their survival characteristics against 365-nm radiation in the absence of zinc acetate. 14 refs., 3 figs.

  12. Correlation between BOLD-MRI and HIF expression level in renal carcinoma.

    PubMed

    Li, Dong; Wang, Xingming; Wang, Shuai; Cheng, Jie

    2015-01-01

    Occupying about 2%~3% of all malignant tumors, renal carcinoma is the most common primary cancer in kidney. The oxidative level of tumor cells is of vital role for optimizing treatment plan, evaluating efficacy and predicting prognosis. This study thus investigated the R2(*) value in mouse renal carcinoma model and the correlation between tumor hypoxia and expression level of hypoxia inducible factor-1 (HIF-1). A total of 20 BALB/C nude mice (4~6 weeks old) were inoculated with human ACHN renal carcinoma cells to generate renal cancer model. After the tumor diameter reached 0.5 cm, all animals were examined by BOLD-MRI, both under normal inhalation (R2a(*)) and carbogen treatment (R2b(*)). The alternation of R2(*) values (ΔR2(*)=R2a(*) - R2b(*)) was calculated. Mice were then sacrificed for Immunohistochemical (IHC) staining targeting HIF-1α and HIF-2α. The positive score of HIF was then analyzed for its correlation with R2(*) value. In 18 mice finished both experiments, Pearson correlation analysis revealed significant negative correlation between R2a(*) and ΔR2(*) (r=-0.48, P<0.05) and positive relationship between ΔR2(*) and HIF-2α (r=0.38, P<0.05). HIF-1α level, however, did not correlated with tumor R(*) values. The positive correlation between ΔR2(*) and HIF-2α, but not HIF-1α, suggested potential role of combined BOLD-MRI technique and HIF-1α staining in clinical diagnosis of renal carcinoma. HIF-2α may work as biological marker for renal cancer.

  13. MicroRNA 203 expression in keratinocytes is dependent on regulation of p53 levels by E6.

    PubMed

    McKenna, Declan J; McDade, Simon S; Patel, Daksha; McCance, Dennis J

    2010-10-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

  14. Is There a Link Between Expression Levels of Histone Deacetylase/Acetyltransferase in Mouse Sperm and Subsequent Blastocyst Development?

    PubMed

    Kim, Jayeon; Kim, Ji-Hee; Jee, Byung-Chul; Suh, Chang-Suk; Kim, Seok-Hyun

    2015-11-01

    Histone acetylation has been known to be significant in spermatogenesis. Histone acetylation is regulated by the act of histone deacetylases (HDACs) and histone acetyltransferases (HATs). We investigated the link between expression levels of HDACs and HATs in mouse sperm and subsequent blastocyst formation rate. In the univariate analysis, expression levels of HDAC1 and HAT were generally not associated with the blastocyst formation rate. When divided by the mature oocyte number category, a significant positive association was observed between the expression levels of HDAC1 and the blastocyst-forming rate in the highest (> 75th) percentile group (a group with ≥34 mature oocytes). In conclusion, expression of sperm HDAC1 could be considered as a possible predictor of embryo development in mice with high ovarian response.

  15. Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

    PubMed

    Erez-Roman, Racheli; Pienik, Reut; Futerman, Anthony H

    2010-01-01

    Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

  16. Role of serum TRAIL level and TRAIL apoptosis gene expression in multiple sclerosis and relation to brain atrophy.

    PubMed

    Tawdy, Mohamed H; Abd El Nasser, Maged M; Abd El Shafy, Sanaa S; Nada, Mona A F; El Sirafy, Mohamed Nasr I; Magd, Amany Hussien Abol

    2014-09-01

    One of the presumed pathological mechanisms of multiple sclerosis (MS) is the failure of apoptosis of autoreactive T lymphocytes. This study aimed to determine the relationship of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA gene expression ratio and serum TRAIL levels with MS and brain atrophy. This study was conducted on 53 relapsing-remitting Egyptian MS patients and 25 matched healthy volunteers. The expression of TRAIL in peripheral blood lymphocytes was analyzed by reverse transcription polymerase chain reaction, serum levels of soluble TRAIL (sTRAIL) were determined by enzyme-linked immunosorbent assay and brain MRI measured "black holes" and the bicaudate ratio as a measure of brain atrophy in all patients. The serum TRAIL level was lower in MS patients compared to controls but no difference was seen in the TRAIL mRNA gene expression ratio. No significant correlation was detected between the serum TRAIL level and the TRAIL mRNA expression ratio in either group. No statistically significant correlation was found between serum TRAIL levels or the TRAIL mRNA expression ratio with the number of black holes or the bicaudate ratio on MRI. Apoptosis of T lymphocytes is decreased in MS patients, which could be useful when designing treatments. There was no difference in the TRAIL mRNA gene expression ratio between MS patients and controls.

