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Sample records for genotype dependent expressed

  1. Genotype dependent burst of transposable element expression in crowns of hexaploid wheat (Triticum aestivum L.) during cold acclimation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The expression of 1,613 transposable elements (TEs) represented in the Affymetix Wheat Genome Chip was examined during cold treatment in crowns of 4 hexaploid wheat genotypes that vary in tolerance to cold and in flowering time. The TE expression profiles showed a constant level of expression throug...

  2. Genotype-dependent expression of specific members of potato protease inhibitor gene families in different tissues and in response to wounding and nematode infection.

    PubMed

    Turrà, David; Bellin, Diana; Lorito, Matteo; Gebhardt, Christiane

    2009-05-01

    Protease inhibitors (PIs) are small ubiquitous proteins with a variety of biological functions in plants, including protein stabilization, modulation of apoptosis and defense against pathogens. Kunitz-like inhibitors (PKPIs) and proteinase inhibitors 1 (PI-1) are abundant in storage organs of potato plants and are up-regulated in other tissues in response to biotic and abiotic stress. However, little information is available on genotype-dependent regulation of individual PKPI group- and PI-1 genes. We isolated, sequenced and characterized four novel full-length PI-1 cDNAs (PPI3A2, PPI3A4, PPI2C4 and PPI2C1A) from Solanum tuberosum cv. Desirée. Specific primers were developed for PI-1 genes PPI3A2, PPI3B2 and PPI2C4 and the three PKPI homology groups A, B and C. Their expression profiles were studied by semi-quantitative RT-PCR in comparison with transcripts of the PI-1, Pin2 and PR1 gene families in various tissues, after wounding and Globodera rostochiensis infection of nematode-resistant genotypes P40 and LB7/4/c-I-7, and susceptible cv. Desirée. Individual PI-1 genes and PKPI homology groups were expressed in a tissue- and genotype-dependent manner after wounding and nematode infection. The differences in PI expression patterns were related to the intensity, type of inhibitors produced, and the kinetics of induction. Therefore, different genotype-environment combinations produce different sets of PI transcripts. Potato plants reacted to G. rostochiensis infection by modulating PKPI, PI-1 and Pin2, but not PR1 gene expression, suggesting that the jasmonic acid but not the salicylic acid defense signaling pathway is activated. PI expression profiles were not correlated with the resistance status of the potato genotype infected with G. rostochiensis. PMID:19095329

  3. Comparative analyses of genotype dependent expressed sequence tags and stress-responsive transcriptome of chickpea wilt illustrate predicted and unexpected genes and novel regulators of plant immunity

    PubMed Central

    Ashraf, Nasheeman; Ghai, Deepali; Barman, Pranjan; Basu, Swaraj; Gangisetty, Nagaraju; Mandal, Mihir K; Chakraborty, Niranjan; Datta, Asis; Chakraborty, Subhra

    2009-01-01

    Background The ultimate phenome of any organism is modulated by regulated transcription of many genes. Characterization of genetic makeup is thus crucial for understanding the molecular basis of phenotypic diversity, evolution and response to intra- and extra-cellular stimuli. Chickpea is the world's third most important food legume grown in over 40 countries representing all the continents. Despite its importance in plant evolution, role in human nutrition and stress adaptation, very little ESTs and differential transcriptome data is available, let alone genotype-specific gene signatures. Present study focuses on Fusarium wilt responsive gene expression in chickpea. Results We report 6272 gene sequences of immune-response pathway that would provide genotype-dependent spatial information on the presence and relative abundance of each gene. The sequence assembly led to the identification of a CaUnigene set of 2013 transcripts comprising of 973 contigs and 1040 singletons, two-third of which represent new chickpea genes hitherto undiscovered. We identified 209 gene families and 262 genotype-specific SNPs. Further, several novel transcription regulators were identified indicating their possible role in immune response. The transcriptomic analysis revealed 649 non-cannonical genes besides many unexpected candidates with known biochemical functions, which have never been associated with pathostress-responsive transcriptome. Conclusion Our study establishes a comprehensive catalogue of the immune-responsive root transcriptome with insight into their identity and function. The development, detailed analysis of CaEST datasets and global gene expression by microarray provide new insight into the commonality and diversity of organ-specific immune-responsive transcript signatures and their regulated expression shaping the species specificity at genotype level. This is the first report on differential transcriptome of an unsequenced genome during vascular wilt. PMID:19732460

  4. Fatty acid composition of chicken breast meat is dependent on genotype-related variation of FADS1 and FADS2 gene expression and desaturating activity.

    PubMed

    Boschetti, E; Bordoni, A; Meluzzi, A; Castellini, C; Dal Bosco, A; Sirri, F

    2016-04-01

    In Western countries the dietary guidance emphasizes the need to decrease the intake of saturated fatty acids and to replace them with polyunsaturated fatty acids (PUFA), particularly long chain n-3 PUFA (LC-PUFA). The production of poultry meat having a lower fat content and healthier fatty acid (FA) profile is a hot topic for the poultry industry, and the possibility to identify genotypes able to produce meat with a higher LC-PUFA content deserves attention. The aims of the present study were to evidence in chicken (i) a genotype-related different expression of the desaturating enzymes delta-6 (Δ6, EC 1.14.99.25), delta-5 (Δ5, EC 1.14.19.) and delta-9 (Δ9, EC 1.14.19.1); (ii) the impact of the hypothesized different expression on the meat FA composition; (iii) the distribution of desaturase products in the different lipid classes. Slow (SG), medium (MG) and fast (FG) growing chickens fed the same diet were evaluated either for the relative expression of FADS1, FADS2 and SCD1 genes in liver (by q-PCR), or for the FA composition of breast meat. MG and particularly SG birds showed a greater expression of FADS2 and FADS1 genes, a higher Δ6 and Δ5 activity (estimated using desaturase indices), and consequently a higher LC-PUFA content in the breast meat than FG birds. The relationship between genotype and desaturating ability was demonstrated, with a significant impact on the PUFA content of breast meat. Due to the high consumption rate of avian meat, the identification of the best genotypes for meat production could represent an important goal not only for the food industry, but also for the improvement of human nutrition. PMID:26670346

  5. Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle.

    PubMed

    Liu, Y; Kong, X; Li, F; Tan, B; Li, Y; Duan, Y; Yin, Y; He, J; Hu, C; Blachier, F; Wu, Guoyao

    2016-01-01

    A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner. PMID:26255284

  6. Comparative analysis of gene expression in response to cold stress in diverse rice genotypes.

    PubMed

    Moraes de Freitas, Gabriela Peres; Basu, Supratim; Ramegowda, Venkategowda; Braga, Eugenia Bolacel; Pereira, Andy

    2016-02-26

    Cold stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution worldwide. Rice production is affected primarily due to its vulnerability to cold stress at seedling stage, as well as reproductive stage leading to spikelet sterility. We report here the analysis of 21 diverse rice genotypes from the USDA mini-core collection for cold tolerance and categorized their tolerance levels on the basis of reduction in growth measured by root and shoot length. The screening identified 12 cold tolerant genotypes from which six tolerant genotypes were characterized at the vegetative stage for cold tolerance and gas-exchange parameters. Two tolerant and two sensitive genotypes were used further for gene expression analysis. Lipid Transfer Protein (LTP) genes showed a clear difference in expression between cold tolerant and sensitive genotypes suggesting that they are good candidates for engineering cold tolerance in rice. Nipponbare was identified as a cold tolerant genotype with stress tolerance mechanism potentially operating via both ABA dependent and independent pathways. PMID:26855133

  7. Genotype-dependent lifespan effects in peptone deprived Caenorhabditis elegans

    PubMed Central

    Stastna, Jana J.; Snoek, L. Basten; Kammenga, Jan E.; Harvey, Simon C.

    2015-01-01

    Dietary restriction appears to act as a general non-genetic mechanism that can robustly prolong lifespan. There have however been reports in many systems of cases where restricted food intake either shortens, or does not affect, lifespan. Here we analyze lifespan and the effect of food restriction via deprived peptone levels on lifespan in wild isolates and introgression lines (ILs) of the nematode Caenorhabditis elegans. These analyses identify genetic variation in lifespan, in the effect of this variation in diet on lifespan and also in the likelihood of maternal, matricidal, hatching. Importantly, in the wild isolates and the ILs, we identify genotypes in which peptone deprivation mediated dietary restriction reduces lifespan. We also identify, in recombinant inbred lines, a locus that affects maternal hatching, a phenotype closely linked to dietary restriction in C. elegans. These results indicate that peptone deprivation mediated dietary restriction affects lifespan in C. elegans in a genotype-dependent manner, reducing lifespan in some genotypes. This may operate by a mechanism similar to dietary restriction. PMID:26539794

  8. Apolipoprotein C-I is an APOE genotype-dependent suppressor of glial activation

    PubMed Central

    2012-01-01

    Background Inheritance of the human ϵ4 allele of the apolipoprotein (apo) E gene (APOE) significantly increases the risk of developing Alzheimer’s disease (AD), in addition to adversely influencing clinical outcomes of other neurologic diseases. While apoE isoforms differentially interact with amyloid β (Aβ), a pleiotropic neurotoxin key to AD etiology, more recent work has focused on immune regulation in AD pathogenesis and on the mechanisms of innate immunomodulatory effects associated with inheritance of different APOE alleles. APOE genotype modulates expression of proximal genes including APOC1, which encodes a small apolipoprotein that is associated with Aβ plaques. Here we tested the hypothesis that APOE-genotype dependent innate immunomodulation may be mediated in part by apoC-I. Methods ApoC-I concentration in cerebrospinal fluid from control subjects of differing APOE genotypes was quantified by ELISA. Real-time PCR and ELISA were used to analyze apoC-I mRNA and protein expression, respectively, in liver, serum, cerebral cortex, and cultured primary astrocytes derived from mice with targeted replacement of murine APOE for human APOE ϵ3 or ϵ4. ApoC-I direct modulation of innate immune activity was investigated in cultured murine primary microglia and astrocytes, as well as human differentiated macrophages, using specific toll-like receptor agonists LPS and PIC as well as Aβ. Results ApoC-I levels varied with APOE genotype in humans and in APOE targeted replacement mice, with ϵ4 carriers showing significantly less apoC-I in both species. ApoC-I potently reduced pro-inflammatory cytokine secretion from primary murine microglia and astrocytes, and human macrophages, stimulated with LPS, PIC, or Aβ. Conclusions ApoC-I is immunosuppressive. Our results illuminate a novel potential mechanism for APOE genotype risk for AD; one in which patients with an ϵ4 allele have decreased expression of apoC-I resulting in increased innate immune activity. PMID

  9. Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury*

    PubMed Central

    Asleh, Rabea; Ward, John; Levy, Nina S.; Safuri, Shady; Aronson, Doron; Levy, Andrew P.

    2014-01-01

    The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb. PMID:24778180

  10. Gene expression profiling of HCV genotype 3a initial liver fibrosis and cirrhosis patients using microarray

    PubMed Central

    2012-01-01

    Background Hepatitis C virus (HCV) causes liver fibrosis that may lead to liver cirrhosis or hepatocellular carcinoma (HCC), and may partially depend on infecting viral genotype. HCV genotype 3a is being more common in Asian population, especially Pakistan; the detail mechanism of infection still needs to be explored. In this study, we investigated and compared the gene expression profile between initial fibrosis stage and cirrhotic 3a genotype patients. Methods Gene expression profiling of human liver tissues was performed containing more than 22000 known genes. Using Oparray protocol, preparation and hybridization of slides was carried out and followed by scanning with GeneTAC integrator 4.0 software. Normalization of the data was obtained using MIDAS software and Significant Microarray Analysis (SAM) was performed to obtain differentially expressed candidate genes. Results Out of 22000 genes studied, 219 differentially regulated genes found with P ≤ 0.05 between both groups; 107 among those were up-regulated and 112 were down-regulated. These genes were classified into 31 categories according to their biological functions. The main categories included: apoptosis, immune response, cell signaling, kinase activity, lipid metabolism, protein metabolism, protein modulation, metabolism, vision, cell structure, cytoskeleton, nervous system, protein metabolism, protein modulation, signal transduction, transcriptional regulation and transport activity. Conclusion This is the first study on gene expression profiling in patients associated with genotype 3a using microarray analysis. These findings represent a broad portrait of genomic changes in early HCV associated fibrosis and cirrhosis. We hope that identified genes in this study will help in future to act as prognostic and diagnostic markers to differentiate fibrotic patients from cirrhotic ones. PMID:22397681

  11. Genotype-dependency of butyrate efficacy in children with congenital chloride diarrhea

    PubMed Central

    2013-01-01

    Background Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by life-long, severe diarrhea with intestinal Cl- malabsorption. It results from a reduced activity of the down regulated in adenoma exchanger (DRA), due to mutations in the solute carrier family 26, member 3 (SLC26A3) gene. Currently available therapies are not able to limit the severity of diarrhea in CLD. Conflicting results have been reported on the therapeutic efficacy of oral butyrate. Methods We investigated the effect of oral butyrate (100 mg/kg/day) in seven CLD children with different SLC26A3 genotypes. Nasal epithelial cells were obtained to assess the effect of butyrate on the expression of the two main Cl- transporters: DRA and putative anion transporter-1 (PAT-1). Results A variable clinical response to butyrate was observed regarding the stool pattern and fecal ion loss. The best response was observed in subjects with missense and deletion mutations. Variable response to butyrate was also observed on SLC26A3 (DRA) and SLC26A6 (PAT1) gene expression in nasal epithelial cells of CLD patients. Conclusions We demonstrate a genotype-dependency for butyrate therapeutic efficacy in CLD. The effect of butyrate is related in part on a different modulation of the expression of the two main apical membrane Cl- exchangers of epithelial cells, members of the SLC26 anion family. Trial registration Australian New Zealand Clinical trial Registry ACTRN12613000450718. PMID:24350656

  12. Dissection of the Phenotypic and Genotypic Associations With Nicotinic Dependence

    PubMed Central

    Baker, Timothy B.; Grucza, Richard; Wang, Jen C.; Johnson, Eric O.; Breslau, Naomi; Hatsukami, Dorothy; Smith, Stevens S.; Saccone, Nancy; Saccone, Scott; Rice, John P.; Goate, Alison M.; Bierut, Laura J.

    2012-01-01

    Introduction: Strong evidence demonstrates that nicotine dependence is associated with 4 genetic variants rs16969968, rs6474412, rs3733829, and rs1329650 in large-scale Genome-Wide Association Studies. We examined how these identified genetic variants relate to nicotine dependence defined by different categorical and dimensional measures. Methods: Four genetic variants were analyzed in 2,047 subjects of European descent (1,062 cases and 985 controls). Nicotine dependence was assessed with multiple smoking measures, including the Fagerström Test for Nicotine Dependence, the Diagnostic and Statistical Manual for Mental Disorders-IV (DSM-IV) nicotine dependence, the Nicotine Dependence Syndrome Scale, and the Wisconsin Inventory of Smoking Dependence Motives. Single-item measures of cigarettes per day (CPD) and time to first cigarette (TTF) in the morning were also examined. Results: Among the variants, association effect sizes were largest for rs16969968, with measures of craving and heavy smoking, especially cigarettes smoked per day, showing the largest effects. Significant but weaker associations were found for rs6474412 and rs3733729 but not for rs1329650. None of the more comprehensive measures of smoking behaviors yielded stronger genetic associations with these variants than did CPD. Conclusions: CPD is an important simple measure that captures in part the genetic associations of CHRNA5 and nicotine dependence, even when other more comprehensive measures of smoking behaviors are examined. The CHRNA5 gene is associated with heavy compulsive smoking and craving; this should inform the mission to improve the diagnostic validity of DSM-V. PMID:22102629

  13. Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and Inflammation

    PubMed Central

    Rhainds, David; Brodeur, Mathieu; Feroz Zada, Yassamin; Fouodjio, René; Provost, Sylvie; Boulé, Marie; Alem, Sonia; Grégoire, Jean C.; L’Allier, Philippe L.; Ibrahim, Reda; Guertin, Marie-Claude; Mongrain, Ian; Olsson, Anders G.; Schwartz, Gregory G.; Rhéaume, Eric

    2016-01-01

    Background— Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. Methods and Results— Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was −1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. Conclusions— Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. Clinical Trials Registration— ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682. PMID:27418594

  14. Environmental dependency of amphibian-ranavirus genotypic interactions: evolutionary perspectives on infectious diseases.

    PubMed

    Echaubard, Pierre; Leduc, Joel; Pauli, Bruce; Chinchar, V Gregory; Robert, Jacques; Lesbarrères, David

    2014-08-01

    The context-dependent investigations of host-pathogen genotypic interactions, where environmental factors are explicitly incorporated, allow the assessment of both coevolutionary history and contemporary ecological influences. Such a functional explanatory framework is particularly valuable for describing mortality trends and identifying drivers of disease risk more accurately. Using two common North American frog species (Lithobates pipiens and Lithobates sylvaticus) and three strains of frog virus 3 (FV3) at different temperatures, we conducted a laboratory experiment to investigate the influence of host species/genotype, ranavirus strains, temperature, and their interactions, in determining mortality and infection patterns. Our results revealed variability in host susceptibility and strain infectivity along with significant host-strain interactions, indicating that the outcome of an infection is dependent on the specific combination of host and virus genotypes. Moreover, we observed a strong influence of temperature on infection and mortality probabilities, revealing the potential for genotype-genotype-environment interactions to be responsible for unexpected mortality in this system. Our study thus suggests that amphibian hosts and ranavirus strains genetic characteristics should be considered in order to understand infection outcomes and that the investigation of coevolutionary mechanisms within a context-dependent framework provides a tool for the comprehensive understanding of disease dynamics. PMID:25469155

  15. CYP2D6 Genotype Dependent Oxycodone Metabolism in Postoperative Patients

    PubMed Central

    Stamer, Ulrike M.; Zhang, Lan; Book, Malte; Lehmann, Lutz E.; Stuber, Frank; Musshoff, Frank

    2013-01-01

    Background The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone's metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption. Methods Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA) for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele), HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity), EM (extensive metabolizers, normal CYP2D6 activity) and UM (ultrarapid metabolizers, increased CYP2D6 activity). Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide. Results Metabolism differed between CYP2D6 genotypes. Mean (95%-CI) oxymophone/oxycodone ratios were 0.10 (0.02/0.19), 0.13 (0.11/0.16), 0.18 (0.16/0.20) and 0.28 (0.07/0.49) in PM, HZ/IM, EM and UM, respectively (p = 0.005). Oxycodone consumption up to the 12th hour was highest in PM (p = 0.005), resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8); EM and UM 2.2 (2.1/2.3); p<0.001). Pain scores did not differ between genotypes. Conclusions In this postoperative setting, the number of

  16. Genotype and gene expression associations with immune function in Drosophila.

    PubMed

    Sackton, Timothy B; Lazzaro, Brian P; Clark, Andrew G

    2010-01-01

    It is now well established that natural populations of Drosophila melanogaster harbor substantial genetic variation associated with physiological measures of immune function. In no case, however, have intermediate measures of immune function, such as transcriptional activity of immune-related genes, been tested as mediators of phenotypic variation in immunity. In this study, we measured bacterial load sustained after infection of D. melanogaster with Serratia marcescens, Providencia rettgeri, Enterococcus faecalis, and Lactococcus lactis in a panel of 94 third-chromosome substitution lines. We also measured transcriptional levels of 329 immune-related genes eight hours after infection with E. faecalis and S. marcescens in lines from the phenotypic tails of the test panel. We genotyped the substitution lines at 137 polymorphic markers distributed across 25 genes in order to test for statistical associations among genotype, bacterial load, and transcriptional dynamics. We find that genetic polymorphisms in the pathogen recognition genes (and particularly in PGRP-LC, GNBP1, and GNBP2) are most significantly associated with variation in bacterial load. We also find that overall transcriptional induction of effector proteins is a significant predictor of bacterial load after infection with E. faecalis, and that a marker upstream of the recognition gene PGRP-SD is statistically associated with variation in both bacterial load and transcriptional induction of effector proteins. These results show that polymorphism in genes near the top of the immune system signaling cascade can have a disproportionate effect on organismal phenotype due to the amplification of minor effects through the cascade. PMID:20066029

  17. Vector Competence in West African Aedes aegypti Is Flavivirus Species and Genotype Dependent

    PubMed Central

    Dickson, Laura B.; Sanchez-Vargas, Irma; Sylla, Massamba; Fleming, Karen; Black, William C.