  17. Dinitrophenol modulates gene expression levels of angiogenic, cell survival and cardiomyogenic factors in bone marrow derived mesenchymal stem cells.

    PubMed

    Ali, Anwar; Akhter, Muhammad Aleem; Haneef, Kanwal; Khan, Irfan; Naeem, Nadia; Habib, Rakhshinda; Kabir, Nurul; Salim, Asmat

    2015-01-25

    Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for

  18. Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone

    PubMed Central

    O’Neill, Helen C.; Koopman, Peter; Lovell-Badge, Robin

    2017-01-01

    During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly. PMID:28045957

  19. Expression levels of JNK associated with polymorphic lactotransferrin haplotypes in human nasopharyngeal carcinoma.

    PubMed

    Luo, Gengqiu; Zhou, Yanhong; Yi, Wei; Yi, Hong

    2016-08-01

    Lactotransferrin (LTF), a member of the transferrin family, serves a role in the innate immune response and is involved in anti-inflammatory, anti-microbial and anti-tumor activity. Alterations in the LTF gene are associated with an increased incidence of cancer. The LTF gene is polymorphic, and several common alleles may be observed in the general population. Our previous study identified a lower rate of occurrence of the 'A-G-G-T' haplotype (constructed with rs1126477, rs1126478, rs2073495 and rs9110) in nasopharyngeal carcinoma (NPC) patients compared with controls. In the present study, in order to elucidate a possible mechanism of LTF-mediated anti-tumor activity in NPC, the protein profiles of NPC and non-tumorous nasopharyngeal epithelium tissues with/without the 'A-G-G-T' haplotype were constructed using LTQ Orbitrap technology. The results revealed that c-Jun N-terminal kinase 2 (JNK2) was highly expressed in NPC tissues and non-tumor nasopharyngeal epithelium tissues without the 'A-G-G-T' haplotype. These results were confirmed by western blot analysis. Furthermore, microRNA (miRNA) microarray analysis was conducted to investigate the differential miRNA profiles of NPC and non-tumor nasopharyngeal epithelium tissues with/without the 'A-G-G-T' haplotype. It was observed that hsa-miR-1256 and hsa-miR-659, which are potentially targeted to the JNK2 gene, were downregulated in NPC tissues without the 'A-G-G-T' haplotype. Hsa-miR-298, another miRNA potentially targeted to the JNK2 gene, was downregulated in non-tumor nasopharyngeal epithelium tissues without the 'A-G-G-T' haplotype. In summary, these results suggested that the expression levels of JNK2 may be associated with polymorphic LTF haplotypes in human NPC.

  20. The Expression of Pre- and Postcopulatory Sexually Selected Traits Reflects Levels of Dietary Stress in Guppies

    PubMed Central

    Rahman, Md. Moshiur; Turchini, Giovanni M.; Gasparini, Clelia; Norambuena, Fernando; Evans, Jonathan P.

    2014-01-01

    Environmental and ecological conditions can shape the evolution of life history traits in many animals. Among such factors, food or nutrition availability can play an important evolutionary role in moderating an animal's life history traits, particularly sexually selected traits. Here, we test whether diet quantity and/or composition in the form of omega-3 long chain polyunsaturated fatty acids (here termed ‘n3LC’) influence the expression of pre- and postcopulatory traits in the guppy (Poecilia reticulata), a livebearing poeciliid fish. We assigned males haphazardly to one of two experimental diets supplemented with n3LC, and each of these diet treatments was further divided into two diet ‘quantity’ treatments. Our experimental design therefore explored the main and interacting effects of two factors (n3LC content and diet quantity) on the expression of precopulatory (sexual behaviour and sexual ornamentation, including the size, number and spectral properties of colour spots) and postcopulatory (the velocity, viability, number and length of sperm) sexually selected traits. Our study revealed that diet quantity had significant effects on most of the pre- and postcopulatory traits, while n3LC manipulation had a significant effect on sperm traits and in particular on sperm viability. Our analyses also revealed interacting effects of diet quantity and n3LC levels on courtship displays, and the area of orange and iridescent colour spots in the males’ colour patterns. We also confirmed that our dietary manipulations of n3LC resulted in the differential uptake of n3LC in body and testes tissues in the different n3LC groups. This study reveals the effects of diet quantity and n3LC on behavioural, ornamental and ejaculate traits in P. reticulata and underscores the likely role that diet plays in maintaining the high variability in these condition-dependent sexual traits. PMID:25170940

  1. Expression levels of JNK associated with polymorphic lactotransferrin haplotypes in human nasopharyngeal carcinoma

    PubMed Central

    Luo, Gengqiu; Zhou, Yanhong; Yi, Wei; Yi, Hong

    2016-01-01

    Lactotransferrin (LTF), a member of the transferrin family, serves a role in the innate immune response and is involved in anti-inflammatory, anti-microbial and anti-tumor activity. Alterations in the LTF gene are associated with an increased incidence of cancer. The LTF gene is polymorphic, and several common alleles may be observed in the general population. Our previous study identified a lower rate of occurrence of the ‘A-G-G-T’ haplotype (constructed with rs1126477, rs1126478, rs2073495 and rs9110) in nasopharyngeal carcinoma (NPC) patients compared with controls. In the present study, in order to elucidate a possible mechanism of LTF-mediated anti-tumor activity in NPC, the protein profiles of NPC and non-tumorous nasopharyngeal epithelium tissues with/without the ‘A-G-G-T’ haplotype were constructed using LTQ Orbitrap technology. The results revealed that c-Jun N-terminal kinase 2 (JNK2) was highly expressed in NPC tissues and non-tumor nasopharyngeal epithelium tissues without the ‘A-G-G-T’ haplotype. These results were confirmed by western blot analysis. Furthermore, microRNA (miRNA) microarray analysis was conducted to investigate the differential miRNA profiles of NPC and non-tumor nasopharyngeal epithelium tissues with/without the ‘A-G-G-T’ haplotype. It was observed that hsa-miR-1256 and hsa-miR-659, which are potentially targeted to the JNK2 gene, were downregulated in NPC tissues without the ‘A-G-G-T’ haplotype. Hsa-miR-298, another miRNA potentially targeted to the JNK2 gene, was downregulated in non-tumor nasopharyngeal epithelium tissues without the ‘A-G-G-T’ haplotype. In summary, these results suggested that the expression levels of JNK2 may be associated with polymorphic LTF haplotypes in human NPC. PMID:27446399

  2. The expression of pre- and postcopulatory sexually selected traits reflects levels of dietary stress in guppies.

    PubMed

    Rahman, Md Moshiur; Turchini, Giovanni M; Gasparini, Clelia; Norambuena, Fernando; Evans, Jonathan P

    2014-01-01

    Environmental and ecological conditions can shape the evolution of life history traits in many animals. Among such factors, food or nutrition availability can play an important evolutionary role in moderating an animal's life history traits, particularly sexually selected traits. Here, we test whether diet quantity and/or composition in the form of omega-3 long chain polyunsaturated fatty acids (here termed 'n3LC') influence the expression of pre- and postcopulatory traits in the guppy (Poecilia reticulata), a livebearing poeciliid fish. We assigned males haphazardly to one of two experimental diets supplemented with n3LC, and each of these diet treatments was further divided into two diet 'quantity' treatments. Our experimental design therefore explored the main and interacting effects of two factors (n3LC content and diet quantity) on the expression of precopulatory (sexual behaviour and sexual ornamentation, including the size, number and spectral properties of colour spots) and postcopulatory (the velocity, viability, number and length of sperm) sexually selected traits. Our study revealed that diet quantity had significant effects on most of the pre- and postcopulatory traits, while n3LC manipulation had a significant effect on sperm traits and in particular on sperm viability. Our analyses also revealed interacting effects of diet quantity and n3LC levels on courtship displays, and the area of orange and iridescent colour spots in the males' colour patterns. We also confirmed that our dietary manipulations of n3LC resulted in the differential uptake of n3LC in body and testes tissues in the different n3LC groups. This study reveals the effects of diet quantity and n3LC on behavioural, ornamental and ejaculate traits in P. reticulata and underscores the likely role that diet plays in maintaining the high variability in these condition-dependent sexual traits.

  3. High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

    PubMed

    Guertin, Amy D; O'Neil, Jennifer; Stoeck, Alexander; Reddy, Joseph A; Cristescu, Razvan; Haines, Brian B; Hinton, Marlene C; Dorton, Ryan; Bloomfield, Alicia; Nelson, Melissa; Vetzel, Marilynn; Lejnine, Serguei; Nebozhyn, Michael; Zhang, Theresa; Loboda, Andrey; Picard, Kristen L; Schmidt, Emmett V; Dussault, Isabelle; Leamon, Christopher P

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has