    2014-01-01

    Background Vector competence of Aedes aegypti mosquitoes is a quantitative genetic trait that varies among geographic locations and among different flavivirus species and genotypes within species. The subspecies Ae. aegypti formosus, found mostly in sub-Saharan Africa, is considered to be refractory to both dengue (DENV) and yellow fever viruses (YFV) compared to the more globally distributed Ae. aegypti aegypti. Within Senegal, vector competence varies with collection site and DENV-2 viral isolate, but knowledge about the interaction of West African Ae. aegypti with different flaviviruses is lacking. The current study utilizes low passage isolates of dengue-2 (DENV-2-75505 sylvatic genotype) and yellow fever (YFV BA-55 -West African Genotype I, or YFV DAK 1279-West African Genotype II) from West Africa and field derived Ae. aegypti collected throughout Senegal to determine whether vector competence is flavivirus or virus genotype dependent. Methodology/Principal Findings Eight collections of 20–30 mosquitoes from different sites were fed a bloodmeal containing either DENV-2 or either isolate of YFV. Midgut and disseminated infection phenotypes were determined 14 days post infection. Collections varied significantly in the rate and intensity of midgut and disseminated infection among the three viruses. Conclusions/Significance Overall, vector competence was dependent upon both viral and vector strains. Importantly, contrary to previous studies, sylvatic collections of Ae. aegypti showed high levels of disseminated infection for local isolates of both DENV-2 and YFV. PMID:25275366

  18. Water deficit effects on tomato quality depend on fruit developmental stage and genotype.

    PubMed

    Ripoll, Julie; Urban, Laurent; Brunel, Béatrice; Bertin, Nadia

    2016-01-15

    Many studies have advocated that water deficit (WD) may exert beneficial effects on fruit quality. However, the fruit response to WD at specific developmental stages was seldom investigated, although different mechanisms could be involved at each stage and lead to different effects on final fruit quality. In the present study, a moderate WD (-60% of water supply compared to control) was applied during each of the three major phases of fruit development, namely cell division (CD), cell expansion (CE) and maturation (MT). Two cocktail tomato (Solanum lycopersicum L.) genotypes were studied, one producing poor quality fruits (LA1420), and the other one producing tasty fruits (PlovdivXXIVa named Plovdiv). Contrasted responses were observed between the two genotypes. For both of them, fruit fresh mass and size were not significantly reduced by WD, whatever the developmental phase affected. Osmotic regulations were likely involved in the CD treatment for LA1420 fruits, which accumulated more sugars (both on a dry and fresh matter basis) and less acids (on a dry matter basis). In the CE treatment, other adaptive strategies involving sugar metabolism and sub-cellular compartmentation were suggested. In contrast, the composition of Plovdiv fruits changed only under the MT treatment, with less sugars, acids and carotenoids compared to control fruits (both on a dry and fresh matter basis). Total ascorbic acid (AsA) was not significantly influenced by treatments in both genotypes. On their whole, results suggest that, depending on genotypes, fruits are sweeter and less acidic under WD, but that the nutritive value related to vitamin and carotenoid contents may be lessened. The sensitivity of each developmental phase highly depends on the genotype. All phases were sensitive to WD for LA1420, but only the ripening phase for Plovdiv. Interestingly, major changes in fruit composition were observed in LA1420 which presents poor fruit quality under control conditions. This suggests

  19. Gene expression and physiological responses to salinity and water stress of contrasting durum wheat genotypes.

    PubMed

    Yousfi, Salima; Márquez, Antonio J; Betti, Marco; Araus, José Luis; Serret, Maria Dolores

    2016-01-01

    Elucidating the relationships between gene expression and the physiological mechanisms remains a bottleneck in breeding for resistance to salinity and drought. This study related the expression of key target genes with the physiological performance of durum wheat under different combinations of salinity and irrigation. The candidate genes assayed included two encoding for the DREB (dehydration responsive element binding) transcription factors TaDREB1A and TaDREB2B, another two for the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), and one for the specific Na(+) /H(+) vacuolar antiporter (TaNHX1). Expression of these genes was related to growth and different trait indicators of nitrogen metabolism (nitrogen content, stable nitrogen isotope composition, and glutamine synthetase and nitrate reductase activities), photosynthetic carbon metabolism (stable carbon isotope composition and different gas exchange traits) and ion accumulation. Significant interaction between genotype and growing conditions occurred for growth, nitrogen content, and the expression of most genes. In general terms, higher expression of TaGS1, TaGS2, TaDREB2B, and to a lesser extent of TaNHX1 were associated with a better genotypic performance in growth, nitrogen, and carbon photosynthetic metabolism under salinity and water stress. However, TaDREB1A was increased in expression under stress compared with control conditions, with tolerant genotypes exhibiting lower expression than susceptible ones. PMID:25869057

  20. The genomic determinants of genotype × environment interactions in gene expression.

    PubMed

    Grishkevich, Vladislav; Yanai, Itai

    2013-08-01

    Predicting phenotype from genotype is greatly complicated by the polygenic nature of most traits and by the complex interactions between phenotype and the environment. Here, we review recent whole-genome approaches to understand the underlying principles, mechanisms, and evolutionary impacts of genotype × environment (G×E) interactions, defined as genotype-specific phenotypic responses to different environments. There is accumulating evidence that G×E interactions are ubiquitous, accounting perhaps for the greater part of the phenotypic variation seen across genotypes. Such interactions appear to be the consequence of changes to upstream regulators as opposed to local changes to promoters. Moreover, genes are not equally likely to exhibit G×E interactions; promoter architecture, expression level, regulatory complexity, and essentiality correlate with the differential regulation of a gene by the environment. One implication of this correlation is that expression variation across genotypes alone could be used as a proxy for G×E interactions in those experimental cases where identifying environmental variation is costly or impossible. PMID:23769209

  1. COMT genotype is associated with differential expression of muscarinic M1 receptors in human cortex.

    PubMed

    Dean, Brian; Scarr, Elizabeth

    2016-09-01

    Catechol-O-methyltransferase (COMT) genotype has been associated with varying levels of cognitive functioning and an altered risk of schizophrenia. COMT regulates the breakdown of catecholamines, particularly dopamine, which is thought critical in maintaining cognitive function and the aetiology of schizophrenia. This hypothesis gained support from reports that the VAL allele at rs4680 was associated with poorer performance on cognitive tests and a slightly increased risk of schizophrenia. More recently, genotype at rs4818, part of a hapblock with rs4680, has been shown to impact on cognitive ability more than genotype at rs4680 but, as yet, not the risk for schizophrenia. Here, we determined if COMT genotype at rs4680 or rs4818, as well as rs165519 and rs737865, two synonymous single nucleotide polymorphisms (SNPs) with no known functional consequences, were associated with an altered risk of schizophrenia and if genotype at the four COMT SNPs was related to expression of the cortical muscarinic M1 receptor (CHRM1) because the expression of the cortical CHRM1 has been reported to be lower in schizophrenia and is important in maintaining cognitive functioning in humans. We report that the variation in gene sequence at the four COMT SNPs studied was not associated with an altered the risk of schizophrenia but genotype at rs4680 and rs4818, but not rs165519 and rs737865, were associated with varying levels of cortical CHRM1 expression in the human dorsolateral prefrontal cortex (DLPFC). These data are the first to suggest that levels of CHRM1 in the human DLPFC are, in part, determined by COMT gene sequence. © 2016 Wiley Periodicals, Inc. PMID:26954460

  2. Haptoglobin genotype- and diabetes-dependent differences in iron-mediated oxidative stress in vitro and in vivo.

    PubMed

    Asleh, Rabea; Guetta, Julia; Kalet-Litman, Shiri; Miller-Lotan, Rachel; Levy, Andrew P

    2005-03-01

    We have recently demonstrated in multiple independent population-based longitudinal and cross sectional analyses that the haptoglobin 2-2 genotype is associated with an increased risk for diabetic cardiovascular disease. The chief function of haptoglobin (Hp) is to bind to hemoglobin and thereby prevent hemoglobin-induced oxidative tissue damage. This antioxidant function of haptoglobin is mediated in part by the ability of haptoglobin to prevent the release of iron from hemoglobin on its binding. We hypothesized that there may be diabetes- and haptoglobin genotype-dependent differences in the amount of catalytically active redox active iron derived from hemoglobin. We tested this hypothesis using several complementary approaches both in vitro and in vivo. First, measuring redox active iron associated with haptoglobin-hemoglobin complexes in vitro, we demonstrate a marked increase in redox active iron associated with Hp 2-2-glycohemoglobin complexes. Second, we demonstrate increased oxidative stress in tissue culture cells exposed to haptoglobin 2-2-hemoglobin complexes as opposed to haptoglobin 1-1-hemoglobin complexes, which is inhibitable by desferrioxamine by either a chelation or reduction mechanism. Third, we demonstrate marked diabetes-dependent differences in the amount of redox active iron present in the plasma of mice genetically modified expressing the Hp 2 allele as compared with the Hp 1 allele. Taken together these data implicate redox active iron in the increased susceptibility of individuals with the Hp 2 allele to diabetic vascular disease. PMID:15662028

  3. Data on IL-6 c.-174 G>C genotype and allele frequencies in patients with coronary heart disease in dependence of cardiovascular outcome.

    PubMed

    Reichert, Stefan; Schlitt, Axel; Benten, Ann-Christin; Hofmann, Britt; Schaller, Hans-Günter; Schulz, Susanne

    2016-09-01

    In this data article we present data on the distribution of alleles and genotypes of the interleukin (IL)-6 c.-174 G>C polymorphism (rs 1800795) in patients with coronary heart disease (CHD) in dependence of the incidence of new cardiovascular events (combined endpoint: myocardial infarction, stroke/TIA, cardiac death, death according to stroke) within three years follow-up. Moreover, we investigated putative associations between individual expression of IL-6 genotypes and IL-6 serum level. This investigation is a subanalysis of the article entitled "The Interleukin 6 c.-174 CC genotype is a predictor for new cardiovascular events in patients with coronary heart disease within three years follow-up" (ClinicalTrials.gov identifier: NCT01045070) (Reichert et al., 2016) [1]. PMID:27570807

  4. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

    PubMed

    Khankhet, Jordan; Vanderwolf, Karen J; McAlpine, Donald F; McBurney, Scott; Overy, David P; Slavic, Durda; Xu, Jianping

    2014-01-01

    Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America. PMID:25122221

  5. Clonal Expansion of the Pseudogymnoascus destructans Genotype in North America Is Accompanied by Significant Variation in Phenotypic Expression

    PubMed Central

    Khankhet, Jordan; Vanderwolf, Karen J.; McAlpine, Donald F.; McBurney, Scott; Overy, David P.; Slavic, Durda; Xu, Jianping

    2014-01-01

    Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition - dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America. PMID:25122221

  6. Low-level sequence variation in Toxoplasma gondii calcium-dependent protein kinases among different genotypes.

    PubMed

    Wang, J L; Zhang, N Z; Huang, S Y; Xu, Y; Wang, R A; Zhu, X Q

    2015-01-01

    The causative agent of toxoplasmosis, Toxoplasma gondii, can infect virtually all nucleated cell types of warm-blooded animals. In this study, we examined the sequence variation in calcium-dependent protein kinase 2 (CDPK2) genes among 13 T. gondii strains from different hosts and geographical locations. The results showed that the lengths of the complete CDPK2 DNA and cDNA sequences were 3671-3673 and 2136 bp, respectively, and the sequence variation was 0-0.9% among different T. gondii strains. Phylogenetic analysis based on the CDPK2 gene sequences revealed that T. gondii strains of the same genotypes were clustered in different clades. Further analysis of all the other T. gondii CDPK genes in genotype I (GT1), II (ME49), or III (VEG) strains indicated the T. gondii CDPK gene family is quite conserved, with sequence variation ranging from 0 to 1.40%. We concluded that CDPK2 as well as all the other CDPK genes in T. gondii cannot be used as proper markers for studying the variants of different T. gondii genotypes from different hosts and geographical locations, but their sequence conservation may be a useful feature promoting them as anti-T. gondii vaccine candidates in further studies. PMID:25966270

  7. Phenotype and Genotype in a Cohort of 312 Adult Patients with Nontransfusion-Dependent Thalassemia in Northeast Thailand.

    PubMed

    Prayalaw, Patcharawadee; Teawtrakul, Nattiya; Jetsrisuparb, Arunee; Pongudom, Saranya; Fucharoen, Goonnapa; Fucharoen, Supan

    2016-01-01

    Patients with nontransfusion-dependent thalassemia (NTDT) do not require regular blood transfusion for survival but may encounter several complications that contribute to morbidity and mortality. We report the molecular heterogeneity and hematological features of NTDT in 312 adult patients in northeast Thailand. Hemoglobin (Hb) and DNA analyses identified 177 subjects with Hb E-β-thalassemia, 1 with homozygous β0-thalassemia and 134 with Hb H, AEBart's and EEBart's diseases. For β-thalassemia, 12 different mutations including both β0- and β+-thalassemias were detected. Coinheritance of α-thalassemia as an ameliorating factor was observed in 18 of 178 cases (10.1%) with β-thalassemia. The α-globin gene triplicated haplotype (αααanti3.7) was observed in 1 case of Hb E-β0-thalassemia. The presence of the -158 (Cx2192;T) Gx03B3;-XmnI polymorphism (+/+) was found to be associated with increased Hb F expression, but its frequency in the studied subjects was low. Those with α-thalassemia included 17 with deletional and 51 nondeletional Hb H, and 63 with AEBart's and 3 with EEBart's diseases. The hematological parameters of these NTDT and genotype-phenotype relationships are presented. The diverse molecular heterogeneity of NTDT underlines the importance of complete genotyping of the patient. These results should prove useful for management planning, the prediction of clinical outcome and to improve genetic counseling for NTDT patients. PMID:26303193

  8. Reduced Expression of α-Synuclein in Alcoholic Brain: Influence of SNCA-Rep1 Genotype

    PubMed Central

    Janeczek, Paulina; MacKay, Rachel K.; Lea, Rodney A.; Dodd, Peter R.; Lewohl, Joanne M.

    2012-01-01

    α-Synuclein has recently been implicated in the pathophysiology of alcohol abuse due to its role in dopaminergic neurotransmission. In these studies, genetic variability in the α-synuclein gene influences its expression which may contribute to susceptibility to chronic alcohol abuse. Real-time PCR was used to quantify α-synuclein mRNA expression in autopsy samples of human dorsolateral prefrontal cortex. Because of the association between length of the α-synuclein-repeat 1 microsatellite marker and expression levels of the gene, this marker was genotyped in a Caucasian sample of 126 controls and 117 alcoholics using capillary gel electrophoresis. The allele and genotype frequencies of α-synuclein-repeat 1 marker differed significantly between alcoholics and controls. Alcoholics had greater frequencies of the shortest allele found (267 bp). The shortest allele of the α-synuclein-repeat 1 marker was associated with decreased expression of α-synuclein in prefrontal cortex. Individuals with at least one copy of the 267 bp allele were more likely to exhibit an alcohol abuse phenotype. These results suggest that individuals with the 267 bp allele may be at increased risk of developing alcoholism and that genetic variation at the α-synuclein-repeat 1 locus may influence α-synuclein expression in the prefrontal cortex. PMID:22974310

  9. Identification of differentially expressed genes between sorghum genotypes with contrasting nitrogen stress tolerance by genome-wide transcriptional profiling

    PubMed Central

    2014-01-01

    Background Sorghum is an important cereal crop, which requires large quantities of nitrogen fertilizer for achieving commercial yields. Identification of the genes responsible for low-N tolerance in sorghum will facilitate understanding of the molecular mechanisms of low-N tolerance, and also facilitate the genetic improvement of sorghum through marker-assisted selection or gene transformation. In this study we compared the transcriptomes of root tissues from seven sorghum genotypes having differential response to low-N stress. Results Illumina RNA-sequencing detected several common differentially expressed genes (DEGs) between four low-N tolerant sorghum genotypes (San Chi San, China17, KS78 and high-NUE bulk) and three sensitive genotypes (CK60, BTx623 and low-NUE bulk). In sensitive genotypes, N-stress increased the abundance of DEG transcripts associated with stress responses including oxidative stress and stimuli were abundant. The tolerant genotypes adapt to N deficiency by producing greater root mass for efficient uptake of nutrients. In tolerant genotypes, higher abundance of transcripts related to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5, and NRT2.6) and lysine histidine transporter 1 (LHT1), may suggest an improved uptake efficiency of inorganic and organic forms of nitrogen. Higher abundance of SEC14 cytosolic factor family protein transcript in tolerant genotypes could lead to increased membrane stability and tolerance to N-stress. Conclusions Comparison of transcriptomes between N-stress tolerant and sensitive genotypes revealed several common DEG transcripts. Some of these DEGs were evaluated further by comparing the transcriptomes of genotypes grown under full N. The DEG transcripts showed higher expression in tolerant genotypes could be used for transgenic over-expression in sensitive genotypes of sorghum and related crops for increased tolerance to N-stress, which results in increased nitrogen use efficiency for sustainable

  10. Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

    PubMed Central

    Aleza, Pablo; Froelicher, Yann; Schwarz, Sergio; Agustí, Manuel; Hernández, María; Juárez, José; Luro, François; Morillon, Raphael; Navarro, Luis; Ollitrault, Patrick

    2011-01-01

    Background and Aims Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. Methods Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of ‘Tardivo di Ciaculli’ mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (‘Carrizo’ citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries. Key Results Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area. Conclusions Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of

  11. FKBP5 Genotype-Dependent DNA Methylation and mRNA Regulation After Psychosocial Stress in Remitted Depression and Healthy Controls

    PubMed Central

    Höhne, Nina; Poidinger, Maximilian; Merz, Franziska; Pfister, Hildegard; Brückl, Tanja; Zimmermann, Petra; Uhr, Manfred; Holsboer, Florian

    2015-01-01

    Background: Polymorphisms in the FK506 binding protein 5 (FKBP5) gene have been shown to influence glucocorticoid receptor sensitivity, stress response regulation, and depression risk in traumatized subjects, with most consistent findings reported for the functional variant rs1360780. In the present study, we investigated whether the FKBP5 polymorphism rs1360780 and lifetime history of major depression are associated with DNA methylation and FKBP5 gene expression after psychosocial stress. Methods: A total of 116 individuals with a positive (n = 61) and negative (n = 55) lifetime history of major depression participated in the Trier Social Stress Test. We assessed plasma cortisol concentrations, FKBP5 mRNA expression, and CpG methylation of FKBP5 intron 7 in peripheral blood cells. Results: Genotype-dependent plasma cortisol response to psychosocial stress exposure was observed in healthy controls, with the highest and longest-lasting cortisol increase in subjects with the TT genotype of the FKBP5 polymorphism rs1360780, and healthy controls carrying the T risk allele responded with a blunted FKBP5 mRNA expression after psychosocial stress. No genotype effects could be found in remitted depression. Conclusions: The FKBP5 rs1360780 polymorphism is associated with plasma cortisol and FKBP5 mRNA expression after psychosocial stress in healthy controls but not in remitted depression. Preliminary results of the DNA methylation analysis suggest that epigenetic modifications could be involved. PMID:25522420

  12. Effect of genotype on duodenal expression of nutrient transporter genes in dairy cows

    PubMed Central

    2013-01-01

    Background Studies have shown clear differences between dairy breeds in their feed intake and production efficiencies. The duodenum is critical in the coordination of digestion and absorption of nutrients. This study examined gene transcript abundance of important classes of nutrient transporters in the duodenum of non lactating dairy cows of different feed efficiency potential, namely Holstein-Friesian (HF), Jersey (JE) and their F1 hybrid. Duodenal epithelial tissue was collected at slaughter and stored at -80°C. Total RNA was extracted from tissue and reverse transcribed to generate cDNA. Gene expression of the following transporters, namely nucleoside; amino acid; sugar; mineral; and lipid transporters was measured using quantitative real-time RT-PCR. Data were statistically analysed using mixed models ANOVA in SAS. Orthogonal contrasts were used to test for potential heterotic effects and spearman correlation coefficients calculated to determine potential associations amongst gene expression values and production efficiency variables. Results While there were no direct effects of genotype on expression values for any of the genes examined, there was evidence for a heterotic effect (P < 0.05) on ABCG8, in the form of increased expression in the F1 genotype compared to either of the two parent breeds. Additionally, a tendency for increased expression of the amino acid transporters, SLC3A1 (P = 0.072), SLC3A2 (P = 0.081) and SLC6A14 (P = 0.072) was also evident in the F1 genotype. A negative (P < 0.05) association was identified between the expression of the glucose transporter gene SLC5A1 and total lactational milk solids yield, corrected for body weight. Positive correlations (P < 0.05) were also observed between the expression values of genes involved in common transporter roles. Conclusion This study suggests that differences in the expression of sterol and amino acid transporters in the duodenum could contribute towards the

  13. 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. PMID:18311543

  14. Iron overload in non-transfusion-dependent thalassemia: association with genotype and clinical risk factors.

    PubMed

    Tantiworawit, Adisak; Charoenkwan, Pimlak; Hantrakool, Sasinee; Choeyprasert, Worawut; Sivasomboon, Chate; Sanguansermsri, Torpong

    2016-06-01

    In the present study, we sought to determine the prevalence of iron overload in patients with non-transfusion-dependent thalassemia (NTDT) and its association with genotype and other clinical risk factors, and to evaluate the correlation between serum ferritin (SF) and liver iron concentration (LIC). Myocardial and liver iron concentration was measured by MRI using a T2* gradient multi-echo sequence in NTDT patients, aged 10-50 years. Of 91 patients, 54 (59 %) had hepatic iron overload. None had cardiac iron overload. The clinical risk factors for hepatic iron overload were age >20 years (adjusted OR 30.2, 95 % CI 4.5-203, p < 0.001), hemoglobin level <7 g/dL (adjusted OR 6.3, 95 % CI 1.01-39.5, p = 0.049), and cumulative RBC transfusion >10 units (adjusted OR 53.6, 95 % CI 3.2-884, p = 0.005). Beta-thalassemia genotype was associated with higher risk of iron overload by univariate analysis, but the association was not significant when adjusted for other clinical factors. The correlation coefficient between SF and LIC was 0.60 (p < 0.001). In conclusion, the prevalence of hepatic iron overload is high in NTDT. Older age, lower hemoglobin level, and higher cumulative RBC transfusion are significant risk factors. SF and LIC show a significant positive correlation. PMID:27052211

  15. The Genotype-Tissue Expression (GTEx) pilot analysis: Multitissue gene regulation in humans

    PubMed Central

    2015-01-01

    Understanding the functional consequences of genetic variation, and how it affects complex human disease and quantitative traits, remains a critical challenge for biomedicine. We present an analysis of RNA sequencing data from 1641 samples across 43 tissues from 175 individuals, generated as part of the pilot phase of the Genotype-Tissue Expression (GTEx) project. We describe the landscape of gene expression across tissues, catalog thousands of tissue-specific and shared regulatory expression quantitative trait loci (eQTL) variants, describe complex network relationships, and identify signals from genome-wide association studies explained by eQTLs. These findings provide a systematic understanding of the cellular and biological consequences of human genetic variation and of the heterogeneity of such effects among a diverse set of human tissues. PMID:25954001

  16. The curli biogenesis genes expression level is unassociated with Enterobacter cloacae hsp60 clusters and PFGE genotypes.

    PubMed

    Akbari, Majid; Bakhshi, Bita; Najar-Peerayeh, Shahin; Behmanesh, Mehrdad

    2016-09-01

    The objective of this study was to determine the correlation between Enterobacter cloacae complex subspecies and clusters involved in UTI infections and specific pulsotypes, and to assess the contribution of major curli biogenesis genes (csgD, csgA) expression level to pathogenesis of clusters and genotypes. Based on the PFGE analysis, 37 different profiles were observed among which 8 profiles were common types. Real time PCR of csgD and csgA genes of 50 E. cloacae complex in relation to PFGE and hsp60 genotypes showed that all the genetic clusters are not equally involved in pathogenesis of urinary tract infections. It was elucidated in this study that isolates with common PFGE genotypes belonged to identical hsp60 clusters, and the foremost clusters (VI, III, and V) mainly comprised within PFGE common types. In our study, no significant correlation was detected between the specific hsp60 clusters or PFGE genotypes and the expression level of csgD and csgA genes (P-value > 0.05). This is the first study describing that unequivalent contribution of E. cloacae genotypes and clusters in pathogenesis of UTI, is not owing to varied curli biogenesis expression potential. The PFGE genotyping showed more discriminatory power than hsp60 genotyping for epidemiological studies and source tracking purpose. PMID:27354208

  17. Adenomatous polyposis coli genotype-dependent toll-like receptor 4 activity in colon cancer

    PubMed Central

    Wang, Wei; Li, Meng; Guo, Fuchun; Sang, Yaxiong; Qin, Qing; Wang, Yongsheng; Li, Qiu

    2016-01-01

    Toll-like receptors (TLRs)/NF-κB activation stimulated by lipopolysaccharide (LPS) was associated with diverse biological response in colon cancer, but the underlying mechanism was largely unknown. In the current study, we reported cell proliferation was elevated in adenomatous polyposis coli (APC) mutated- and APC knockdown cell lines, while the proliferation was inhibited in APC wild-type cell lines. Besides, in vivo experiments showed that LPS promoted APC knockdown tumor growth while inhibited proliferation of APC wild type. Further study confirmed that activation of TLRs/NF-κB signaling pathway by LPS cross regulated with APC/GSK-3β/β-catenin pathway, which were depend on APC status of cell lines. Taken together, APC genotypes play a key role in LPS induced different colon cancer biological response by cross-regulating β-catenin and NF-κB, which may provide a novel strategy for carcinogenesis prevention. PMID:26760960

  18. Comparative Transcriptome Analysis of Two Ipomoea aquatica Forsk. Cultivars Targeted To Explore Possible Mechanism of Genotype-Dependent Accumulation of Cadmium.

    PubMed

    Huang, Ying-Ying; Shen, Chuang; Chen, Jing-Xin; He, Chun-Tao; Zhou, Qian; Tan, Xiao; Yuan, Jian-Gang; Yang, Zhong-Yi

    2016-06-29

    A low-shoot-Cd (QLQ) and a high-shoot-Cd cultivar (T308) of water spinach (Ipomoea aquatica Forsk.) were used to investigate molecular mechanism of the genotype difference in cadmium (Cd) accumulation. RNA-Seq under 9 and 72 h cadmium exposures (5 mg L(-1)) were undertaken to explore Cd induced genotype differences in molecular processes. In total, 253 747 540 clean reads were assembled into 57 524 unigenes. Among them, 6136 and 10 064 unigenes were differentially expressed in QLQ and T308, respectively. Cell wall biosynthesis genes, such as GAUT and laccase, and three Cd efflux genes (Nramp5, MATE9, and YSL7) had higher expression levels in QLQ, while the genes in sulfur and glutathione metabolism pathway, e.g., sulfate transporter and cysteine synthase, showed higher expression levels in T308. These findings would be useful for further understanding of the mechanisms related to genotype-dependent Cd accumulation and developing the molecular assisted screening and breeding of low-shoot-Cd cultivars for water spinach. PMID:27267580

  19. Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content.

    PubMed

    Pandurangaiah, Shilpa; Ravishankar, Kundapura V; Shivashankar, Kodthalu S; Sadashiva, Avverahally T; Pillakenchappa, Kavitha; Narayanan, Sunil Kumar

    2016-06-01

    Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene beta-cyclases can be used in marker-assisted breeding. PMID:27240986

  20. Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different Verticillium Host Plants

    PubMed Central

    Berg, Gabriele; Roskot, Nicolle; Steidle, Anette; Eberl, Leo; Zock, Angela; Smalla, Kornelia

    2002-01-01

    To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards Verticillium. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere. PMID:12089011

  1. Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants.

    PubMed

    Berg, Gabriele; Roskot, Nicolle; Steidle, Anette; Eberl, Leo; Zock, Angela; Smalla, Kornelia

    2002-07-01

    To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere. PMID:12089011

  2. Estrogen, SNP-Dependent Chemokine Expression and Selective Estrogen Receptor Modulator Regulation.

    PubMed

    Ho, Ming-Fen; Bongartz, Tim; Liu, Mohan; Kalari, Krishna R; Goss, Paul E; Shepherd, Lois E; Goetz, Matthew P; Kubo, Michiaki; Ingle, James N; Wang, Liewei; Weinshilboum, Richard M

    2016-03-01

    We previously reported, on the basis of a genome-wide association study for aromatase inhibitor-induced musculoskeletal symptoms, that single-nucleotide polymorphisms (SNPs) near the T-cell leukemia/lymphoma 1A (TCL1A) gene were associated with aromatase inhibitor-induced musculoskeletal pain and with estradiol (E2)-induced TCL1A expression. Furthermore, variation in TCL1A expression influenced the downstream expression of proinflammatory cytokines and cytokine receptors. Specifically, the top hit genome-wide association study SNP, rs11849538, created a functional estrogen response element (ERE) that displayed estrogen receptor (ER) binding and increased E2 induction of TCL1A expression only for the variant SNP genotype. In the present study, we pursued mechanisms underlying the E2-SNP-dependent regulation of TCL1A expression and, in parallel, our subsequent observations that SNPs at a distance from EREs can regulate ERα binding and that ER antagonists can reverse phenotypes associated with those SNPs. Specifically, we performed a series of functional genomic studies using a large panel of lymphoblastoid cell lines with dense genomic data that demonstrated that TCL1A SNPs at a distance from EREs can modulate ERα binding and expression of TCL1A as well as the expression of downstream immune mediators. Furthermore, 4-hydroxytamoxifen or fulvestrant could reverse these SNP-genotype effects. Similar results were found for SNPs in the IL17A cytokine and CCR6 chemokine receptor genes. These observations greatly expand our previous results and support the existence of a novel molecular mechanism that contributes to the complex interplay between estrogens and immune systems. They also raise the possibility of the pharmacological manipulation of the expression of proinflammatory cytokines and chemokines in a SNP genotype-dependent fashion. PMID:26866883

  3. Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

    PubMed

    Hogarth, Marshall W; Garton, Fleur C; Houweling, Peter J; Tukiainen, Taru; Lek, Monkol; Macarthur, Daniel G; Seto, Jane T; Quinlan, Kate G R; Yang, Nan; Head, Stewart I; North, Kathryn N

    2016-03-01

    A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations. PMID:26681802

  4. T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora.

    PubMed

    Dugé De Bernonville, Thomas; Gaucher, Matthieu; Flors, Victor; Gaillard, Sylvain; Paulin, Jean-Pierre; Dat, James F; Brisset, Marie-Noëlle

    2012-06-01

    Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea. PMID:22525238

  5. Bacterial histo-blood group antigens contributing to genotype-dependent removal of human noroviruses with a microfiltration membrane.

    PubMed

    Amarasiri, Mohan; Hashiba, Satoshi; Miura, Takayuki; Nakagomi, Toyoko; Nakagomi, Osamu; Ishii, Satoshi; Okabe, Satoshi; Sano, Daisuke

    2016-05-15

    We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:B7, and Staphylococcus epidermidis), respectively, and the mixture was filtered with an MF membrane having a nominal pore size of 0.45 μm. All NoVLP genotypes were rejected by the MF membrane in the presence of Enterobacter sp. SENG-6, which excreted HBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:B7, but the removal of EPS of E. coli O86:K61:B7 increased the removal efficiency due to the interaction of NoVLPs with the exposed B-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:B7. No MF membrane removal of all three genotypes was observed when S. epidermidis, an HBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane. PMID:27095709

  6. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

    PubMed

    Witteveldt, Jeroen; Martin-Gans, Marion; Simmonds, Peter

    2016-05-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  7. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing

    PubMed Central

    Witteveldt, Jeroen; Martin-Gans, Marion

    2016-01-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  8. Comparative Transcriptional Profiling of Two Wheat Genotypes, with Contrasting Levels of Minerals in Grains, Shows Expression Differences during Grain Filling

    PubMed Central

    Singh, Sudhir P.; Jeet, Raja; Kumar, Jitendra; Shukla, Vishnu; Srivastava, Rakesh; Mantri, Shrikant S.; Tuli, Rakesh

    2014-01-01

    Wheat is one of the most important cereal crops in the world. To identify the candidate genes for mineral accumulation, it is important to examine differential transcriptome between wheat genotypes, with contrasting levels of minerals in grains. A transcriptional comparison of developing grains was carried out between two wheat genotypes- Triticum aestivum Cv. WL711 (low grain mineral), and T. aestivum L. IITR26 (high grain mineral), using Affymetrix GeneChip Wheat Genome Array. The study identified a total of 580 probe sets as differentially expressed (with log2 fold change of ≥2 at p≤0.01) between the two genotypes, during grain filling. Transcripts with significant differences in induction or repression between the two genotypes included genes related to metal homeostasis, metal tolerance, lignin and flavonoid biosynthesis, amino acid and protein transport, vacuolar-sorting receptor, aquaporins, and stress responses. Meta-analysis revealed spatial and temporal signatures of a majority of the differentially regulated transcripts. PMID:25364903

  9. [Genotype-dependent mice behavior in cognitive tasks. Effect of noopept].

    PubMed

    Bel'nik, A P; Ostrovskaia, R U; Poletaeva, I I

    2007-01-01

    The interstrain differences in performance of C57BL/6J, BALB/c and DBA/2J male mice in two cognitive tasks were found. Mice C57BL/6J showed good learning ability and preservation of memory traces tested 10 days after performance in a simplified version of Morris water maze. Mice BALB/c learned the task but, virtually, no long-term memory traces were revealed, whereas DBA/2J demonstrated poor learning. The effect of nootropic drug Noopept (GVS-111, N-phenil-acetyl-L-prolylglycin ethyl ether) was shown to be genotype-dependent. Its administration (0.5 mg/kg i.p., 15 min before learning) improved the long-term memory in Morris test in BALB/c mice but failed to produce any improvement in C57BL/6J. The ability of mice for extrapolation of the direction of stimulus movement differently changed after Noopept injections: the proportion of correct task solutions increased in C57BL/6J and BALB/c mice, whereas the performance of DBA/2J did not change. PMID:18592707

  10. Identification of Salmonella enterica Serovar Typhi Genotypes by Use of Rapid Multiplex Ligation-Dependent Probe Amplification

    PubMed Central

    Pham Thanh, Duy; Tran Vu Thieu, Nga; Tran Thuy, Chau; Lodén, Martin; Tuin, Kiki; Campbell, James I.; Van Minh Hoang, Nguyen; Voong Vinh, Phat; Farrar, Jeremy J.; Holt, Kathryn E.; Dougan, Gordon

    2013-01-01

    Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic. PMID:23824765

  11. Expression of zinc and cadmium responsive genes in leaves of willow (Salix caprea L.) genotypes with different accumulation characteristics

    PubMed Central

    Konlechner, Cornelia; Türktaş, Mine; Langer, Ingrid; Vaculík, Marek; Wenzel, Walter W.; Puschenreiter, Markus; Hauser, Marie-Theres

    2013-01-01

    Salix caprea is well suited for phytoextraction strategies. In a previous survey we showed that genetically distinct S. caprea plants isolated from metal-polluted and unpolluted sites differed in their zinc (Zn) and cadmium (Cd) tolerance and accumulation abilities. To determine the molecular basis of this difference we examined putative homologues of genes involved in heavy metal responses and identified over 200 new candidates with a suppression subtractive hybridization (SSH) screen. Quantitative expression analyses of 20 genes in leaves revealed that some metallothioneins and cell wall modifying genes were induced irrespective of the genotype's origin and metal uptake capacity while a cysteine biosynthesis gene was expressed constitutively higher in the metallicolous genotype. The third and largest group of genes was only induced in the metallicolous genotype. These data demonstrate that naturally adapted woody non-model species can help to discover potential novel molecular mechanisms for metal accumulation and tolerance. PMID:23562959

  12. Influence of genotype-dependent effects of covariates on the outcome of segregation analysis of the body mass index.

    PubMed Central

    Borecki, I B; Bonney, G E; Rice, T; Bouchard, C; Rao, D C

    1993-01-01

    Several recent studies of the body mass index (BMI) have provided support for a recessive major gene influencing heaviness in humans. Segregation analysis of the BMI was carried out recently in a series of randomly sampled French-Canadian families to determine whether we could replicate the major gene finding by using a residual phenotype adjusted for the effects of age and sex. The best model included a recessive major effect for high BMI values with residual familial resemblance; however, Mendelian transmission could not be confirmed, and the no-transmission hypothesis (where all the tau's are constrained to be equal) was not rejected. Considering that the BMI is a complex phenotype affected by many factors and that there are known variations in body composition during growth and aging, we undertook a reanalysis of the data, using a model that allowed the estimation of genotype-specific age and gender effects. New tests on the transmission parameters satisfy the criteria for interfering Mendelian segregation. The results suggest that individuals with the "high" recessive genotype show the greatest degree of heaviness at birth, with a subsequent trend toward lower values throughout life, while individuals with the dominant "normal" genotypes show no appreciable trends with age. In addition, the "high" genotype appears to confer a greater degree of heaviness in females as compared with males. These results, along with other observations from the data, suggest that, while a recessive single gene influence may be discernible, the phenotypic expression of the BMI is likely to be complicated by genotype x environment interactions and, possibly, by the action of other loci. Further, the data also are consistent with the hypothesis that modifying factors may include the adoption of a more prudent life-style by individuals genetically predisposed to heaviness and a secular increase in the incidence, prevalence, and potency of environmentally based triggers leading to a

  13. Genotype-by-environment interactions underlie the expression of pre- and post-copulatory sexually selected traits in guppies.

    PubMed

    Evans, J P; Rahman, M M; Gasparini, C

    2015-04-01

    The role that genotype-by-environment interactions (GEIs) play in sexual selection has only recently attracted the attention of evolutionary biologists. Yet GEIs can have profound evolutionary implications by compromising the honesty of sexual signals, maintaining high levels of genetic variance underlying their expression and altering the patterns of genetic covariance among fitness traits. In this study, we test for GEIs in a highly sexually dimorphic freshwater fish, the guppy Poecilia reticulata. We conducted an experimental quantitative genetic study in which male offspring arising from a paternal half-sibling breeding design were assigned to differing nutritional 'environments' (either high or low feed levels). We then determined whether the manipulation of diet quantity influenced levels of additive genetic variance and covariance for several highly variable and condition-dependent pre- and post-copulatory sexual traits. In accordance with previous work, we found that dietary limitation had strong phenotypic effects on numerous pre- and post-copulatory sexual traits. We also report evidence for significant GEI for several of these traits, which in some cases (area of iridescence and sperm velocity) reflected a change in the rank order of genotypes across different nutritional environments (i.e. ecological crossover). Furthermore, we show that genetic correlations vary significantly between nutritional environments. Notably, a highly significant negative genetic correlation between iridescent coloration and sperm viability in the high food treatment broke down under dietary restriction. Taken together, these findings are likely to have important evolutionary implications for guppies; ecological crossover may influence sexual signal reliability in unstable (nutritional) environments and contribute towards the extreme levels of polymorphism in sexual traits typically reported for this species. Furthermore, the presence of environment-specific genetic covariance

  14. Genotype-dependent tumor regression in Marek's disease mediated at the level of tumor immunity.

    PubMed

    Kumar, Shyamesh; Buza, Joram J; Burgess, Shane C

    2009-12-01

    Marek's disease (MD) of chickens is a unique natural model of Hodgkin's and Non Hodgkin's lymphomas in which the neoplastically-transformed cells over-express CD30 (CD30(hi)) antigen. All chicken genotypes can be infected with MD virus and develop microscopic lymphomas. From 21 days post infection (dpi) microscopic lymphomas regress in resistant chickens but, in contrast, they progress to gross lymphomas in susceptible chickens. Here we test our hypothesis that in resistant chickens at 21 dpi the tissue microenvironment is pro T-helper (Th)-1 and compatible with cytotoxic T lymphocyte (CTL) immunity but in susceptible lines it is pro Th-2 or pro T-regulatory (T-reg) and antagonistic to CTL immunity. We used the B2, non-MHC-associated, MD resistance/susceptibility system (line [L]6(1)/line [L]7(2)) and quantified the levels of key mRNAs that can be used to define Th-1 (IL-2, IL-12, IL-18, IFNgamma), Th-2 (IL-4, IL-10) and T-reg (TGFbeta, GPR-83, CTLA-4, SMAD-7) lymphocyte phenotypes. We measured gene expression in both whole tissues (represents tissue microenvironment and tumor microenvironment) and in the lymphoma lesions (tumor microenvironment) themselves. Gene ontology-based modeling of our results shows that the dominant phenotype in whole tissue as well as in microscopic lymphoma lesions, is pro T-reg in both L6(1) and L7(2) but a minor pro Th-1 and anti Th-2 tissue microenvironment exists in L6(1) whereas there is an anti Th-1 and pro Th-2 tissue microenvironment in L7(2). The tumor microenvironment per se is pro T-reg, anti Th-1 and pro Th-2 in both L6(1) and L7(2). Together our data suggests that the neoplastic transformation is essentially the same in both L6(1) and L7(2) and that resistance/susceptibility is mediated at the level of tumor immunity in the tissues. PMID:19308678

  15. Towards Systems Genetic Analyses in Barley: Integration of Phenotypic, Expression and Genotype Data into GeneNetwork

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A typical genetical genomics experiment results in three separate data sets: genotype, gene expression, and higher-order phenotypic data. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. The predictive power of these experiments is largely d...

  16. Stably Expressed D Genome-derived HMW Glutenin Subunit Genes Transformed Into Different Durum Wheat Genotypes Change Dough Mixing Properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glutenin subunits 1Dx5 and 1Dy10 are encoded by chromosome 1D and associated with higher dough strength in hexaploid bread wheats. In order to study the effects of their expression in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area were co-transformed, vi...

  17. Nitrate Starvation Induced Changes in Root System Architecture, Carbon:Nitrogen Metabolism, and miRNA Expression in Nitrogen-Responsive Wheat Genotypes.

    PubMed

    Sinha, Subodh Kumar; Rani, Manju; Bansal, Niketa; Gayatri; Venkatesh, K; Mandal, P K

    2015-11-01

    Improvement of nutrient use efficiency in cereal crops is highly essential not only to reduce the cost of cultivation but also to save the environmental pollution, reduce energy consumption for production of these chemical fertilizers, improve soil health, and ultimately help in mitigating climate change. In the present investigation, we have studied the morphological (with special emphasis on root system architecture) and biochemical responses (in terms of assay of the key enzymes involved in N assimilation) of two N-responsive wheat genotypes, at the seedling stage, under nitrate-optimum and nitrate-starved conditions grown in hydroponics. Expression profile of a few known wheat micro RNAs (miRNAs) was also studied in the root tissue. Total root size, primary root length, and first- and second-order lateral root numbers responded significantly under nitrate-starved condition. Morphological parameters in terms of root and shoot length and fresh and dry weight of roots and shoots have also been observed to be significant between N-optimum and N-starved condition for each genotypes. Nitrate reductase (NR), glutamine synthatase (GS), and glutamate dehydrogenase (GDH) activity significantly decreased under N-starved condition. Glutamine oxoglutarate amino transferase (GOGAT) and pyruvate kinase (PK) activity was found to be genotype dependent. Most of the selected miRNAs were expressed in root tissues, and some of them showed their differential N-responsive expression. Our studies indicate that one of the N-responsive genotype (NP-890) did not get affected significantly under nitrogen starvation at seedling stage. PMID:26315134

  18. Molecular Phylogeny of the Psittacid Herpesviruses Causing Pacheco's Disease: Correlation of Genotype with Phenotypic Expression

    PubMed Central

    Tomaszewski, Elizabeth K.; Kaleta, Erhard F.; Phalen, David N.

    2003-01-01

    Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species. PMID:14512573

  19. Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression.

    PubMed

    Tomaszewski, Elizabeth K; Kaleta, Erhard F; Phalen, David N

    2003-10-01

    Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species. PMID:14512573

  20. Genotype and Tumor Locus Determine Expression Profile of Pseudohypoxic Pheochromocytomas and Paragangliomas12

    PubMed Central

    Shankavaram, Uma; Fliedner, Stephanie M J; Elkahloun, Abdel G; Barb, Jenifer J; Munson, Peter J; Huynh, Thanh T; Matro, Joey C; Turkova, Hana; Linehan, W Marston; Timmers, Henri J; Tischler, Arthur S; Powers, James F; de Krijger, Ronald; Baysal, Bora E; Takacova, Martina; Pastorekova, Silvia; Gius, David; Lehnert, Hendrik; Camphausen, Kevin; Pacak, Karel

    2013-01-01

    Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies. PMID:23555188

  1. Genotype and tumor locus determine expression profile of pseudohypoxic pheochromocytomas and paragangliomas.

    PubMed

    Shankavaram, Uma; Fliedner, Stephanie M J; Elkahloun, Abdel G; Barb, Jenifer J; Munson, Peter J; Huynh, Thanh T; Matro, Joey C; Turkova, Hana; Linehan, W Marston; Timmers, Henri J; Tischler, Arthur S; Powers, James F; de Krijger, Ronald; Baysal, Bora E; Takacova, Martina; Pastorekova, Silvia; Gius, David; Lehnert, Hendrik; Camphausen, Kevin; Pacak, Karel

    2013-04-01

    Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies. PMID:23555188

  2. Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

    PubMed

    Asselman, Jana; De Coninck, Dieter I M; Vandegehuchte, Michiel B; Jansen, Mieke; Decaestecker, Ellen; De Meester, Luc; Vanden Bussche, Julie; Vanhaecke, Lynn; Janssen, Colin R; De Schamphelaere, Karel A C

    2015-05-01

    The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype × environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia. PMID:25639773

  3. Sex-specific genotype-by-environment interactions for cuticular hydrocarbon expression in decorated crickets, Gryllodes sigillatus: implications for the evolution of signal reliability.

    PubMed

    Weddle, C B; Mitchell, C; Bay, S K; Sakaluk, S K; Hunt, J

    2012-10-01

    Phenotypic traits that convey information about individual identity or quality are important in animal social interactions, and the degree to which such traits are influenced by environmental variation can have profound effects on the reliability of these cues. Using inbred genetic lines of the decorated cricket, Gryllodes sigillatus, we manipulated diet quality to test how the cuticular hydrocarbon (CHC) profiles of males and females respond across two different nutritional rearing environments. There were significant differences between lines in the CHC profiles of females, but the effect of diet was not quite statistically significant. There was no significant genotype-by-environment interaction (GEI), suggesting that environmental effects on phenotypic variation in female CHCs are independent of genotype. There was, however, a significant effect of GEI for males, with changes in both signal quantity and content, suggesting that environmental effects on phenotypic expression of male CHCs are dependent on genotype. The differential response of male and female CHC expression to variation in the nutritional environment suggests that these chemical cues may be under sex-specific selection for signal reliability. Female CHCs show the characteristics of reliable cues of identity: high genetic variability, low condition dependence and a high degree of genetic determination. This supports earlier work showing that female CHCs are used in self-recognition to identify previous mates and facilitate polyandry. In contrast, male CHCs show the characteristics of reliable cues of quality: condition dependence and a relatively higher degree of environmental determination. This suggests that male CHCs are likely to function as cues of underlying quality during mate choice and/or male dominance interactions. PMID:22900500

  4. Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion

    PubMed Central

    Carlson, Paula; Acosta, Andres; Busciglio, Irene

    2015-01-01

    The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT2 PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls (P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 (P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion. PMID:25930081

  5. Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion.

    PubMed

    Camilleri, Michael; Carlson, Paula; Acosta, Andres; Busciglio, Irene

    2015-07-01

    The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT(2) PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥ 2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls (P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 (P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion. PMID:25930081

  6. The Blood Group A Genotype Determines the Level of Expression of the Blood Group A on Platelets But Not the Anti-B Isotiter

    PubMed Central

    Lehner, Barbara; Eichelberger, Beate; Jungbauer, Christof; Panzer, Simon

    2015-01-01

    Summary Background The extent of expression of the blood group A on platelets is controversial. Further, the relation between platelets' blood group A expression and the titers of isoagglutinins has not been thoroughly investigated, so far. Methods We evaluated the relation between the genotype with platelets' blood group A and H expression estimated by flow cytometry and the titers of isoagglutinins. Results The A expression varied between genotypes and within genotypes. However, the expression in A1 was stronger than in all other genotypes (p < 0.0001). An overlap of expression levels was apparent between homozygous A1A1 and heterozygous A1 individuals. Still, The A1A1 genotype is associated with a particularly high antigen expression (p = 0.009). Platelets' A expression in A2 versus blood group O donors was also significant (p = 0.007), but there was again an overlap of expression. The secretor status had only little influence on the expression (p = 0.18). Also, isoagglutinin titers were not associated with genotypes. Conclusion: To distinguish between A1 and A2 donors may reduce incompatible platelet transfusions and therefore be favorable on platelet transfusion increment. Clinical data are needed to support this notion. PMID:26733767

  7. Osteoradionecrosis in Head-and-Neck Cancer Has a Distinct Genotype-Dependent Cause

    SciTech Connect

    Lyons, Andrew J.; West, Catharine M.; Risk, Janet M.; Slevin, Nick J.; Chan, Clara; Crichton, Siobhan; Rinck, Gabrielle; Howell, Dawn; Shaw, Richard J.

    2012-03-15

    Purpose: We performed a case-control study to establish whether the development of osteoradionecrosis (ORN) was related to a variant allele substituting T for C at -509 of the transforming growth factor-{beta}1 gene (TGF-{beta}1). Patients and Methods: A total of 140 patients, 39 with and 101 without ORN, who underwent radiotherapy for head-and-neck cancer with a minimum of 2 years follow-up, were studied. None of the patients had clinical evidence of recurrence at this time. DNA extracted from blood was genotyped for the -509 C-T variant allele of the TGF-{beta}1 gene. Results: There were no significant differences in patient, cancer treatment, or tumor characteristics between the two groups. Of the 39 patients who developed ORN, 9 were homozygous for the common CC allele, 19 were heterozygous, and 11 were homozygous for the rare TT genotype. Of the 101 patients without ORN, the distribution was 56 (CC), 33 (CT), and 12 (TT). The difference in distribution was significant, giving an increased risk of ORN of 5.7 (95% CI, 1.7-19.2) for homozygote TT patients (p = 0.001) and 3.6 (95% CI, 1.3-10.0) for heterozygotes (p = 0.004) when compared with patients with the CC genotype. Postradiotherapy dentoalveolar surgery preceding the development of ORN was associated with the CC genotype (p = 0.02). Conclusions: Our findings support the postulate that the development of ORN is related to the presence of the T variant allele at -509 within the TGF-{beta}1 gene.

  8. Effects of Lactobacillus helveticus on murine behavior are dependent on diet and genotype and correlate with alterations in the gut microbiome.

    PubMed

    Ohland, Christina L; Kish, Lisa; Bell, Haley; Thiesen, Aducio; Hotte, Naomi; Pankiv, Evelina; Madsen, Karen L

    2013-09-01

    Modulation of the gut microbiota with diet and probiotic bacteria can restore intestinal homeostasis in inflammatory conditions and alter behavior via the gut-brain axis. The purpose of this study was to determine whether the modulatory effects of probiotics differ depending on diet and mouse genotype. At weaning, wild type (WT) and IL-10 deficient (IL-10(-/-)) 129/SvEv mice were placed on a standard mouse chow or a Western-style diet (fat 33%, refined carbohydrate 49%)±Lactobacillus helveticus ROO52 (10(9)cfu/d) for 21 days. Animal weight and food eaten were monitored weekly. Intestinal immune function was analysed for cytokine expression using the Meso Scale Discovery platform. Spatial memory and anxiety-like behavior was assessed in a Barnes maze. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze the fecal microbiota. Both WT and IL-10(-/-) mice on a Western diet had increased weight gain along with changes in gut microbiota and cytokine expression and altered anxiety-like behavior. The ability of L. helveticus to modulate these factors was genotype- and diet-dependent. Anxiety-like behavior and memory were negatively affected by Western-style diet depending on inflammatory state, but this change was prevented with L. helveticus administration. However, probiotics alone decreased anxiety-like behavior in WT mice on a chow diet. Mice on the Western diet had decreased inflammation and fecal corticosterone, but these markers did not correlate with changes in behavior. Analysis of bacterial phyla from WT and IL-10(-/-)mice showed discrete clustering of the groups to be associated with both diet and probiotic supplementation, with the diet-induced shift normalized to some degree by L. helveticus. These findings suggest that the type of diet consumed by the host and the presence or absence of active inflammation may significantly alter the ability of probiotics to modulate host physiological function. PMID:23566632

  9. Dependence of deodorant usage on ABCC11 genotype: scope for personalized genetics in personal hygiene.

    PubMed

    Rodriguez, Santiago; Steer, Colin D; Farrow, Alexandra; Golding, Jean; Day, Ian N M

    2013-07-01

    Earwax type and axillary odor are genetically determined by rs17822931, a single-nucleotide polymorphism (SNP) located in the ABCC11 gene. The literature has been concerned with the Mendelian trait of earwax, although axillary odor is also Mendelian. Ethnic diversity in rs17822931 exists, with higher frequency of allele A in east Asians. Influence on deodorant usage has not been investigated. In this work, we present a detailed analysis of the rs17822931 effect on deodorant usage in a large (N∼17,000 individuals) population cohort (the Avon Longitudinal Study of Parents and Children (ALSPAC)). We found strong evidence (P=3.7 × 10(-20)) indicating differential deodorant usage according to the rs17822931 genotype. AA homozygotes were almost 5-fold overrepresented in categories of never using deodorant or using it infrequently. However, 77.8% of white European genotypically nonodorous individuals still used deodorant, and 4.7% genotypically odorous individuals did not. We provide evidence of a behavioral effect associated with rs17822931. This effect has a biological basis that can result in a change in the family's environment if an aerosol deodorant is used. It also indicates potential cost saving to the nonodorous and scope for personalized genetics usage in personal hygiene choices, with consequent reduction of inappropriate chemical exposures for some. PMID:23325016

  10. Differential Expression of Matrix Metalloproteinase-9 Gene in Wounds of Type 2 Diabetes Mellitus Cases With Susceptible -1562C>T Genotypes and Wound Severity.

    PubMed

    Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Mohan, Gyanendra; Chaturvedi, Sunanda; Singh, Kiran

    2014-05-25

    Coordinated extracellular matrix deposition is a prerequisite for proper wound healing which is mainly orchestrated by matrix metalloproteinases (MMPs). Diabetic wounds generally show compromised wound healing cascade and abnormal MMP9 concentration is one of the cause. Our group have recently shown that the polymorphism -1562 C>T in the promoter region of MMP9 gene is associated with pathogenesis of wound healing impairment in T2DM patients. In present study we have done expression profiling of MMP9 gene in the wound biopsy of DFU cases. Expression level of MMP9 mRNA was then compared with susceptible -1562 C>T genotypes (TT and CT) as well as with different grades of wounds. We also screened the promoter region of MMP9 gene to see the methylation state of CpGs present there. Our study suggests that levels of MMP9 mRNA increase significantly with the wound grades. Moreover, the MMP9 levels in diabetic wounds were also dependent on -1562 C>T polymorphism in the promoter region of MMP9. Diabetic wounds also showed a significant unmethylated status of MMP9 promoter compared to control wounds. In conclusion, The risk genotypes of -1562 C>T polymorphism along with lack of methylation of CpG sites in MMP9 gene promoter may result in altered expression of MMP9 in wounds of T2DM cases resulting into nonhealing chronic ulcers in them. PMID:24861096

  11. Dose-dependent effect of the CYP2D6 genotype on the steady-state fluvoxamine concentration.

    PubMed

    Watanabe, Junzo; Suzuki, Yutaro; Fukui, Naoki; Sugai, Takuro; Ono, Shin; Inoue, Yoshimasa; Someya, Toshiyuki

    2008-12-01

    Several studies have reported that the cytochrome P450 (CYP) 2D6 plays an important role in the fluvoxamine metabolism. However, some other studies have reported that the CYP2D6 genotype has no major impact on the fluvoxamine concentration. This study investigated the dose-dependent effect of CYP2D6-variant alleles on the steady-state fluvoxamine concentration. There were 23 patients whose plasma concentrations of fluvoxamine were measured at 4 doses (50, 100, 150, and 200 mg/d). The differences in the plasma fluvoxamine concentration were analyzed between 2 genotype groups divided by the number of CYP2D6-variant alleles (with 0 and 1 or 2 variant alleles). The results demonstrated the nonlinear kinetics of fluvoxamine metabolism, and the degree of nonlinear kinetics decreased as the dose was increased. Significant differences in fluvoxamine concentration were observed between the subjects with 0 variant alleles and the subjects with 1 or 2 variant alleles (P = 0.044) when they were treated by 50 mg of fluvoxamine. There were no significant differences in the plasma concentration of fluvoxamine at 100, 150, and 200 mg/d. The present study suggests that the effect of the CYP2D6 genotype on fluvoxamine metabolism is greater at lower doses of fluvoxamine. PMID:18978520

  12. Nitrogen-Deficiency Stress Induces Protein Expression Differentially in Low-N Tolerant and Low-N Sensitive Maize Genotypes

    PubMed Central

    Nazir, Muslima; Pandey, Renu; Siddiqi, Tariq O.; Ibrahim, Mohamed M.; Qureshi, Mohammad I.; Abraham, Gerard; Vengavasi, Krishnapriya; Ahmad, Altaf

    2016-01-01

    Nitrogen (N) is essential for proper plant growth and its application has proven to be critical for agricultural produce. However, for unavoidable economic and environmental problems associated with excessive use of N-fertilizers, it is an urgent demand to manage application of fertilizers. Improving the N-use efficiency (NUE) of crop plants to sustain productivity even at low N levels is the possible solution. In the present investigation, contrasting low-N sensitive (HM-4) and low-N tolerant (PEHM-2) genotypes were identified and used for comparative proteome-profiling of leaves under optimum and low N as well as restoration of low N on 3rd (NR3) and 5th (NR5) days after re-supplying N. The analysis of differential expression pattern of proteins was performed by 2-D gel electrophoresis. Significant variations in the expression of proteins were observed under low N, which were genotype specific. In the leaf proteome, 25 spots were influenced by N treatment and four spots were different between the two genotypes. Most of the proteins that were differentially accumulated in response to N level and were involved in photosynthesis and metabolism, affirming the relationship between N and carbon metabolism. In addition to this, greater intensity of some defense proteins in the low N tolerant genotype was found that may have a possible role in imparting it tolerance under N starvation conditions. The new insights generated on maize proteome in response to N-starvation and restoration would be useful toward improvement of NUE in maize. PMID:27047497

  13. Nitrogen-Deficiency Stress Induces Protein Expression Differentially in Low-N Tolerant and Low-N Sensitive Maize Genotypes.

    PubMed

    Nazir, Muslima; Pandey, Renu; Siddiqi, Tariq O; Ibrahim, Mohamed M; Qureshi, Mohammad I; Abraham, Gerard; Vengavasi, Krishnapriya; Ahmad, Altaf

    2016-01-01

    Nitrogen (N) is essential for proper plant growth and its application has proven to be critical for agricultural produce. However, for unavoidable economic and environmental problems associated with excessive use of N-fertilizers, it is an urgent demand to manage application of fertilizers. Improving the N-use efficiency (NUE) of crop plants to sustain productivity even at low N levels is the possible solution. In the present investigation, contrasting low-N sensitive (HM-4) and low-N tolerant (PEHM-2) genotypes were identified and used for comparative proteome-profiling of leaves under optimum and low N as well as restoration of low N on 3rd (NR3) and 5th (NR5) days after re-supplying N. The analysis of differential expression pattern of proteins was performed by 2-D gel electrophoresis. Significant variations in the expression of proteins were observed under low N, which were genotype specific. In the leaf proteome, 25 spots were influenced by N treatment and four spots were different between the two genotypes. Most of the proteins that were differentially accumulated in response to N level and were involved in photosynthesis and metabolism, affirming the relationship between N and carbon metabolism. In addition to this, greater intensity of some defense proteins in the low N tolerant genotype was found that may have a possible role in imparting it tolerance under N starvation conditions. The new insights generated on maize proteome in response to N-starvation and restoration would be useful toward improvement of NUE in maize. PMID:27047497

  14. Galanin-expression and galanin-dependent sensory neurons are not required for itch

    PubMed Central

    2012-01-01

    Background Galanin is a key modulator of nociception, and it is also required for the developmental survival of a subset of C-fibre sensory neurons which are critical to the mediation of neuropathic and inflammatory pain. However, the potential modulatory roles played by galanin, or the galanin-dependent neurons, in pruritoceptive mechanisms underlying the sensation of itch have not been investigated. Findings Here we report that mice carrying a loss-of-function mutation in the galanin gene (Gal-KO) show no differences in spontaneous behavioural itch responses compared to wild-type (WT) controls. Similarly, the responses to a range of pruritogens are not significantly different between the two genotypes. Conclusions These results suggest that neither galanin expression, nor the galanin-dependent subpopulation of sensory neurons is required for itch-related behaviours. PMID:23216829

  15. The Change of Expression Configuration Affects Identity-Dependent Expression Aftereffect but Not Identity-Independent Expression Aftereffect

    PubMed Central

    Song, Miao; Shinomori, Keizo; Qian, Qian; Yin, Jun; Zeng, Weiming

    2015-01-01

    The present study examined the influence of expression configuration on cross-identity expression aftereffect. The expression configuration refers to the spatial arrangement of facial features in a face for conveying an emotion, e.g., an open-mouth smile vs. a closed-mouth smile. In the first of two experiments, the expression aftereffect is measured using a cross-identity/cross-expression configuration factorial design. The facial identities of test faces were the same or different from the adaptor, while orthogonally, the expression configurations of those facial identities were also the same or different. The results show that the change of expression configuration impaired the expression aftereffect when the facial identities of adaptor and tests were the same; however, the impairment effect disappears when facial identities were different, indicating the identity-independent expression representation is more robust to the change of the expression configuration in comparison with the identity-dependent expression representation. In the second experiment, we used schematic line faces as adaptors and real faces as tests to minimize the similarity between the adaptor and tests, which is expected to exclude the contribution from the identity-dependent expression representation to expression aftereffect. The second experiment yields a similar result as the identity-independent expression aftereffect observed in Experiment 1. The findings indicate the different neural sensitivities to expression configuration for identity-dependent and identity-independent expression systems. PMID:26733922

  16. Global Gene Expression Profiles of Resistant and Susceptible Genotypes of Glycine tomentella During Phakopsora pachyrhizi Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean rust, caused by Phakopsora pachyrhizi, is a destructive foliar disease that occurs in many soybean-producing countries. Towards the goal of identifying genes controlling resistance to soybean rust, transcriptome profiling was conducted in resistant and susceptible Glycine tomentella genotype...

  17. Transcriptomics of salinity tolerance capacity in Arctic charr (Salvelinus alpinus): a comparison of gene expression profiles between divergent QTL genotypes

    PubMed Central

    Ferguson, Moira M.; Danzmann, Roy G.

    2013-01-01

    Osmoregulatory capabilities have played an important role in the evolution, dispersal, and diversification of vertebrates. To better understand the genetic architecture of hypo-osmoregulation in fishes and to determine which genes and biological processes affect intraspecific variation in salinity tolerance, we used mRNA sequence libraries from Arctic charr gill tissue to compare gene expression profiles in fish exhibiting divergent salinity tolerance quantitative trait locus (QTL) genotypes. We compared differentially expressed genes with QTL positions to gain insight about the nature of the underlying polymorphisms and examined gene expression within the context of genome organization to gain insight about the evolution of hypo-osmoregulation in fishes. mRNA sequencing of 18 gill tissue libraries produced 417 million reads, and the final reduced de novo transcriptome assembly consisted of 92,543 contigs. Families contained a similar number of differentially expressed contigs between high and low salinity tolerance capacity groups, and log2 expression ratios ranged from 10.4 to −8.6. We found that intraspecific variation in salinity tolerance capacity correlated with differential expression of immune response genes. Some differentially expressed genes formed clusters along linkage groups. Most clusters comprised gene pairs, though clusters of three, four, and eight genes were also observed. We postulated that conserved synteny of gene clusters on multiple ancestral and teleost chromosomes may have been preserved via purifying selection. Colocalization of QTL with differentially expressed genes suggests that polymorphisms in cis-regulatory elements are part of a majority of QTL. PMID:24368751

  18. APOE genotype-dependent modulation of astrocyte chemokine CCL3 production

    PubMed Central

    Cudaback, Eiron; Yang, Yue; Montine, Thomas J.; Keene, C. Dirk

    2014-01-01

    Apolipoprotein E (apoE) is well known as a regulator of cholesterol homeostasis, and is increasingly recognized to play a prominent role in the modulation of innate immune response, including cell-to-cell communication and migration. Three common alleles Alzheimer’s disease (AD) is a slowly progressive neurodegenerative disorder characterized by neuroinflammation that appears to be an important component of the pathophysiology of the disease. Astrocytes are the majority cell type in brain, exerting significant influence over a range of central nervous system activities, including microglial-mediated neuroinflammatory responses. As the resident innate immune effector cells of the brain, microglia respond to soluble chemical signals released from tissue during injury and disease by mobilizing to lesion sites, clearing toxic molecules, and releasing chemical signals of their own. While microglial-mediated neuroinflammation in the AD brain remains an area of intense investigation, the mechanisms underlying reinforcement and regulation of these aberrant microglial responses by astrocytes are largely unstudied. Moreover, although inheritance of APOE ε4 represents the greatest genetic risk factor for sporadic AD, the mechanism by which apoE isoforms differentially influence AD pathophysiology is unknown. Here we show that APOE ε4 genotype specifically modulates astrocyte secretion of potent microglial chemotactic agents, including CCL3, thus providing evidence that APOE modulation of central nervous system (CNS) innate immune response is mediated through astrocytes. PMID:25092803

  19. Identification of Genes in a Partially Resistant Genotype of Avena sativa Expressed in Response to Puccinia coronata Infection.

    PubMed

    Loarce, Yolanda; Navas, Elisa; Paniagua, Carlos; Fominaya, Araceli; Manjón, José L; Ferrer, Esther

    2016-01-01

    Cultivated oat (Avena sativa), an important crop in many countries, can suffer significant losses through infection by the fungus Puccinia coronata, the causal agent of crown rust disease. Understanding the molecular basis of existing partial resistance to this disease might provide targets of interest for crop improvement programs. A suppressive subtractive hybridization (SSH) library was constructed using cDNA from the partially resistant oat genotype MN841801-1 after inoculation with the pathogen. A total of 929 genes returned a BLASTx hit and were annotated under different GO terms, including 139 genes previously described as participants in mechanisms related to the defense response and signal transduction. Among these were genes involved in pathogen recognition, cell-wall modification, oxidative burst/ROS scavenging, and abscisic acid biosynthesis, as well genes related to inducible defense responses mediated by salicylic and jasmonic acid (although none of which had been previously reported involved in strong responses). These findings support the hypothesis that basal defense mechanisms are the main systems operating in oat partial resistance to P. coronata. When the expression profiles of 20 selected genes were examined at different times following inoculation with the pathogen, the partially resistant genotype was much quicker in mounting a response than a susceptible genotype. Additionally, a number of genes not previously described in oat transcriptomes were identified in this work, increasing our molecular knowledge of this crop. PMID:27303424

  20. Identification of Genes in a Partially Resistant Genotype of Avena sativa Expressed in Response to Puccinia coronata Infection

    PubMed Central

    Loarce, Yolanda; Navas, Elisa; Paniagua, Carlos; Fominaya, Araceli; Manjón, José L.; Ferrer, Esther

    2016-01-01

    Cultivated oat (Avena sativa), an important crop in many countries, can suffer significant losses through infection by the fungus Puccinia coronata, the causal agent of crown rust disease. Understanding the molecular basis of existing partial resistance to this disease might provide targets of interest for crop improvement programs. A suppressive subtractive hybridization (SSH) library was constructed using cDNA from the partially resistant oat genotype MN841801-1 after inoculation with the pathogen. A total of 929 genes returned a BLASTx hit and were annotated under different GO terms, including 139 genes previously described as participants in mechanisms related to the defense response and signal transduction. Among these were genes involved in pathogen recognition, cell-wall modification, oxidative burst/ROS scavenging, and abscisic acid biosynthesis, as well genes related to inducible defense responses mediated by salicylic and jasmonic acid (although none of which had been previously reported involved in strong responses). These findings support the hypothesis that basal defense mechanisms are the main systems operating in oat partial resistance to P. coronata. When the expression profiles of 20 selected genes were examined at different times following inoculation with the pathogen, the partially resistant genotype was much quicker in mounting a response than a susceptible genotype. Additionally, a number of genes not previously described in oat transcriptomes were identified in this work, increasing our molecular knowledge of this crop. PMID:27303424

  1. A meta-analysis strategy for gene prioritization using gene expression, SNP genotype, and eQTL data.

    PubMed

    Che, Jingmin; Shin, Miyoung

    2015-01-01

    In order to understand disease pathogenesis, improve medical diagnosis, or discover effective drug targets, it is important to identify significant genes deeply involved in human disease. For this purpose, many earlier approaches attempted to prioritize candidate genes using gene expression profiles or SNP genotype data, but they often suffer from producing many false-positive results. To address this issue, in this paper, we propose a meta-analysis strategy for gene prioritization that employs three different genetic resources--gene expression data, single nucleotide polymorphism (SNP) genotype data, and expression quantitative trait loci (eQTL) data--in an integrative manner. For integration, we utilized an improved technique for the order of preference by similarity to ideal solution (TOPSIS) to combine scores from distinct resources. This method was evaluated on two publicly available datasets regarding prostate cancer and lung cancer to identify disease-related genes. Consequently, our proposed strategy for gene prioritization showed its superiority to conventional methods in discovering significant disease-related genes with several types of genetic resources, while making good use of potential complementarities among available resources. PMID:25874220

  2. A Meta-Analysis Strategy for Gene Prioritization Using Gene Expression, SNP Genotype, and eQTL Data

    PubMed Central

    2015-01-01

    In order to understand disease pathogenesis, improve medical diagnosis, or discover effective drug targets, it is important to identify significant genes deeply involved in human disease. For this purpose, many earlier approaches attempted to prioritize candidate genes using gene expression profiles or SNP genotype data, but they often suffer from producing many false-positive results. To address this issue, in this paper, we propose a meta-analysis strategy for gene prioritization that employs three different genetic resources—gene expression data, single nucleotide polymorphism (SNP) genotype data, and expression quantitative trait loci (eQTL) data—in an integrative manner. For integration, we utilized an improved technique for the order of preference by similarity to ideal solution (TOPSIS) to combine scores from distinct resources. This method was evaluated on two publicly available datasets regarding prostate cancer and lung cancer to identify disease-related genes. Consequently, our proposed strategy for gene prioritization showed its superiority to conventional methods in discovering significant disease-related genes with several types of genetic resources, while making good use of potential complementarities among available resources. PMID:25874220

  3. Integrative properties of retinal ganglion cell electrical responsiveness depend on neurotrophic support and genotype in the mouse.

    PubMed

    Chou, Tsung-Han; Feuer, William J; Schwartz, Odelia; Rojas, Mario J; Roebber, Jennifer K; Porciatti, Vittorio

    2016-04-01

    Early stages of glaucoma and optic neuropathies are thought to show inner retina remodeling and functional changes of retinal ganglion cells (RGCs) before they die. To assess RGC functional plasticity, we investigated the contrast-gain control properties of the pattern electroretinogram (PERG), a sensitive measure of RGC function, as an index of spatio-temporal integration occurring in the inner retina circuitry subserving PERG generators. We studied the integrative properties of the PERG in mice exposed to different conditions of neurotrophic support. We also investigated the effect of genotypic differences among mouse strains with different susceptibility to glaucoma (C57BL/6J, DBA/2J, DBA/2.Gpnmb(+)). Results show that the integrative properties of the PERG recorded in the standard C57BL/6J inbred mouse strain are impaired after deficit of neurotrophic support and partially restored after exogenous neurotrophic administration. Changes in PERG amplitude, latency, and contrast-dependent responses differ between mouse strains with different susceptibility to glaucoma. Results represent a proof of concept that the PERG could be used as a tool for in-vivo monitoring of RGC functional plasticity before RGC death, the effect of neuroactive treatments, as well as for high-throughput tool for phenotypic screening of different mouse genotypes. PMID:26614910

  4. A time- and dose-dependent STAT1 expression system

    PubMed Central

    Leitner, Nicole R; Strobl, Birgit; Bokor, Marion; Painz, Ronald; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Karaghiosoff, Marina

    2006-01-01

    Background The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. Results The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. Conclusion These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner. PMID:17184522

  5. Hierarchical Modeling and Differential Expression Analysis for RNA-seq Experiments with Inbred and Hybrid Genotypes

    PubMed Central

    Lithio, Andrew; Nettleton, Dan

    2016-01-01

    The performance of inbred and hybrid genotypes is of interest in plant breeding and genetics. High-throughput sequencing of RNA (RNA-seq) has proven to be a useful tool in the study of the molecular genetic responses of inbreds and hybrids to environmental stresses. Commonly used experimental designs and sequencing methods lead to complex data structures that require careful attention in data analysis. We demonstrate an analysis of RNA-seq data from a split-plot design involving drought stress applied to two inbred genotypes and two hybrids formed by crosses between the inbreds. Our generalized linear modeling strategy incorporates random effects for whole-plot experimental units and uses negative binomial distributions to allow for overdispersion in count responses for split-plot experimental units. Variations in gene length and base content, as well as differences in sequencing intensity across experimental units, are also accounted for. Hierarchical modeling with thoughtful parameterization and prior specification allows for borrowing of information across genes to improve estimation of dispersion parameters, genotype effects, treatment effects, and interaction effects of primary interest. PMID:27110090

  6. Array-based genotyping and expression analysis of barley cv. Maythorpe and Golden Promise

    PubMed Central

    Walia, Harkamal; Wilson, Clyde; Condamine, Pascal; Ismail, Abdelbagi M; Xu, Jin; Cui, Xinping; Close, Timothy J

    2007-01-01

    Background Golden Promise is a salt-tolerant spring barley closely related to Maythorpe. Salt tolerance in Golden Promise has been attributed to a single mutation at the Ari-e locus (on 5H) resulting from irradiation of Maythorpe. Golden Promise accumulates lower shoot Na+ compared to Maythorpe when growing under saline conditions. This study focused on elucidating the genetic basis and mechanisms involved in this difference. Results The level of polymorphism between the two genotypes was explored using the Barley1 GeneChip for single feature polymorphisms (SFPs) and an oligonucleotide pool assay for single nucleotide polymorphisms (SNPs). Polymorphism analyses revealed three haplotype blocks spanning 6.4 cM on chromosome 1H, 23.7 cM on chromosome 4H and 3.0 cM on 5H. The Barley1 GeneChip was used to examine transcript abundance in different tissues and stages during development. Several genes within the polymorphic haplotype blocks were differentially regulated. Additionally, a more global difference in the jasmonic acid pathway regulation was detected between the two genotypes. Conclusion The results confirm that Golden Promise and Maythorpe are genetically very closely related but establish that they are not isogenic, as previously reported, due to three polymorphic haplotype blocks. Transcriptome analysis indicates that the response of the two genotypes to salinity stress is quite different. Additionally, the response to salinity stress in the roots and shoot tissue is strikingly different. PMID:17394671

  7. Cumulative discounted expressions of sire genotypes for the complex vertebral malformation and beta-casein loci in commercial dairy herds.

    PubMed

    Kearney, J F; Amer, P R; Villanueva, B

    2005-12-01

    Based on discounted gene-flow principles, a set of recursive equations was developed to quantify the value of using sires with a specific genotype for an identified gene in a commercial dairy herd. Two examples were used to demonstrate the usefulness of the method. The first example deals with the implications of using sires that are known carriers of the lethal recessive genetic defect, complex vertebral malformation (CVM). The second example examines the value of using sires homozygous for the A2 allele of beta-casein. Results are presented in terms of cumulative discounted expressions. These are then multiplied by the economic values of specific genotypes to determine the cost or benefit of using these sires. In general, the degree of mortality and the required price reduction for carrier sires increased as the proportion of carrier sires used, the duration of sire use, and the initial frequency in the cow herd increased. A semen discount of 3.10 pound sterling per CVM straw used would be required to offset the expected mortality when 20% of CVM carrier sires are used for 3 yr when 5% of cows are carriers. The cumulative discounted expressions' of using sires homozygous for the A2 allele of beta-casein also increased when the proportion and duration of carrier sire use and the initial frequency of the A2 allele increased. Assuming an A2A2 cow is worth 160 pound sterling more than a non-A2A2 cow, the expected benefit of using A2A2 sires in a 100-cow herd for 5 yr would be 57 pound sterling,120 for a 20-yr planning horizon. The results of this study demonstrate how the starting gene frequency in the herd, and the proportion and duration of use of sires of particular genotypes are critical to the economic implications of using single genes in commercial dairy farms. PMID:16291634

  8. Pupal diapause termination in Bactrocera minax: an insight on 20-hydroxyecdysone induced phenotypic and genotypic expressions.

    PubMed

    Chen, Zhenzhong; Dong, Yongcheng; Wang, Yaohui; Andongma, Awawing A; Rashid, Muhammad A; Krutmuang, Patcharin; Niu, Changying

    2016-01-01

    The Chinese citrus fruit fly, Bactrocera minax, is an economically important pest of citrus. It exhibits pupal diapause from November to May to combat harsh environmental conditions. Such a long pupal diapause is a barrier for laboratory rearing and development of control strategies against this pest. In the present study, 20-hydroxyecdysone (20E) was used to break pupal diapause of B. minax by topical application. After diapause termination by 20E treated, the pupal ontogenetic processes were observed along the temporal trajectory. The pupal response time to 20E was estimated by detecting the relative expression of 20E responsive genes at different times after 20E-treatment. Results revealed that 20E could effectively terminate the pupal diapause in a dose-dependent manner and significantly shorten the time for 50% adult emergence (Et50). 20E response genes, including ecr, broad and foxo, were up-regulated within 72h, indicating these genes are involved in pupal metamorphosis and diapause termination processes. Morphological changes showed the pupal metamorphosis began ~7 days after 20E-treatment at 22 °C. This study does not only pave the way for artificial rearing in the laboratory through manipulating of pupal diapause termination, but also deepens our understanding of the underlying pupal diapause termination mechanism of B. minax. PMID:27273028

  9. Pupal diapause termination in Bactrocera minax: an insight on 20-hydroxyecdysone induced phenotypic and genotypic expressions

    PubMed Central

    Chen, Zhenzhong; Dong, Yongcheng; Wang, Yaohui; Andongma, Awawing A.; Rashid, Muhammad A.; Krutmuang, Patcharin; Niu, Changying

    2016-01-01

    The Chinese citrus fruit fly, Bactrocera minax, is an economically important pest of citrus. It exhibits pupal diapause from November to May to combat harsh environmental conditions. Such a long pupal diapause is a barrier for laboratory rearing and development of control strategies against this pest. In the present study, 20-hydroxyecdysone (20E) was used to break pupal diapause of B. minax by topical application. After diapause termination by 20E treated, the pupal ontogenetic processes were observed along the temporal trajectory. The pupal response time to 20E was estimated by detecting the relative expression of 20E responsive genes at different times after 20E-treatment. Results revealed that 20E could effectively terminate the pupal diapause in a dose-dependent manner and significantly shorten the time for 50% adult emergence (Et50). 20E response genes, including ecr, broad and foxo, were up-regulated within 72h, indicating these genes are involved in pupal metamorphosis and diapause termination processes. Morphological changes showed the pupal metamorphosis began ~7 days after 20E-treatment at 22 °C. This study does not only pave the way for artificial rearing in the laboratory through manipulating of pupal diapause termination, but also deepens our understanding of the underlying pupal diapause termination mechanism of B. minax. PMID:27273028

  10. Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes.

    PubMed

    Ikoma, Yoshinori; Matsumoto, Hikaru; Kato, Masaya

    2016-01-01

    Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as β-cryptoxanthin, violaxanthin, lycopene, and β-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown. PMID:27069398

  11. Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes

    PubMed Central

    Ikoma, Yoshinori; Matsumoto, Hikaru; Kato, Masaya

    2016-01-01

    Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as β-cryptoxanthin, violaxanthin, lycopene, and β-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown. PMID:27069398

  12. The genotype-dependent influence of functionalized multiwalled carbon nanotubes on fetal development.

    PubMed

    Huang, Xinglu; Zhang, Fan; Sun, Xiaolian; Choi, Ki-Young; Niu, Gang; Zhang, Guofeng; Guo, Jinxia; Lee, Seulki; Chen, Xiaoyuan

    2014-01-01

    In many cases cancer is caused by gene deficiency that is being passed along from generation to generation. Soluble carbon nanotubes (CNTs) have shown promising applications in the diagnosis and therapy of cancer, however, the potential relationship between cancer-prone individuals and response to CNT exposure as a prerequisite for development of personalized nanomedicine, is still poorly understood. Here we report that intravenous injections of multi-walled carbon nanotubes into p53 (a well-known cancer-susceptible gene) heterozygous pregnant mice can induce p53- dependent responses in fetal development. Larger sized multi-walled carbon nanotubes moved across the blood-placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas single-walled and smaller sized multi-walled carbon nanotubes showed no or less fetotoxicity. A molecular mechanism study found that multi-walled carbon nanotubes directly triggered p53-dependent apoptosis and cell cycle arrest in response to DNA damage. Based on the molecular mechanism, we also incorporated N-acetylcysteine (NAC), an FDA approved antioxidant, to prevent CNTs induced nuclear DNA damage and reduce brain development abnormalities. Our findings suggest that CNTs might have genetic background-dependent toxic effect on the normal development of the embryo, and provide new insights into protection against nanoparticle-induced toxicity in potential clinical applications. PMID:24344357

  13. The genotype-dependent influence of functionalized multiwalled carbon nanotubes on fetal development

    PubMed Central

    Huang, Xinglu; Zhang, Fan; Sun, Xiaolian; Choi, Ki Young; Niu, Gang; Zhang, Guofeng; Guo, Jinxia; Lee, Seulki; Chen, Xiaoyuan

    2013-01-01

    In many cases cancer is caused by gene deficiency that is being passed along from generation to generation. Soluble carbon nanotubes (CNTs) have shown promising applications in the diagnosis and therapy of cancer, however, the potential relationship between cancer-prone individuals and response to CNT exposure as a prerequisite for development of personalized nanomedicine, is still poorly understood. Here we report that intravenous injections of multi-walled carbon nanotubes into p53 (a well-known cancer susceptible gene) heterozygous pregnant mice can induce p53- dependent responses in fetal development. Larger sized multi-walled carbon nanotubes moved across the blood-placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas single-walled and smaller sized multi-walled carbon nanotubes showed no or less fetotoxicity. A molecular mechanism study found that multi-walled carbon nanotubes directly triggered p53-dependent apoptosis and cell cycle arrest in response to DNA damage. Based on the molecular mechanism, we also incorporated N-acetylcysteine (NAC), a FDA approved antioxidant, to prevent CNTs induced nuclear DNA damage and reduce brain development abnormalities. Our findings suggest that CNTs might have genetic background-dependent toxic effect on the normal development of the embryo, and provide new insights into protection against nanoparticle-induced toxicity in potential clinical applications. PMID:24344357

  14. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera.

    PubMed

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality. PMID:26507253

  15. Effects of ploidy and sex-locus genotype on gene expression patterns in the fire ant Solenopsis invicta

    PubMed Central

    Nipitwattanaphon, Mingkwan; Wang, John; Ross, Kenneth G.; Riba-Grognuz, Oksana; Wurm, Yannick; Khurewathanakul, Chitsanu; Keller, Laurent

    2014-01-01

    Males in many animal species differ greatly from females in morphology, physiology and behaviour. Ants, bees and wasps have a haplodiploid mechanism of sex determination whereby unfertilized eggs become males while fertilized eggs become females. However, many species also have a low frequency of diploid males, which are thought to develop from diploid eggs when individuals are homozygous at one or more sex determination loci. Diploid males are morphologically similar to haploids, though often larger and typically sterile. To determine how ploidy level and sex-locus genotype affect gene expression during development, we compared expression patterns between diploid males, haploid males and females (queens) at three developmental timepoints in Solenopsis invicta. In pupae, gene expression profiles of diploid males were very different from those of haploid males but nearly identical to those of queens. An unexpected shift in expression patterns emerged soon after adult eclosion, with diploid male patterns diverging from those of queens to resemble those of haploid males, a pattern retained in older adults. The finding that ploidy level effects on early gene expression override sex effects (including genes implicated in sperm production and pheromone production/perception) may explain diploid male sterility and lack of worker discrimination against them during development. PMID:25355475

  16. The genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy due to mutations in ALDH7A1

    PubMed Central

    Scharer, Gunter; Brocker, Chad; Vasiliou, Vasilis; Creadon-Swindell, Geralyn; Gallagher, Renata C.; Spector, Elaine

    2011-01-01

    Pyridoxine-dependent epilepsy is a disorder associated with severe seizures that may be caused by deficient activity of α-aminoadipic semialdehyde dehydrogenase, encoded by the ALDH7A1 gene, with accumulation of α-aminoadipic semialdehyde and piperideine-6-carboxylic acid. The latter reacts with pyridoxal-phosphate, explaining the effective treatment with pyridoxine. We report the clinical phenotype of three patients, their mutations and those of 12 additional patients identified in our clinical molecular laboratory. There were six missense, one nonsense, and five splice-site mutations, and two small deletions. Mutations c.1217_1218delAT, I431F, IVS-1(+2)T>G, IVS-2(+1)G>A, and IVS-12(+1)G>A are novel. Some disease alleles were recurring: E399Q (eight times), G477R (six times), R82X (two times), and c.1217_1218delAT (two times). A systematic review of mutations from the literature indicates that missense mutations cluster around exons 14, 15, and 16. Nine mutations represent 61% of alleles. Molecular modeling of missense mutations allows classification into three groups: those that affect NAD+binding or catalysis, those that affect the substrate binding site, and those that affect multimerization. There are three clinical phenotypes: patients with complete seizure control with pyridoxine and normal developmental outcome (group 1) including our first patient; patients with complete seizure control with pyridoxine but with developmental delay (group 2), including our other two patients; and patients with persistent seizures despite pyridoxine treatment and with developmental delay (group 3). There is preliminary evidence for a genotype-phenotype correlation with patients from group 1 having mutations with residual activity. There is evidence from patients with similar genotypes for nongenetic factors contributing to the phenotypic spectrum. PMID:20814824

  17. A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

    PubMed Central

    2014-01-01

    Background Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community. PMID:24456189

  18. A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

    SciTech Connect

    Shakoor, N; Nair, R; Crasta, O; Morris, G; Feltus, A; Kresovich, S

    2014-01-23

    Background: Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results: This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e. g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions: Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

  19. Molecular Cloning, Expression Pattern and Genotypic Effects on Glucoraphanin Biosynthetic Related Genes in Chinese Kale (Brassica oleracea var. alboglabra Bailey).

    PubMed

    Yin, Ling; Chen, Changming; Chen, Guoju; Cao, Bihao; Lei, Jianjun

    2015-01-01

    Glucoraphanin is a plant secondary metabolite that is involved in plant defense and imparts health-promoting properties to cruciferous vegetables. In this study, three genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4 (BCAT4), methylthioalkylmalate synthase 1 (MAM1) and dihomomethionine N-hydroxylase (CYP79F1), were cloned from Chinese kale (Brassica oleracea var. alboglabra Bailey). Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The transcript levels of BCAT4, MAM1 and CYP79F1 were higher in cotyledon, leaf and stem compared with flower and silique. BCAT4, MAM1 and CYP79F1 were expressed throughout leaf development with lower transcript levels during the younger stages. Glucoraphanin content varied extensively among different varieties, which ranged from 0.25 to 2.73 µmol·g(-1) DW (dry weight). Expression levels of BCAT4 and MAM1 were high at vegetative-reproductive transition phase, while CYP79F1 was expressed high at reproductive phase. BCAT4, MAM1 and CYP79F1 were expressed significantly high in genotypes with high glucoraphanin content. All the results provided a better understanding of the roles of BCAT4, MAM1 and CYP79F1 in the glucoraphanin biosynthesis of Chinese kale. PMID:26569208

  20. Evidence of Allelic Suppression for Transcripts Expressed in Day 30 Pig Embryos by SNP Genotyping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic imprinting results in alleles being differentially expressed in a parent-of-origin specific manner. Parthenogenetic and biparental pig embryo gene expression profiles were compared using three cDNA microarray platforms. Comparison of the profiles of the two tissue types indicated different...

  1. Nonlinear Dependence in the Discovery of Differentially Expressed Genes

    PubMed Central

    Deller, J. R.; Radha, Hayder; McCormick, J. Justin; Wang, Huiyan

    2012-01-01

    Microarray data are used to determine which genes are active in response to a changing cell environment. Genes are “discovered” when they are significantly differentially expressed in the microarray data collected under the differing conditions. In one prevalent approach, all genes are assumed to satisfy a null hypothesis, ℍ0, of no difference in expression. A false discovery (type 1 error) occurs when ℍ0 is incorrectly rejected. The quality of a detection algorithm is assessed by estimating its number of false discoveries, 𝔉. Work involving the second-moment modeling of the z-value histogram (representing gene expression differentials) has shown significantly deleterious effects of intergene expression correlation on the estimate of 𝔉. This paper suggests that nonlinear dependencies could likewise be important. With an applied emphasis, this paper extends the “moment framework” by including third-moment skewness corrections in an estimator of 𝔉. This estimator combines observed correlation (corrected for sampling fluctuations) with the information from easily identifiable null cases. Nonlinear-dependence modeling reduces the estimation error relative to that of linear estimation. Third-moment calculations involve empirical densities of 3 × 3 covariance matrices estimated using very few samples. The principle of entropy maximization is employed to connect estimated moments to 𝔉 inference. Model results are tested with BRCA and HIV data sets and with carefully constructed simulations. PMID:25937940

  2. The Liver MicroRNA Expression Profiles Associated With Chronic Hepatitis C Virus (HCV) Genotype-4 Infection: A Preliminary Study

    PubMed Central

    El-Guendy, Nadia Mohamed; Helwa, Reham; El-Halawany, Medhat Salah; Abdel Rahman Ali, Shimaa; Tantawy Aly, Marwa; Hasan Alieldin, Nelly; Fouad, Shawky Abdel Hamid; Saeid, Hany; Abdel-Wahab, Abdel-Hady Ali

    2016-01-01

    Background MicroRNAs (miRNAs) have been repeatedly shown to play important roles in liver pathologies, including hepatitis, liver cirrhosis, and liver cancer. Egypt has the highest hepatitis C virus (HCV) infection rate worldwide, predominantly involving genotype-4. Objectives In this study, we attempted to characterize the miRNA profile of the poorly studied genotype 4 of HCV in chronically infected Egyptian patients to obtain a better understanding of the disease and its complications and help in the design of better management protocols. Patients and Methods We analyzed the expression levels of a selected panel of 94 miRNAs in fresh liver biopsies collected from 50 Egyptian patients diagnosed with chronic HCV infection using quantitative real-time polymerase chain reaction (PCR) assay. Non-parametric tests were used to analyze the expression level of each miRNA and association with the clinicopathological features of enrolled patients in this study. Results Our results revealed differential expression levels of the analyzed miRNAs compared to the normal controls. Twenty-seven miRNAs (including miR-105, miR-147, miR-149-3p, and miR-196b) showed up-regulation, while 17 miRNAs (including miR-21, miR-122, miR-199a-3p, and miR-223) showed down-regulation. An inverse correlation was observed between levels of miR-95, miR-130a, and miR-142-5p with the blood albumin level. Increased expression levels of seven miRNAs (miR-29c, miR-30c, miR-126, miR-145, miR-199a, miR-199a-3p, and miR-222) were observed with severe chronic hepatic inflammation. Several deregulated miRNAs found in this study have been previously linked to chronic liver inflammation and the risk of hepatocellular carcinoma (HCC) development. Conclusions The identified expression profiles of some examined miRNAs might offer important points to consider for the treatment of naive patients and the management of chronically infected HCV patients in Egypt and around the world.

  3. Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species.

    PubMed

    Farnham, M W; Griffith, S M; Miller, S S; Vance, C P

    1990-12-01

    Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species. PMID:16667896

  4. Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork

    PubMed Central

    Druka, Arnis; Druka, Ilze; Centeno, Arthur G; Li, Hongqiang; Sun, Zhaohui; Thomas, William TB; Bonar, Nicola; Steffenson, Brian J; Ullrich, Steven E; Kleinhofs, Andris; Wise, Roger P; Close, Timothy J; Potokina, Elena; Luo, Zewei; Wagner, Carola; Schweizer, Günther F; Marshall, David F; Kearsey, Michael J; Williams, Robert W; Waugh, Robbie

    2008-01-01

    Background A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. Description Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork . GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. Conclusion By integrating barley genotypic, phenotypic and mRNA abundance data

  5. Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

    PubMed

    Agger, Teit; Petersen, Jesper B; O'Connor, Susan M; Murphy, Rachael L; Kelly, Joan M; Nielsen, Jens

    2002-01-18

    The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation. PMID:11689252

  6. Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

    PubMed

    Adamczyk, J J; Adams, L C; Hardee, D D

    2001-12-01

    Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hübner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P < 0.05) in Bollgard II plots compared with Bollgard. Population numbers for fall army worms, Spodoptera frugiperda (J. E. Smith), and salt marsh caterpillars, Estigmene acrea (Drury), were lower in Bollgard II plots compared with Bollgard but means did not differ significantly (P > 0.05). Single and dual-toxin genotypes remained superior (P < 0.05) compared with conventional cotton against the tobacco budworm, Heliothis virescens (F.). The addition of Cry2Ab had no significant (P > 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed. PMID:11777069

  7. DETERMINATION OF GENOTYPE COMBINATIONS THAT CAN PREDICT THE OUTCOME OF THE TREATMENT OF ALCOHOL DEPENDENCE USING THE 5-HT3 ANTAGONIST ONDANSETRON

    PubMed Central

    Johnson, Bankole A.; Seneviratne, Chamindi; Wang, Xin-Qun; Ait-Daoud, Nassima; Li, Ming D.

    2013-01-01

    Objective Previously, we reported that the 5′-HTTLPR-LL and rs1042173-TT (SLC6A4-LL/TT) genotypes in the serotonin transporter gene predicted a significant reduction in the severity of alcohol consumption among alcoholics receiving the 5-HT3 antagonist ondansetron. In this study, we explored additional markers of ondansetron treatment response in alcoholics by examining polymorphisms in the HTR3A and HTR3B genes, which regulate directly the function and binding of 5-HT3 receptors to ondansetron. Method We genotyped 1 rare and 18 common single-nucleotide polymorphisms in HTR3A and HTR3B in the same sample that we had genotyped for SLC6A4-LL/TT in the previous randomized, double-blind, 11-week clinical trial. Participants were 283 European Americans who received oral ondansetron (4 μg/kg twice daily) or placebo along with weekly cognitive behavioral therapy. Associations of individual and combined genotypes with treatment response on drinking outcomes were analyzed. Results Individuals carrying one or more of genotypes rs1150226-AG and rs1176713-GG in HTR3A and rs17614942-AC in HTR3B showed a significant overall mean difference between ondansetron and placebo in drinks per drinking day (−2.50; effect size (ES)=0.867), percentage of heavy drinking days (−20.58%; ES=0.780), and percentage of days abstinent (18.18%; ES=0.683). Combining these HTR3A/HTR3B and SLC6A4-LL/TT genotypes increased the target cohort from approaching 20% (identified in our previous study) to 34%. Conclusions We present initial evidence suggesting that a combined 5-marker genotype panel can be used to predict the outcome of treatment of alcohol dependence with ondansetron. Additional, larger pharmacogenetic studies would help to validate our results. PMID:23897038

  8. Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

    PubMed Central

    Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

    2014-01-01

    ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine

  9. Androgen-Dependent Regulation of Human MUC1 Mucin Expression

    PubMed Central

    Mitchell, Stephen; Abel, Paul; Madaan, Sanjeev; Jeffs, James; Chaudhary, Khurram; Stamp, Gordon; Lalani, El-Nasir

    2002-01-01

    Abstract MUC1 mucin is transcriptionally regulated by estrogen, progesterone, and glucocorticoids. Our objective was to determine whether androgen receptor (AR) activation regulates expression of MUC1. The following breast and prostatic cell lines were phenotyped and grouped according to AR and MUC1 protein expression: 1) AR+MUC1+ [DAR17+19 (AR transfectants of DU-145), ZR-75-1, MDA-MB-453, and T47D]; 2) AR-MUC1+ [DZeo1 (AR-vector control), DU-145, BT20,MDA-MB-231, and MCF7]; 3) AR+MUC1- (LNCaP and LNCaP-r). Cell proliferation was determined using the MTT assay in the presence of synthetic androgen R1881, 0.1 pM to 1 µM. Cell surface MUC1 expression was determined by flow cytometry in the presence or absence of oestradiol, medroxy progesterone acetate or R1881, with and without 4 hydroxy-flutamide (4-OH), a nonsteroidal AR antagonist. The functional significance of MUC1 expression was investigated with a cell-cell aggregation assay. Only AR+ MUC1+ cell lines showed a significant increase in MUC1 expression with AR activation (P (range) =.01 to.0001), reversed in the presence of 4-OHF. Cell proliferation was unaffected. Increased expression of MUC1 was associated with a significant (P (range) =.002 to.001) reduction in cell-cell adhesion. To our knowledge, this is the first description of androgen-dependent regulation of MUC1 mucin. This is also functionally associated with decreased cell-cell adhesion, a recognised feature of progressive malignancy. These findings have important implications for physiological and pathological processes. PMID:11922395

  10. Genotypic diversity among rhizospheric bacteria of three legumes assessed by cultivation-dependent and cultivation-independent techniques.

    PubMed

    Pongsilp, Neelawan; Nimnoi, Pongrawee; Lumyong, Saisamorn

    2012-02-01

    The genotypic diversity of rhizospheric bacteria of 3 legumes including Vigna radiata, Arachis hypogaea and Acacia mangium was compared by using cultivation-dependent and cultivation-independent methods. For cultivation-dependent method, Random amplified polymorphic DNA (RAPD) profiles revealed that the bacterial genetic diversity of V. radiata and A. mangium rhizospheres was higher than that of A. hypogaea rhizosphere. For cultivation-independent method, Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes revealed the difference in bacterial community and diversity of rhizospheres collected from 3 legumes. The ribotype richness which indicates species diversity, was highest in V. radiata rhizosphere, followed by A. hypogaea and A. mangium rhizospheres, respectively. Three kinds of media were used to cultivate different target groups of bacteria. The result indicates that the communities of cultivable bacteria in 3 rhizospheres recovered from nutrient agar (NA) medium were mostly different from each other, while Bradyrhizobium selective medium (BJSM) and nitrogen-free medium shaped the communities of cultivable bacteria. Nine isolates grown on BJSM were identified by 16S rRNA gene sequence analysis. These isolates were very closely related (with 96% to 99% identities) to either one of the three groups including Cupriavidus-Ralstonia group, Bacillus group and Bradyrhizobium-Bosea-Afipia group. The rhizospheres were also examined for their enzymatic patterns. Of 19 enzymes tested, 3 rhizospheres were distinguishable by the presence or the absence of leucine acrylamidase and acid phosphatase. The selected cultivable bacteria recovered from NA varied in their abilities to produce indole-acetic acid and ammnonia. The resistance to 10 antibiotics was indistinguishable among bacteria isolated from different rhizospheres. PMID:22806857

  11. Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

    PubMed Central

    Marraccini, Pierre; Vinecky, Felipe; Alves, Gabriel S.C.; Ramos, Humberto J.O.; Elbelt, Sonia; Vieira, Natalia G.; Carneiro, Fernanda A.; Sujii, Patricia S.; Alekcevetch, Jean C.; Silva, Vânia A.; DaMatta, Fábio M.; Ferrão, Maria A.G.; Leroy, Thierry; Pot, David; Vieira, Luiz G.E.; da Silva, Felipe R.; Andrade, Alan C.

    2012-01-01

    The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora. PMID:22511801

  12. The hairless gene of the mouse: relationship of phenotypic effects with expression profile and genotype.

    PubMed

    Cachón-González, M B; San-José, I; Cano, A; Vega, J A; García, N; Freeman, T; Schimmang, T; Stoye, J P

    1999-10-01

    Various mutations of the hairless (hr) gene of mice result in hair loss and other integument defects. To examine the role of the hr gene in mouse development, the expression profile of hr has been determined by in situ hybridisation and correlated to the nature of genetic changes and morphological abnormalities in different mutant animals. Four variant alleles have been characterised at the molecular level. hr/hr mice produce reduced, but significant, levels of hr mRNA whereas other alleles contain mutations which would be expected to preclude the synthesis of functional product, demonstrating a correlation between allelic variation at the hr locus and phenotypic severity. hr expression was shown to be widespread and temporally regulated. It was identified in novel tissues such as cartilage, developing tooth, inner ear, retina, and colon as well as in skin and brain. Analysis of mice homozygous for the rhino allele of hairless revealed that, although no morphological defects were detectable in many tissues normally expressing hr, previously undescribed abnormalities were present in several tissues including inner ear, retina, and colon. These findings indicate that the hairless gene product plays a wider role in development than previously suspected. Dev Dyn 1999;216:113-126. PMID:10536052

  13. Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

    PubMed

    Ishikawa, Tomohiro; Abe, Makoto; Masuda, Michiaki

    2015-01-01

    Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V. PMID:25451067

  14. Identifying differentially expressed genes in trophozoites and cysts of Acanthamoeba T4 genotype: Implications for developing new treatments for Acanthamoeba keratitis.

    PubMed

    Abedkhojasteh, Hoda; Niyyati, Maryam; Rezaei, Sasan; Mohebali, Mehdi; Farnia, Shohreh; Kazemi-Rad, Elham; Roozafzoon, Reza; Sianati, Hamed; Rezaeian, Mostafa; Heidari, Mansour

    2015-02-01

    Acanthamoeba T4 genotype is the most prevalent genotype associated with amoebic keratitis. Acanthamoeba keratitis therapy is difficult due to transformation of trophozoite to cyst stage, which hinders the treatment of the disease. Although encystation assists the organism to survive against the chemotherapeutic compounds, the precise mechanism of encystation remains poorly understood. The purpose of this work was to identify differentially expressed genes in Acanthamoeba T4 genotype which might be useful for understanding of the encystment process and may thus help develop more efficient treatment. The mRNA profile of trophozoite and cyst of Acanthamoeba T4 genotype isolated from a soft contact lens wearer were analyzed using a cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. Subsequently, a real time reverse transcriptase-PCR was performed to validate the cDNA-AFLP results. Three genes, heat shock protein70 (hsp70), actin-I and elongation factor-1alpha (EF-1α) were differentially expressed during Acanthamoeba differentiation. An in silico result predicted that transformation of trophozoite to cyst could be mediated through their cooperation with the protein partners interaction. Taken together, our experimental and bioinformatics findings suggested potential functions of hsp70, EF-1α and actin-I in differentiation of Acanthamoeba T4 genotype which may be useful in the design of an efficient therapeutic strategy in AK. PMID:25543551

  15. Analysis of Antioxidant Enzyme Activity and Antioxidant Genes Expression During Germination of Two Different Genotypes of Lolium multiflorum Under Salt Tolerance.

    PubMed

    Wang, Xia; Ma, Xiao; Xinquan-Zhang; Linkai-Huang; Li, Zhou; Nie, Wenzhi-Xu Gang

    2016-01-01

    Annual ryegrass (Lolium multiflorum) is widely used as a cool-season forage grass for its luxuriant growth, palatable and high digestible. To investigate the salt tolerance mechanism in annual ryegrass under salt stress, salt-tolerant genotype 'R102-3' and salt-sensitive genotype 'Tetragold' were subject to 300mmol/L NaCl in a controlled growth chamber for 12 days. The results showed high concentrations of NaCl decreased relative water content (RWC), and increased the electrolyte leakage (EL) in both genotypes. However the 'Tetragold' had a greater increased extent of malondialdehyde (MDA) and EL than in 'R102-3', in contrast, the activities of Superoxide (SOD), Peroxidase (POD), Catalase (CAT) and Ascorbate peroxidase (APX) were higher in salt resistant compared to sensitive ones. For ensure the accurate of qRT-PCR, we used RefFinder to choose the most stably reference genes eEF1A(s) and GAPDH to normalize the antioxidant genes expression data. The results indicated that higher expression of Fe-SOD, Mn-SOD, Chl-Cu/Zn SOD, Cyt-Cu/Zn SOD, POD and CAT in 'R102-3' when compared with 'Tetragold', which may play an important role in defensed damage of Reactive oxygen species (ROS) under salt stress. Thus, the salt-tolerant genotype could effectively resist oxidative damage induced by salt tress relative to salt-sensitive genotype. PMID:26972970

  16. A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

    PubMed Central

    Van Gelder, R N; Krasnow, M A

    1996-01-01

    The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila. Images PMID:8612586

  17. Influence of Populus Genotype on Gene Expression by the Wood Decay Fungus Phanerochaete chrysosporium

    PubMed Central

    Gaskell, Jill; Marty, Amber; Mozuch, Michael; Kersten, Philip J.; Splinter BonDurant, Sandra; Sabat, Grzegorz; Azarpira, Ali; Ralph, John; Skyba, Oleksandr; Mansfield, Shawn D.; Blanchette, Robert A.

    2014-01-01

    We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chrysosporium gene expression patterns are substantially influenced by lignin composition. PMID:25015893

  18. The phenotype masks the genotype: A possible new expression of diabetes

    PubMed Central

    Mimbacas, Adriana; Vitarella, Graciela; Souto, Jorge; Reyes, Ana Laura; Farias, Joaquina; Fernández, Mariana; Fabregat, Matias; Javiel, Gerardo

    2012-01-01

    The concept of a new form of diabetes, with signs of both types 1 and 2, has not been often considered, until recently. It is of immense interest to explore the role of the admixture that characterizes the Uruguayan population (higher and different from other Latin America countries) for the presence of such expression of that particular disease. We describe here a child who possibly presents with this expression. He had typical signs of both diabetic conditions: type 1 (young age, positive immunologic and genetic markers, ketoacidosis) and type 2 (obesity [body mass index = 36 kg/m2] and acanthosis nigricans). In spite of complying with the established guidelines, therapeutic and nutritional control, quality of life and good metabolic control, the patient's obesity had been continually increasing. Looking for a genetic explanation, we studied three single nucleotide polymorphisms involved in three different metabolic pathways (peroxisome proliferator-activated receptor gamma 2, insulin receptor substrate-1 and uncoupling protein-2) associated with insulin resistance. Our patient showed three mutations, GG, GA, GG, associated with insulin resistance that explains obesity associated with limited response to the commonly used drugs. According to the clinical presentation and the genetic and immunological background, we considered that this patient presents with a new form of diabetes. We have termed this particular disease “hybrid diabetes” because of the involvement of genes associated with both the classical type of diabetes. However, at least in an admixed population such as in Uruguay, clinical classification would not strictly dictate the choice of treatment.

  19. [ESTIMATING THE EFFECTIVENESS OF HYPOLIPIDEMIC THERAPY WITH ROSUVASTATIN IN PATIENTS WITH CORONARY HEART DISEASE DEPENDING ON THE GENOTYPE OF LIPOPROTEIN LIPASE].

    PubMed

    Zvyagina, M V; Mal, G S; Bushueva, O Yu; Alymenko, M A; Bykanova, M A; Letova, I M; Gribovskaya, I A; Churnosov, M I; Solodilova, M A; Polonikov, A V

    2016-01-01

    Taking into account the genetic heterogeneity of hyperlipidemias, polymorphic genes involved in the regulation of lipid metabolism may explain differences in the efficacy of hypolipidemic therapy. In the present prospective and randomized study, we have investigated the efficacy of rosuvastatin (10 mg/day) in the therapy of atherogenic hyperlipidemias in a group of 62 patients with coronary heart disease (CHD), depending on the genotype of lipoprotein lipase (LPL). The pharmacological correction was carried out during one year under control of lipid metabolism parameters (total cholesterol, LDL-C, HDL-C, HDL-unrelated cholesterol, triglycerides, atherogenic index) at the baseline and on 4th, 8th, 24th and 48th week. The HindIII polymorphism (+495T > G, rs320) of the LPL gene was genotyped in all patients studied through a real-time PCR TaqMan assay. Rosuvastatin produced a significant hypolipidemic effect with respect to all investigated lipid metabolism parameters for 24 weeks of treatment. Changes in the parameters of lipid metabolism upon rosuvastatin treatment differed in patients with genotype +495GG as compared to the rest LPL genotypes. In comparison to the +495TT and TG genotypes, the genotype +495GG showed a greater reduction in total cholesterol on 8th week, and in LDL-C, HDL-unrelated cholesterol, and atherogenic index on the 48th week of rosuvastatin therapy (p <0.01). It can be suggested that the pronounced hypolipidemic effect of rosuvastatin in homozygotes +495GG of the LPL gene is associated with modulation of the LPL activity, as it has been previously reported for other statin drugs. PMID:27159952

  20. herg1b expression as a potential specific marker in pediatric acute myeloid leukemia patients with HERG 897K/K genotype.

    PubMed

    Erdem, Merve; Tekiner, Tugce Ayca; Fejzullahu, Arta; Akan, Gokce; Anak, Sema; Saribeyoglu, Ebru Tugrul; Ozbek, Ugur; Atalar, Fatmahan

    2015-04-01

    Human ether-a-go-go related gene (herg) encoding HERG K(+) channel has been demonstrated in many previous studies with its association to cell cycle progression and growth in tumor cells. The upregulated expression of HERG K+ channels was determined in different tumor types. Furthermore, not only full-length transcript herg1 but also a truncated isoform herg1b was shown to be expressed in cancer cells. In this study, the expression levels of herg1 and herg1b and the impact of K897T mutation on their expressions were investigated in pediatric acute myeloid leukemia (pAML). Expression levels of herg1 and herg1b isoforms were analyzed by quantitative real time polymerase chain reaction (PCR) in pAML patients together with healthy donors, and their expressions were confirmed by western blotting. The 2690 A>C nucleotide variation in KCNH2 gene corresponding to K897T amino acid change was analyzed by PCR followed by restriction enzyme digestion. herg1b overexpression was observed in tumor cells compared to healthy controls (P = .0024). However, herg1 expression was higher in healthy control cells than tumor cells (P = .001). The prevalence of polymorphic allele 897T was 26% in our patient group and 897T carriers showed increased herg1b expression compared to wild-type allele carriers. Our results demonstrate the presence of the increased levels of herg1b expression in pAML. In addition, we report for the first time that, pAML subgroup with HERG 897K/K genotype compared to 897K/T and T/T genotypes express increased levels of herg1b. In conclusion, HERG 897K/K genotype with increased level of herg1b expression might well be a prognostic marker for pAML. PMID:25247487

  1. Effect of sucrose concentration on sucrose-dependent adhesion and glucosyltransferase expression of S. mutans in children with severe early-childhood caries (S-ECC).

    PubMed

    Zhao, Wei; Li, Wenqing; Lin, Jiacheng; Chen, Zhuoyu; Yu, Dongsheng

    2014-09-01

    Sucrose, extracellular polysaccharide, and glucosyltransferases (GTFs) are key factors in sucrose-dependent adhesion and play important roles in the process of severe early-childhood caries (S-ECC). However, whether sucrose concentration regulates gtf expression, extracellular polysaccharide synthesis, and sucrose-dependent adhesion is related to the different genotypes of S. mutans isolated from ECC in children and still needs to be investigated. In this study, 52 strains of S. mutans were isolated from children with S-ECC and caries-free (CF) children. Water-insoluble glucan (WIG) synthesis was detected by the anthrone method, adhesion capacity by the turbidimetric method, and expression of gtf by RT-PCR in an in vitro model containing 1%-20% sucrose. The genotypes of S. mutans were analyzed by AP-PCR. The results showed that WIG synthesis, adhesion capacity, and gtf expression increased significantly when the sucrose concentration was from 1% to 10%. WIG synthesis and gtfB as well as gtfC expression of the 1% and 5% groups were significantly lower than those of the 10% and 20% groups (p < 0.05). There were no significant differences between the 10% and 20% groups. The fingerprints of S. mutans detected from individuals in the S-ECC group exhibited a significant difference in diversity compared with those from CF individuals (p < 0.05). Further, the expression of gtfB and gtfC in the S-ECC group was significantly different among the 1- to 5-genotype groups (p < 0.05). It can be concluded that sucrose-dependent adhesion might be related to the diversity of genotypes of S. mutans, and the 10% sucrose level can be seen as a "turning point" and essential factor for the prevention of S-ECC. PMID:25207825

  2. Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus.

    PubMed

    Feng, Y; Rustan, T D; Ferguson, R J; Doll, M A; Hein, D W

    1994-01-01

    Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures. PMID:8291051

  3. Ondansetron reduces naturalistic drinking in non-treatment seeking alcohol dependent individuals with the LL 5′-HTTLPR genotype: a laboratory study

    PubMed Central

    Kenna, George A.; Zywiak, William H.; Swift, Robert M.; McGeary, John E.; Clifford, James S.; Shoaff, Jessica R.; Vuittonet, Cynthia; Fricchione, Samuel; Brickley, Michael; Beaucage, Kayla; Haass-Koffler, Carolina L.; Leggio, Lorenzo

    2014-01-01

    Background One hypothesis suggests that the differential response to ondansetron and serotonin specific re-uptake inhibitors (SSRIs) may be due to a functional polymorphism of the 5′-HTTLPR promoter region in SLC6A4, the gene that codes for the serotonin transporter (5-HTT). The LL 5′-HTTLPR genotype is postulated to be specifically sensitive to the effects of ondansetron with SS/SL 5′-HTTLPR genotypes sensitive to SSRIs. This study tests this hypothesis by matching non-treatment seeking alcohol dependent (AD) individuals with LL genotype to ondansetron and SS/SL genotypes to the SSRI sertraline, and mis-matching them assessing naturalistic and bar-laboratory alcohol drinking. Methods Seventy-seven AD individuals were randomized to one of two counterbalanced arms to receive sertraline 200mg/day or ondansetron 0.5 mg/day for three weeks followed by an alcohol self-administration experiment (ASAE), then received placebo for three weeks followed by a second ASAE. Individuals then received the alternate drug for three weeks followed by a third ASAE. Drinks per drinking day (DDD with drinks in SDUs) for 7 days prior to each ASAE and milliliters consumed during each ASAE were the primary outcomes. Results Fifty-five participants completed the study. The genotype x order interaction was significant [F(1,47) = 8.42, p = .006] for DDD. Three ANCOVAs were conducted for DDD during the week before each ASAE. Ondansetron compared to sertraline resulted in a significant reduction in DDD during the week before the first [F(1,47) = 7.64, p = .008] but not the third ASAE. There was no difference in milliliters consumed during each ASAE. Conclusion This study modestly supports the hypothesis that ondansetron may reduce DDD in AD individuals with the LL genotype as measured naturalistically. By contrast there was no support that ondansetron reduces drinking during the ASAEs or that sertraline reduces alcohol use in individuals who have SS/SL genotypes. We provide limited

  4. Analysis of MHC class I and II expression in relation to presence of HPV genotypes in premalignant and malignant cervical lesions.

    PubMed Central

    Cromme, F. V.; Meijer, C. J.; Snijders, P. J.; Uyterlinde, A.; Kenemans, P.; Helmerhorst, T.; Stern, P. L.; van den Brule, A. J.; Walboomers, J. M.

    1993-01-01

    Cervical intraepithelial neoplasia (CIN) grades I to III lesions (n = 94) and squamous cell carcinomas of the uterine cervix (n = 27) were analysed for MHC class I and II expression and presence of HPV genotypes. MHC class I and II expression was studied by immunohistochemistry and HPV typing was performed by general primer- and type-specific primer mediated PCR (GP/TS PCR). Both techniques were performed on paraffin embedded tissue sections. Results show disturbed MHC class I heavy chain expression in CIN I to CIN III, as well as in cervical carcinomas. Upregulated MHC class II expression on dysplastic epithelial cells was also found in the different CIN groups and carcinomas. Prevalence of HPV genotypes increased with the severity of the lesion, mainly due to the contribution of the HPV types 16 and 18. No correlation could be established between the presence of specific HPV genotypes and any MHC expression pattern in the different CIN groups or cervical carcinomas. In some cases these data were confirmed by RNA in situ hybridisation showing HPV 16 E7 transcripts in the same dysplastic/neoplastic cells from which MHC status was determined. The results indicate that local differences may exist in the type of cellular immune response to HPV induced lesions. Images Figure 1 Figure 2 Figure 4 PMID:8390286

  5. Experience-dependent gene expression in adult visual cortex.

    PubMed

    Chen, Jiabin; Yamahachi, Homare; Gilbert, Charles D

    2010-03-01

    Experience-dependent plasticity of the adult visual cortex underlies perceptual learning and recovery of function following central nervous system lesions. To reveal the signal transduction cascades involved in adult cortical plasticity, we utilized a model of remapping of cortical topography following binocular retinal lesions. In this model, the lesion projection zone (LPZ) of primary visual cortex (V1) recovers visually driven activity by the sprouting of horizontal axonal connections originating from the cells in the surrounding region. To explore the molecular mechanism underlying this process, we used gene microarrays from an expression library prepared from Macaque V1. By microarray analysis of gene expression levels in the LPZ and the surrounding region, and subsequent confirmation with Quantitative Real-Time polymerase chain reaction and in situ hybridization, the participation of a number of genes was observed, including the Rho GTPase family. Its role in regulation of cytoskeleton assembly provides a possible link between the alteration of neural activity and cortical functional reorganization. PMID:19571270

  6. Estrogen-Dependent Gene Expression in the Mouse Ovary

    PubMed Central

    Liew, Seng H.; Sarraj, Mai A.; Drummond, Ann E.; Findlay, Jock K.

    2011-01-01

    Estrogen (E) plays a pivotal role in regulating the female reproductive system, particularly the ovary. However, the number and type of ovarian genes influenced by estrogen remain to be fully elucidated. In this study, we have utilized wild-type (WT) and aromatase knockout (ArKO; estrogen free) mouse ovaries as an in vivo model to profile estrogen dependent genes. RNA from each individual ovary (n = 3) was analyzed by a microarray-based screen using Illumina Sentrix Mouse WG-6 BeadChip (45,281 transcripts). Comparative analysis (GeneSpring) showed differential expression profiles of 450 genes influenced by E, with 291 genes up-regulated and 159 down-regulated by 2-fold or greater in the ArKO ovary compared to WT. Genes previously reported to be E regulated in ArKO ovaries were confirmed, in addition to novel genes not previously reported to be expressed or regulated by E in the ovary. Of genes involved in 5 diverse functional processes (hormonal processes, reproduction, sex differentiation and determination, apoptosis and cellular processes) 78 had estrogen-responsive elements (ERE). These analyses define the transcriptome regulated by E in the mouse ovary. Further analysis and investigation will increase our knowledge pertaining to how E influences follicular development and other ovarian functions. PMID:21347412

  7. Lignification in Sugarcane: Biochemical Characterization, Gene Discovery, and Expression Analysis in Two Genotypes Contrasting for Lignin Content1[W

    PubMed Central

    Bottcher, Alexandra; Cesarino, Igor; Brombini dos Santos, Adriana; Vicentini, Renato; Mayer, Juliana Lischka Sampaio; Vanholme, Ruben; Morreel, Kris; Goeminne, Geert; Moura, Jullyana Cristina Magalhães Silva; Nobile, Paula Macedo; Carmello-Guerreiro, Sandra Maria; Antonio dos Anjos, Ivan; Creste, Silvana; Boerjan, Wout; Landell, Marcos Guimarães de Andrade; Mazzafera, Paulo

    2013-01-01

    Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes. PMID:24144790

  8. Human platelet antigen genotyping and expression of CD109 (human platelet antigen 15) mRNA in various human cell types.

    PubMed

    Hwang, Sang Mee; Kim, Mi Jung; Chang, Ho Eun; Hong, Yun Ji; Kim, Taek Soo; Song, Eun Young; Park, Kyoung Un; Song, Junghan; Han, Kyou-Sup

    2013-01-01

    CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34- PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs. PMID:23509816

  9. Iron-dependent gene expression in Actinomyces oris

    PubMed Central

    Mulé, Matthew P.; Giacalone, David; Lawlor, Kayla; Golden, Alexa; Cook, Caroline; Lott, Thomas; Aksten, Elizabeth; O'Toole, George A.; Bergeron, Lori J.

    2015-01-01

    Background Actinomyces oris is a Gram-positive bacterium that has been associated with healthy and diseased sites in the human oral cavity. Most pathogenic bacteria require iron to survive, and in order to acquire iron in the relatively iron-scarce oral cavity A. oris has been shown to produce iron-binding molecules known as siderophores. The genes encoding these siderophores and transporters are thought to be regulated by the amount of iron in the growth medium and by the metal-dependent repressor, AmdR, which we showed previously binds to the promoter of proposed iron-regulated genes. Objective The purpose of this study was to characterize siderophore and associated iron transport systems in A. oris. Design We examined gene expression of the putative iron transport genes fetA and sidD in response to low- and high-iron environments. One of these genes, sidD, encoding a putative Fe ABC transporter protein, was insertionally inactivated and was examined for causing growth defects. To gain a further understanding of the role of iron metabolism in oral diseases, clinical isolates of Actinomyces spp. were examined for the presence of the gene encoding AmdR, a proposed global regulator of iron-dependent gene expression in A. oris. Results When A. oris was grown under iron-limiting conditions, the genes encoding iron/siderophore transporters fetA and sidD showed increased expression. One of these genes (sidD) was mutated, and the sidD::Km strain exhibited a 50% reduction in growth in late log and stationary phase cells in media that contained iron. This growth defect was restored when the sidD gene was provided in a complemented strain. We were able to isolate the AmdR-encoding gene in seven clinical isolates of Actinomyces. When these protein sequences were aligned to the laboratory strain, there was a high degree of sequence similarity. Conclusions The growth of the sidD::Km mutant in iron-replete medium mirrored the growth of the wild-type strain grown in iron

  10. LINC00507 Is Specifically Expressed in the Primate Cortex and Has Age-Dependent Expression Patterns.

    PubMed

    Mills, James D; Ward, Melanie; Chen, Bei Jun; Iyer, Anand M; Aronica, Eleonora; Janitz, Michael

    2016-08-01

    Over the past decade, there has been an increase in the appreciation of the role of non-coding RNA in the development of organism phenotype. It is possible to divide the non-coding elements of the transcriptome into three categories: short non-coding RNAs, circular RNAs and long non-coding RNAs. Long non-coding RNAs are those transcripts that are greater than 200 nts in length and lack any significant open reading frames that produce proteins greater then 100 amino acids. Long intervening non-coding RNAs (lincRNAs) are a subclass of long non-coding RNAs. In contrast to protein coding RNAs, lincRNAs are expressed in a more tissue- and species-specific manner. In particular, many lincRNAs are only conserved amongst higher primates. This coupled with the propensity of many lincRNAs to be expressed in the brain, suggests that they are in fact one of the major drivers of organism complexity. We analysed 39 lincRNAs that are expressed in the frontal cortex and identified LINC00507 as being expressed in a cortex-specific manner in non-human primates and humans. The expression patterns of LINC00507 appear to be age-dependent, suggesting it may be involved in brain development of higher primates. Moreover, the analysis of LINC00507 potential to bind ribosomes revealed that this previously identified non-coding transcript may harbour a micropeptide. PMID:27059230

  11. Chilling-Dependent Release of Seed and Bud Dormancy in Peach Associates to Common Changes in Gene Expression

    PubMed Central

    Arbona, Vicent; Gómez-Cadenas, Aurelio; Llácer, Gerardo; Badenes, María Luisa; Ríos, Gabino

    2012-01-01

    Reproductive meristems and embryos display dormancy mechanisms in specialized structures named respectively buds and seeds that arrest the growth of perennial plants until environmental conditions are optimal for survival. Dormancy shows common physiological features in buds and seeds. A genotype-specific period of chilling is usually required to release dormancy by molecular mechanisms that are still poorly understood. In order to find common transcriptional pathways associated to dormancy release, we analyzed the chilling-dependent expression in embryos of certain genes that were previously found related to dormancy in flower buds of peach. We propose the presence of short and long-term dormancy events affecting respectively the germination rate and seedling development by independent mechanisms. Short periods of chilling seem to improve germination in an abscisic acid-dependent manner, whereas the positive effect of longer cold treatments on physiological dwarfing coincides with the accumulation of phenylpropanoids in the seed. PMID:22590512

  12. A functional haplotype implicated in vulnerability to develop cocaine dependence is associated with reduced PDYN expression in human brain.

    PubMed

    Yuferov, Vadim; Ji, Fei; Nielsen, David A; Levran, Orna; Ho, Ann; Morgello, Susan; Shi, Ruijin; Ott, Jurg; Kreek, Mary Jeanne

    2009-04-01

    Dynorphin peptides and the kappa-opioid receptor are important in the rewarding properties of cocaine, heroin, and alcohol. We tested polymorphisms of the prodynorphin gene (PDYN) for association with cocaine dependence and cocaine/alcohol codependence. We genotyped six single nucleotide polymorphisms (SNPs), located in the promoter region, exon 4 coding, and 3' untranslated region, in 106 Caucasians and 204 African Americans who were cocaine dependent, cocaine/alcohol codependent, or controls. In Caucasians, we found point-wise significant associations of 3'UTR SNPs (rs910080, rs910079, and rs2235749) with cocaine dependence and cocaine/alcohol codependence. These SNPs are in high linkage disequilibrium, comprising a haplotype block. The haplotype CCT was significantly experiment-wise associated with cocaine dependence and with combined cocaine dependence and cocaine/alcohol codependence (false discovery rate, q=0.04 and 0.03, respectively). We investigated allele-specific gene expression of PDYN, using SNP rs910079 as a reporter, in postmortem human brains from eight heterozygous subjects, using SNaPshot assay. There was significantly lower expression for C allele (rs910079), with ratios ranging from 0.48 to 0.78, indicating lower expression of the CCT haplotype of PDYN in both the caudate and nucleus accumbens. Analysis of total PDYN expression in 43 postmortem brains also showed significantly lower levels of preprodynorphin mRNA in subjects having the risk CCT haplotype. This study provides evidence that a 3'UTR PDYN haplotype, implicated in vulnerability to develop cocaine addiction and/or cocaine/alcohol codependence, is related to lower mRNA expression of the PDYN gene in human dorsal and ventral striatum. PMID:18923396

  13. Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin.

    PubMed

    Vicente-Rodríguez, Marta; Herradón, Gonzalo; Ferrer-Alcón, Marcel; Uribarri, María; Pérez-García, Carmen

    2015-07-20

    The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN-/-) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations. PMID:26046300

  14. The Study of HFE Genotypes and Its Expression Effect on Iron Status of Iranian Haemochromatosis, Iron Deficiency Anemia Patients, Iron-Taker and Non Iron-Taker Controls.

    PubMed

    Beiranvand, Elham; Abediankenari, Saeid; Rostamian, Mosayeb; Beiranvand, Behnoush; Naazeri, Saeed

    2015-01-01

    The role of HFE gene mutations or its expression in regulation of iron metabolism of hereditary haemochromatosis (HH) patients is remained controversial. Therefore here the correlation between two common HFE genotype (p.C282Y, p.H63D) and HFE gene expression with iron status in HH, iron deficiency anemia (IDA) and healthy Iranian participants was studied. For this purpose genotype determination was done by polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP). Real-Time PCR was applied for evaluation of HFE gene expression. Biochemical parameters and iron consumption were also assessed. Homozygote p.H63D mutation was seen in all HH patients and p.C282Y was not observed in any member of the population. A significant correlation was observed between serum ferritin (SF) level and gender or age of HH patients. p.H63D homozygote was seen to be able to significantly increase SF and transferrin saturation (TS) level without affecting on liver function. Our results also showed that iron consumption affects on TS level increasing. HFE gene expression level of IDA patients was significantly higher than other groups. Also the HFE gene expression was negatively correlated with TS. Finally, the main result of our study showed that loss of HFE function in HH is not derived from its gene expression inhibition and much higher HFE gene expression might lead to IDA. However we propose repeating of the study for more approval of our finding. PMID:25687342

  15. Measles Virus Genotype D Wild Strains Suppress Interferon-Stimulated Gene Expression More Potently than Laboratory Strains in SiHa Cells.

    PubMed

    Jinushi, Masaru; Yamamoto, Soh; Ogasawara, Noriko; Nagano, Hideki; Hashimoto, Shin; Tsutsumi, Hiroyuki; Himi, Tetsuo; Yokota, Shin-Ichi

    2016-06-01

    Changes in interferon (IFN)-stimulated gene (ISG) expression in cells infected with measles virus (MeV), four wild strains (belonging to different genotypes), and the laboratory strain Edmonston were examined. ISGs [MxA, 2'-5'-oligoadenylate synthetase, and interferon regulatory factor-1] were upregulated in an MeV-infection-induced manner and in an IFN-induced manner. In MeV-infected SiHa cell lines, the MeV infection-induced expression levels were in the order of A>H1>D8>D5>D3. On the other hand, all infected cell lines abolished type I and III IFN-induced ISG expression. However, partial type II IFN-mediated induction was observed in the MeV-infected cells. The wild strain of genotype D3 was the most potent inhibitor of MeV infection-induced and IFN-induced ISG expression and generated the highest titer of infectious viral particles. Edmonston triggered the highest levels of MeV infection-induced ISG expression in SiHa cells and produced the lowest titer of infectious particles. Expression of the viral C protein was associated with suppression of MeV infection-induced and type II IFN-induced ISG expression. PMID:27035543

  16. Non-classical HLA-class I expression in serous ovarian carcinoma: Correlation with the HLA-genotype, tumor infiltrating immune cells and prognosis

    PubMed Central

    Andersson, Emilia; Poschke, Isabel; Villabona, Lisa; Carlson, Joseph W; Lundqvist, Andreas; Kiessling, Rolf; Seliger, Barbara; Masucci, Giuseppe V

    2016-01-01

    In our previous studies, we have shown that patients with serous ovarian carcinoma in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A*02 genotype. This represent a stronger prognostic factor than loss or downregulation of the MHC class I heavy chain (HC) on tumor cells. In this study, we investigated the expression of the non-classical, immune tolerogenic HLA -G and -E on the tumor cells along with the infiltration of immune cells in the tumor microenvironment. FFPE primary tumors from 72 patients with advanced stages of serous adenocarcinoma and metastatic cells present in ascites fluid from 8 additional patients were included in this study. Both expression of HLA-G and aberrant expression of HLA-E were correlated to a significant worse prognosis in patients with HLA-A*02, but not with different HLA genotypes. Focal cell expression of HLA-G correlated to a site-specific downregulation of classical MHC class I HC products and aberrant HLA-E expression, showing a poor survival. HLA-G was more frequently expressed in metastatic cells than in primary tumor lesions and the expression of HLA-G inversely correlated with the frequency of tumor infiltrating immune cells. All these parameters can contribute together to identify and discriminate subpopulations of patients with extremely poor prognosis and can give them the opportunity to receive, and benefit of individually tailored treatments. PMID:26942060

  17. The Role of CYP2C8 and CYP2C9 Genotypes in Losartan-Dependent Inhibition of Paclitaxel Metabolism in Human Liver Microsomes.

    PubMed

    Mukai, Yuji; Senda, Asuna; Toda, Takaki; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2016-06-01

    The aim of the present study was to further investigate a previously identified metabolic interaction between losartan and paclitaxel, which is one of the marker substrates of CYP2C8, by using human liver microsomes (HLMs) from donors with different CYP2C8 and CYP2C9 genotypes. Although CYP2C8 and CYP2C9 exhibit genetic linkage, previous studies have yet to determine whether losartan or its active metabolite, EXP-3174 which is specifically generated by CYP2C9, is responsible for CYP2C8 inhibition. Concentrations of 6α-hydroxypaclitaxel and EXP-3174 were measured by high-performance liquid chromatography after incubations with paclitaxel, losartan or EXP-3174 in HLMs from seven donors with different CYP2C8 and CYP2C9 genotypes. The half maximal inhibitory concentration (IC50 ) values were not fully dependent on CYP2C8 genotypes. Although the degree of inhibition was small, losartan significantly inhibited the production of 6α-hydroxypaclitaxel at a concentration of 1 μmol/L in only HL20 with the CYP2C8*3/*3 genotype. HLMs with either CYP2C9*2/*2 or CYP2C9*1/*3 exhibited a lower losartan intrinsic clearance (Vmax /Km ) than other HLMs including those with CYP2C9*1/*1 and CYP2C9*1/*2. Significant inhibition of 6α-hydroxypaclitaxel formation by EXP-3174 could only be found at levels that were 50 times higher (100 μmol/L) than the maximum concentration generated in the inhibition study using losartan. These results suggest that the metabolic interaction between losartan and paclitaxel is dependent on losartan itself rather than its metabolite and that the CYP2C8 inhibition by losartan is not affected by the CYP2C9 genotype. Further study is needed to define the effect of CYP2C8 genotypes on losartan-paclitaxel interaction. PMID:26551762

  18. Association of Interleukin-6 and Interleukin-10 Genotypes With Radiographic Damage in Rheumatoid Arthritis Is Dependent on Autoantibody Status

    PubMed Central

    Marinou, I; Healy, J; Mewar, D; Moore, D J; Dickson, M C; Binks, M H; Montgomery, D S; Walters, K; Wilson, A G

    2007-01-01

    Objective Recent evidence has highlighted a major genetic contribution to radiographic damage in rheumatoid arthritis (RA). The objective of this study was to determine whether genetic variants in the loci for interleukin-1 (IL-1), IL-6, IL-10, protein tyrosine phosphatase N22 (PTPN22), and selenoprotein S are associated with radiographic damage. Methods Modified Larsen scores of radiographic damage were determined in a cross-sectional population of patients with RA (n = 964). Rheumatoid factor (RF) and anti–cyclic citrullinated peptide (anti-CCP) were also assayed. The Kruskal-Wallis nonparametric test was used to compare median radiographic damage scores across genotype groups, followed by the Cuzick nonparametric test for trend to assess gene-dose effects. Results An allele-dose association of IL-6 −174G with increasing radiographic damage was present (P = 0.005), but only in patients who were RF positive (P = 0.004) or anti-CCP positive (P = 0.01). Patients with the IL-10 −592CC genotype had more extensive radiographic damage than did those with the AC or AA genotype (P = 0.006), but this was observed only among patients who were RF negative (P = 0.002) or anti-CCP negative (P = 0.002). However, RF status and anti-CCP status were not associated with the IL-6 or IL-10 genotype. No other genetic associations were detected, apart from a marginal association of PTPN22 +1858T with increased radiographic damage. Conclusion The reported associations of IL-6 −174G with high IL-6 production and IL-10 −592 with low IL-10 production and our own results support a role of genetically determined dysregulated cytokine production in disease severity. The lack of association of these genotypes with RF and anti-CCP antibody status suggests that they act downstream of autoantibody production. We conclude that IL-6 and IL-10 genotypes may be useful in predicting disease severity in autoantibody-positive and autoantibody-negative patients, respectively. PMID:17665434

  19. Large-scale analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner–toward the identification of candidate genes for marker assisted-selection

    PubMed Central

    2014-01-01

    Background A successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach. Results The microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars. Conclusions Our results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response. PMID

  20. Allelic and genotypic associations of DRD2 TaqI A polymorphism with heroin dependence in Spanish subjects: a case control study

    PubMed Central

    Perez de los Cobos, Jose; Baiget, Montserrat; Trujols, Joan; Sinol, Nuria; Volpini, Victor; Banuls, Enrique; Calafell, Francesc; Luquero, Elena; del Rio, Elisabeth; Alvarez, Enric

    2007-01-01

    Background Conflicting associations with heroin dependence have been found involving the A1 allele of dopamine D2 receptor gene (DRD2) TaqI A polymorphism. Methods We compared two samples of unrelated Spanish individuals, all of European origin: 281 methadone-maintained heroin-dependent patients (207 males and 74 females) who frequently used non-opioid substances, and 145 control subjects (98 males and 47 females). Results The A1-A1 genotype was detected in 7.1% of patients and 1.4% of controls (P = 0.011, odds ratio = 5.48, 95% CI 1.26–23.78). Although the A1 allele was not associated with heroin dependence in the entire sample, the frequency of A1 allele was higher in male patients than in male controls (24.4% vs. 16.3%, P = 0.024, odds ratio = 1.65, 95% CI 1.07–2.57). A logistic regression analysis showed an interaction between DRD2 alleles and gender (odds ratio = 1.77, 95% CI 1.15–2.70). Conclusion Our results indicate that, in Spanish individuals, genotypes of the DRD2 TaqI A polymorphism contribute to variations in the risk of heroin dependence, while single alleles contribute only in males. PMID:17543096

  1. Genotyping methods.

    PubMed

    Tümmler, Burkhard

    2014-01-01

    Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray. Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20-70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa. PMID:24818895

  2. Chamber-dependent circadian expression of cardiac natriuretic peptides.

    PubMed

    Goetze, Jens Peter; Georg, Birgitte; Jørgensen, Henrik L; Fahrenkrug, Jan

    2010-02-25

    Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have important local functions within the myocardium, where they protect against accelerated fibrosis. As circadian expression of cardiac natriuretic peptides could be of importance in local cardiac protection against disease, we examined the diurnal changes of the mRNAs encoding ANP, BNP, and their common receptor NPR-A in atrial and ventricular myocardium. Forty eight mice were killed at the following ZT times: 4, 8, 12, 16, 20, and 24, where ZT designates Zeitgeber; ZT 0 corresponds to lights ON and ZT 12 corresponds to lights OFF. Eight animals (4 males and 4 females) were included at each time point. Another 48 animals were killed during the second cycle of dark/dark (designated Circadian Time or CT: CT 4, CT 8, CT 12, CT 16, CT 20, and CT 24). The cellular contents of the clock genes Per1 and Bmal1 as well as ANP, BNP, and their common receptor (NPR-A) were determined using RT-PCR. Per1 and Bmal1 mRNA contents oscillated in antiphase in both atrial and ventricular regions, where Bmal1 mRNA peaked 12h out of phase relative to Per1 mRNA. ANP and NPR-A atrial mRNA contents revealed borderline significant diurnal changes, whereas ventricular BNP mRNA contents exhibited pronounced oscillation during constant darkness with nadir at CT 12 (P<0.0001). In conclusion, we report a chamber-dependent circadian profile of cardiac BNP mRNA contents, which is not paralleled by the related ANP gene. Our findings suggest that the BNP mRNA pattern could be associated with increased cardiac susceptibility and response to disease. PMID:20035806

  3. Genotypes and haplotypes of the methyl-CpG-binding domain 2 modify breast cancer risk dependent upon menopausal status

    PubMed Central

    Zhu, Yong; Brown, Heather N; Zhang, Yawei; Holford, Theodore R; Zheng, Tongzhang

    2005-01-01

    Introduction MBD2, the gene encoding methyl-CpG-binding domain (MBD)2, is a major methylation related gene and functions as a transcriptional repressor that can specifically bind to the methylated regions of other genes. MBD2 may also mediate gene activation because of its potential DNA demethylase activity. The present case-control study investigated associations between two single nucleotide polymorphisms (SNPs) in the MBD2 gene and breast cancer risk. Methods DNA samples from 393 Caucasian patients with breast cancer (cases) and 436 matched control individuals, collected in a recently completed breast cancer case–control study conducted in Connecticut, were included in the study. Because no coding SNPs were found in the MBD2 gene, one SNP in the noncoding exon (rs1259938) and another in the intron 3 (rs609791) were genotyped. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate cancer risk associated with the variant genotypes and the reconstructed haplotypes. Results The variant genotypes at both SNP loci were significantly associated with reduced risk among premenopausal women (OR = 0.41 for rs1259938; OR = 0.54 for rs609791). Further haplotype analyses showed that the two rare haplotypes (A-C and A-G) were significantly associated with reduced breast cancer risk (OR = 0.40, 95% CI = 0.20–0.83 for A-C; OR = 0.47, 95% CI = 0.26–0.84 for A-G) in premenopausal women. No significant associations were detected in the postmenopausal women and the whole population. Conclusion Our results demonstrate a role for the MBD2 gene in breast carcinogenesis in premenopausal women. These findings suggest that genetic variations in methylation related genes may potentially serve as a biomarker in risk estimates for breast cancer. PMID:16168120

  4. IL36RN Mutations Affect Protein Expression and Function: A Basis for Genotype-Phenotype Correlation in Pustular Diseases.

    PubMed

    Tauber, Marie; Bal, Elodie; Pei, Xue-Yuan; Madrange, Marine; Khelil, Amel; Sahel, Houria; Zenati, Akila; Makrelouf, Mohamed; Boubridaa, Khaled; Chiali, Amel; Smahi, Naima; Otsmane, Farida; Bouajar, Bakar; Marrakchi, Slaheddine; Turki, Hamida; Bourrat, Emmanuelle; Viguier, Manuelle; Hamel, Yamina; Bachelez, Hervé; Smahi, Asma

    2016-09-01

    Homozygous or compound heterozygous IL36RN gene mutations underlie the pathogenesis of psoriasis-related pustular eruptions including generalized pustular psoriasis, palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau, and acute generalized exanthematous pustular eruption. We identified two unreported IL36RN homozygous mutations (c.41C>A/p.Ser14X and c.420_426del/p.Gly141MetfsX29) in patients with familial generalized pustular psoriasis. We analyzed the impact of a spectrum of IL36RN mutations on IL-36 receptor antagonist protein by using site-directed mutagenesis and expression in HEK293T cells. This enabled us to differentiate null mutations with complete absence of IL-36 receptor antagonist (the two previously unreported mutations, c.80T>C/p.Leu27Pro, c.28C>T/p.Arg10X, c.280G>T/p.Glu94X, c.368C>G/p.Thr123Arg, c.368C>T/p.Thr123Met, and c.227C>T/p.Pro76Leu) from mutations with decreased (c.95A>G/p.His32Arg, c.142C>T/p.Arg48Trp, and c.308C>T/p.Ser113Leu) or unchanged (c.304C>T/p.Arg102Trp and c.104A>G/p.Lys35Arg) protein expression. Functional assays measuring the impact of mutations on the capacity to repress IL-36-dependent activation of the NF-κB pathway showed complete functional impairment for null mutations, whereas partial or no impairment was observed for other mutations considered as hypomorphic. Finally, null mutations were associated with severe clinical phenotypes (generalized pustular psoriasis, acute generalized exanthematous pustular eruption), whereas hypomorphic mutations were identified in both localized (palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau) and generalized variants. These results provide a preliminary basis for genotype-phenotype correlation in patients with deficiency of the IL-36Ra (DITRA), and suggest the involvement of other factors in the modulation of clinical expression. PMID:27220475

  5. Aging, Alzheimer's, and APOE genotype influence the expression and neuronal distribution patterns of microtubule motor protein dynactin-P50

    PubMed Central

    Aboud, Orwa; Parcon, Paul A.; DeWall, K. Mark; Liu, Ling; Mrak, Robert E.; Griffin, W. Sue T.

    2015-01-01

    Reports from neural cell cultures and experimental animal studies provide evidence of age- and disease-related changes in retrograde transport of spent or misfolded proteins destined for degradation or recycling. However, few studies address these issues in human brain from those who either age without dementia and overt neuropathology, or succumb to Alzheimer's; especially as such propensity may be influenced by APOE genotype. We studied the expression and distribution of the dynein subunit dynactin-P50, the β amyloid precursor protein (βAPP), and hyperphosphorylated tau (P-tau) in tissues and tissue sections of brains from non-demented, neuropathology-free patients and from Alzheimer patients, with either APOE ε3,3 or APOE ε4,4. We found that advanced age in patients without dementia or neuropathological change was associated with coordinated increases in dynactin-P50 and βAPP in neurons in pyramidal layers of the hippocampus. In contrast, in Alzheimer's, βAPP and dynactin were significantly reduced. Furthermore, the dynactin-P50 and βAPP that was present was located primarily in dystrophic neurites in Aβ plaques. Tissues from Alzheimer patients with APOE ε3,3 had less P-tau, more βAPP, dynactin-P50, and synaptophysin than did tissues from Alzheimer patients carrying APOE ε4,4. It is logical to conclude, then, that as neurons age successfully, there is coordination between retrograde delivery and maintenance and repair, as well as between retrograde delivery and degradation and/or recycling of spent proteins. The buildup of proteins slated for repair, synaptic viability, transport, and re-cycling in neuron soma and dystrophic neurites suggest a loss of this coordination in Alzheimer neurons. Inheritance of APOE ε3,3 rather than APOE ε4,4, is associated with neuronal resilience, suggestive of better repair capabilities, more synapses, more efficient transport, and less hyperphosphorylation of tau. We conclude that even in disease the ε3 allele is

  6. Viral load is associated with abnormal serum levels of micronutrients and glutathione and glutathione-dependent enzymes in genotype 3 HCV patients

    PubMed Central

    Razzaq, Zarish; Malik, Arif

    2014-01-01

    Background Oxidative stress in hepatitis C patients has been linked to hepatitis C virus. We verified this assumption in HCV genotype 3 patients by detecting the relationship between viral load and certain specific oxidative stress markers like Cu, Mn, Fe, Se, Zn and glutathione and glutathione-dependent enzymes. Method Subjects (n = 200, average age 24 years) with quantitative HCV RNA polymerase chain reaction-proven genotype 3 hepatitis C were simultaneously evaluated. Cu, Mn, Fe, Se and Zn serum levels were by using atomic absorption spectrophotometer. Internationally accepted methods were used for viral load quantification of glutathione, GR and Gpx serum levels. Result There was a significant correlation between HCV viral load and studied parameters. With the increase of viral load from mild group (200,000–1,000,000 copies/ml) to severe group (5,000,000–25,000,000 copies/ml) the serum levels of Cu, Mn, Zn, and Fe and glutathione reductase were found to be abnormally high. However, in severe viral load group serum concentration of Se and glutathione was less than the healthy controls. Conclusion As a significant correlation was detected between the study parameters in genotype 3 HCV patients, it is concluded that the studied micronutrients and glutathione and glutathione-dependent enzymes are the biomolecular targets of HCV to induce oxidative stress. General significance Constant monitoring and regulation of the recommended biomolecular targets of HCV can improve the plight of more than 170 million patients suffering from hepatitis C virus around the globe. PMID:26674880

  7. Short tandem repeat (STR) genotyping of keratinised hair. Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles.

    PubMed

    McNevin, Dennis; Wilson-Wilde, Linzi; Robertson, James; Kyd, Jennelle; Lennard, Chris

    2005-10-29

    A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFlSTR Profiler Plus PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris-HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFlSTR Profiler Plus STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The

  8. Physiochemical and thermal characteristics of starch isolated from a waxy wheat genotype exhibiting partial expression of Wx proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A unique wheat genotype carrying waxy type allelic composition at the Wx loci, Gunji-1, was developed and its starch properties were evaluated in comparison to parental waxy and wild type wheat varieties. Gunji-1 was null in all three of the Wx genes, but exhibited a lower level of Wx proteins than ...

  9. Leaf aquaporin transcript abundance in peanut genotypes diverging in expression of the limited-transpiration trait when subjected to differing vapor pressure deficits and aquaporin inhibitors.

    PubMed

    Devi, M Jyostna; Sinclair, Thomas R; Jain, Mukesh; Gallo, Maria

    2016-04-01

    A plant trait currently being exploited to decrease crop yield loss under water-deficit conditions is limited-transpiration rate (TRlim ) under high atmospheric vapor pressure deficit (VPD) conditions. Although limited genotype comparisons for the TRlim trait have been performed in peanut (Arachis hypogaea), no detailed study to describe the basis for this trait in peanut has been reported. Since it has been hypothesized that the TRlim trait may be a result of low leaf hydraulic conductance associated with aquaporins (AQPs), the first objective of this study was to examine a possible correlation of TRlim to leaf AQP transcriptional profiles in six peanut cultivars. Five of the studied cultivars were selected because they expressed TRlim while the cultivar York did not. Transcripts of six AQPs were measured. Under exposure to high vapor pressure deficit, cultivar C 76-16 had decreased AQP transcript abundance for four of the six AQPs but in York only one AQP had decreased abundance. The second objective was to explore the influence of AQP inhibitors mercury and silver on expression of TRlim and AQP transcription profiles. Quantitative RT-PCR data were compared in cultivars York and C 76-16, which had the extreme response in TR to VPD. Inhibitor treatment resulted in increased abundance of AQP transcripts in both. The results of these experiments indicate that AQP transcript abundance itself may not be useful in identifying genotypes expressing the TRlim trait under high VPD conditions. PMID:26303261

  10. Plasma homocysteine in adolescents depends on the interaction between methylenetetrahydrofolate reductase genotype, lipids and folate: a seroepidemiological study

    PubMed Central

    Gil-Prieto, Ruth; Hernández, Valentín; Cano, Beatriz; Oya, Manuel; Gil, Ángel

    2009-01-01

    Background Many publications link high homocysteine levels to cardiovascular disease. In Spain there is little information on the prevalence of hyperhomocysteinaemia and associated vitamin factors among the general population, and less still among children. Cardiovascular risk factors in the childhood population may be related to the appearance of cardiovascular disease at adult age. The aim of this study is to establish a definition of hyperhomocysteinaemia in adolescents and to analyze the influence of vitamin and metabolic factors in homocysteine levels in this population group. Methods Descriptive, cross-sectional epidemiological study to estimate serum homocysteine, vitamin B12 and folate levels, as well as plasma total, HDL- and LDL- cholesterol in a schoolgoing population aged 13 to 17 years in Madrid, Spain. Spearman correlation analysis was performed to ascertain quantitative comparison, Pearson's χ2 test (frequency < 5, Fisher) was used for comparison of prevalences, Mann-Whitney U and Kruskal-Wallis test were used for comparison of means and Bonferroni correction was used for post-hoc tests. A multivariate logistic regression model was performed in the multivariate analysis. Results Based on the classic values for definition of hyperhomocysteinaemia in adults, prevalence of hyperhomocysteinaemia in the study population was: 1.26% for 15 μmol/L; and 2.52% for 12 μmol/L. Deficits in HDL cholesterol and serum folate levels yielded adjusted Odds Ratios (OR) for hyperhomocysteinemia of 2.786, 95% CI (1.089-7.126), and 5.140, 95% CI (2.347-11.256) respectively. Mutation of the methylenetetrahydrofolate reductase (MTHFR) C677T genotype also raises the risk of hyperhomocysteinaemia (CC→CT: OR = 2.362; 95% CI (1.107-5.042) CC→TT: OR = 6.124, 95% CI (2.301-16.303)) Conclusion A good definition of hyperhomocysteinaemia in adolescents is the 90th percentile, equivalent to 8.23 μmol/L. Risk factors for hyperhomocysteinaemia are cHDL and folate deficiency, and

  11. Tissue Dependent Limited Pleiotropy Affects Gene Expression in Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-synonymous coding mutations in a gene change the resulting protein no matter where it is expressed, but the effects of cis-regulatory mutations could be spatially or temporally limited, a phenomenon termed limited pleiotropy. Here we report the genome-wide occurrence of limited pleiotropy of cis...

  12. Influences of Gestational Obesity on Associations between Genotypes and Gene Expression Levels in Offspring following Maternal Gastrointestinal Bypass Surgery for Obesity

    PubMed Central

    Guénard, Frédéric; Lamontagne, Maxime; Bossé, Yohan; Deshaies, Yves; Cianflone, Katherine; Kral, John G.; Marceau, Picard; Vohl, Marie-Claude

    2015-01-01

    Maternal obesity and excess gestational weight gain with compromised metabolic fitness predispose offspring to lifelong obesity and its comorbidities. We demonstrated that compared to offspring born before maternal gastrointestinal bypass surgery (BMS) those born after (AMS) were less obese, with less cardiometabolic risk reflected in the expression and methylation of diabetes, immune and inflammatory pathway genes. Here we examine relationships between gestational obesity and offspring gene variations on expression levels. Methods Whole-genome genotyping and gene expression analyses in blood of 22 BMS and 23 AMS offspring from 19 mothers were conducted using Illumina HumanOmni-5-Quad and HumanHT-12 v4 Expression BeadChips, respectively. Using PLINK we analyzed interactions between offspring gene variations and maternal surgical status on offspring gene expression levels. Altered biological functions and pathways were identified and visualized using DAVID and Ingenuity Pathway Analysis. Results Significant interactions (p ≤ 1.22x10-12) were found for 525 among the 16,060 expressed transcripts: 1.9% of tested SNPs were involved. Gene function and pathway analysis demonstrated enrichment of transcription and of cellular metabolism functions and overrepresentation of cellular stress and signaling, immune response, inflammation, growth, proliferation and development pathways. Conclusion We suggest that impaired maternal gestational metabolic fitness interacts with offspring gene variations modulating gene expression levels, providing potential mechanisms explaining improved cardiometabolic risk profiles of AMS offspring related to ameliorated maternal lipid and carbohydrate metabolism. PMID:25603303

  13. Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

    PubMed Central

    Bratt, Jennifer M.; Williams, Keisha; Rabowsky, Michelle F.; Last, Michael S.; Franzi, Lisa M.; Last, Jerold A.; Kenyon, Nicholas J.

    2010-01-01

    Objectives and Design. The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia. PMID:20953358

  14. [Dependence of the genotypic characteristics of Acidithiobacillus ferrooxidans on the physical, chemical, and electrophysical properties of pyrites].

    PubMed

    Tupikina, O V; Kondrat'eva, T F; Karavaĭko, G I

    2005-01-01

    This study focused on the effect of physical, chemical, and electrophysical properties of two pyrites, pyrite 1, which had hole-type (p-type) conductivity, and pyrite 2, with electron-type (n-type) conductivity, on the genotypic characteristics of Acidithiobacillus ferrooxidans strains TFV-1 and TFBk, which were isolated from different substrates. After the adaptation of the strains to the pyrites at a pulp density of 1%, pulsed-field electrophoresis revealed changes in the chromosomal DNA of strain TFV-1 adapted to pyrite 1 and strain TFBk adapted to either of the pyrite types. In pyrite-adapted strain TFBk, the plasmid composition was the same as after growth on a medium containing ferrous iron, whereas, in strain TFV-1, changes in plasmid sizes or both in plasmid sizes and plasmid number occurred. After an increase in the density of the pyrite 2 pulp from 1 to 10%, the plasmid number increased from three to four, and, after an increase in the density of the pyrite 1 pulp from 1 to 7%, the plasmid number increased from two to six. PMID:16315978

  15. In Utero and Lactational Exposure to PCBs in Mice: Adult Offspring Show Altered Learning and Memory Depending on Cyp1a2 and Ahr Genotypes

    PubMed Central

    Curran, Christine P.; Genter, Mary Beth; Patel, Krishna V.; Schaefer, Tori L.; Skelton, Matthew R.; Williams, Michael T.; Vorhees, Charles V.

    2011-01-01

    Background: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2)-uninduced basal levels and > 12-fold variability in aryl hydrocarbon receptor (AHR)affinity; because CYP1A2 is known to sequester coplanar PCBs and because AHR ligands include coplanar PCBs, both genotypes can affect PCB response. Objectives: We aimed to develop a mouse paradigm with extremes in Cyp1a2 and Ahr genotypes to explore genetic susceptibility to PCB-induced developmental neurotoxicity using an environmentally relevant mixture of PCBs. Methods: We developed a mixture of eight PCBs to simulate human exposures based on their reported concentrations in human tissue, breast milk, and food supply. We previously characterized specific differences in PCB congener pharmacokinetics and toxicity, comparing high-affinity–AHR Cyp1a2 wild-type [Ahrb1_Cyp1a2(+/+)], poor-affinity–AHR Cyp1a2 wild-type [Ahrd_Cyp1a2(+/+)], and high-affinity–AHR Cyp1a2 knockout [Ahrb1_Cyp1a2(–/–)] mouse lines [Curran CP, Vorhees CV, Williams MT, Genter MB, Miller ML, Nebert DW. 2011. In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes. Toxicol Sci 119:189–208]. Dams received a mixture of three coplanar and five noncoplanar PCBs on gestational day 10.5 and postnatal day (PND) 5. In the present study we conducted behavioral phenotyping of exposed offspring at PND60, examining multiple measures of learning, memory, and other behaviors. Results: We observed the most significant deficits in response to PCB treatment in Ahrb1_Cyp1a2(–/–) mice, including impaired novel object recognition and increased failure rate in the Morris water maze. However, all PCB-treated genotypes showed significant differences on

  16. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages

    PubMed Central

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A.; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

  17. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    PubMed

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but

  18. Dynamic multiphosphorylation passwords for activity-dependent gene expression.

    PubMed

    Deisseroth, Karl; Tsien, Richard W

    2002-04-11

    Synapse-to-nucleus signaling leading to CREB-mediated transcription is important for neuronal plasticity. Nuclear CREB phosphorylation at Ser133 allows convergence of multiple kinase pathways driven by neuronal activity and links them to transcriptional activation. But, can various pathways share a common effector mechanism (phosphorylating Ser133) while generating distinct patterns of gene expression? We review three Neuron articles that highlight novel ways Ca(2+) signals can trigger multiple phosphorylation events working in combination to control CREB and its interaction with coactivator molecules. PMID:11970860

  19. Children’s Inferential Styles, 5-HTTLPR Genotype, and Maternal Expressed Emotion-Criticism: An Integrated Model for the Intergenerational Transmission of Depression

    PubMed Central

    Gibb, Brandon E.; Uhrlass, Dorothy J.; Grassia, Marie; Benas, Jessica S.; McGeary, John

    2010-01-01

    We tested a model for the intergenerational transmission of depression integrating specific genetic (5-HTTLPR), cognitive (inferential style), and environmental (mother depressive symptoms and expressed-emotion criticism) risk factors. Supporting the hypothesis that maternal depression is associated with elevated levels of stress in children’s lives, mothers with a history of major depressive disorder (MDD) exhibited higher depressive symptoms across a 6-month multi-wave follow-up than mothers with no depression history. In addition, partially supporting our hypothesis, levels of maternal criticism during the follow-up were significantly related to mothers’ current depressive symptoms, but not history of MDD. Finally, we found support for an integrated gene × cognition × environment model of risk. Specifically, among children with negative inferential styles regarding their self-characteristics, there was a clear dose response of 5-HTTLPR genotype moderating the relation between maternal criticism and children’s depressive symptoms, with the highest depressive symptoms during the follow-up observed among children carrying two copies of the 5-HTTLPR lower expressing alleles (S or LG) who also exhibited negative inferential styles for self-characteristics and who experienced high levels of EE-Crit. In contrast, children with positive inferential styles exhibited low depressive symptoms regardless of 5-HTTLPR genotype or level of maternal criticism. PMID:19899843

  20. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure.

    PubMed

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  1. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure

    PubMed Central

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S.; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  2. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  3. Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins

    PubMed Central

    Braun, Ralf J.

    2015-01-01

    Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders. PMID:25814926

  4. Trait Specific Expression Profiling of Salt Stress Responsive Genes in Diverse Rice Genotypes as Determined by Modified Significance Analysis of Microarrays

    PubMed Central

    Hossain, Mohammad R.; Bassel, George W.; Pritchard, Jeremy; Sharma, Garima P.; Ford-Lloyd, Brian V.

    2016-01-01

    Stress responsive gene expression is commonly profiled in a comparative manner involving different stress conditions or genotypes with contrasting reputation of tolerance/resistance. In contrast, this research exploited a wide natural variation in terms of taxonomy, origin and salt sensitivity in eight genotypes of rice to identify the trait specific patterns of gene expression under salt stress. Genome wide transcptomic responses were interrogated by the weighted continuous morpho-physiological trait responses using modified Significance Analysis of Microarrays. More number of genes was found to be differentially expressed under salt stressed compared to that of under unstressed conditions. Higher numbers of genes were observed to be differentially expressed for the traits shoot Na+/K+, shoot Na+, root K+, biomass and shoot Cl−, respectively. The results identified around 60 genes to be involved in Na+, K+, and anion homeostasis, transport, and transmembrane activity under stressed conditions. Gene Ontology (GO) enrichment analysis identified 1.36% (578 genes) of the entire transcriptome to be involved in the major molecular functions such as signal transduction (>150 genes), transcription factor (81 genes), and translation factor activity (62 genes) etc., under salt stress. Chromosomal mapping of the genes suggests that majority of the genes are located on chromosomes 1, 2, 3, 6, and 7. The gene network analysis showed that the transcription factors and translation initiation factors formed the major gene networks and are mostly active in nucleus, cytoplasm and mitochondria whereas the membrane and vesicle bound proteins formed a secondary network active in plasma membrane and vacuoles. The novel genes and the genes with unknown functions thus identified provide picture of a synergistic salinity response representing the potentially fundamental mechanisms that are active in the wide natural genetic background of rice and will be of greater use once their roles

  5. Extracellular matrix protein expression is brain region dependent.

    PubMed

    Dauth, Stephanie; Grevesse, Thomas; Pantazopoulos, Harry; Campbell, Patrick H; Maoz, Ben M; Berretta, Sabina; Parker, Kevin Kit

    2016-05-01

    In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc. PMID:26780384

  6. Expression of tenascin during carcinogenesis and involution of hormone-dependent tissues.

    PubMed

    Vollmer, G

    1994-01-01

    Cytotactin/tenascin/hexabrachion, now referred to as tenascin-C (TN-C), is a hexameric glycoprotein of the extracellular matrix of mesenchymal tissue constituents. A high expression was found in embryonic development and during carcinogenesis of almost all organs. TN-C expression by the mesenchyme thereby appears to be induced by paracrine-acting, epithelial-derived (growth) factors. In normal adult organs there is little, if any, TN-C expression. In the human endometrium for instance, tenascin expression is low in the normal proliferative endometrium and undetectable in the normal secretory endometrium. In this paper, the appearance and expression of TN-C in hormone-dependent tissues, regressing hormone-dependent tissues, and tumors of the endometrium, breast and prostate is reviewed. Further, the regulation of TN-C expression is summarized and possible functions of TN-C during regression and carcinogenesis of hormone-dependent tissues are discussed. PMID:7544587

  7. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    SciTech Connect

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  8. Role of polymorphic Fc gamma receptor IIIa and EGFR expression level in cetuximab mediated, NK cell dependent in vitro cytotoxicity of head and neck squamous cell carcinoma cells.

    PubMed

    López-Albaitero, Andrés; Lee, Steve C; Morgan, Sarah; Grandis, Jennifer R; Gooding, William E; Ferrone, Soldano; Ferris, Robert L

    2009-11-01

    Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P < 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P < 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient

  9. Role of polymorphic Fc gamma receptor IIIa and EGFR expression level in cetuximab mediated, NK cell dependent in vitro cytotoxicity of head and neck squamous cell carcinoma cells

    PubMed Central

    López-Albaitero, Andrés; Lee, Steve C.; Morgan, Sarah; Grandis, Jennifer R.; Gooding, William E.; Ferrone, Soldano

    2012-01-01

    Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10–20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients’ differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcγR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcγR polymorphism. Effector cells expressing the FcγR IIIa-158 VV allele were significantly (P < 0.0001) more effective than those expressing FcγR IIIa VF and VV alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P < 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcγR IIIa 158 FF expressing NK cells. The importance of the FcγR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab

  10. A Calcium-dependent switch in a CREST-BRG1 complex regulates activity-dependent gene expression

    PubMed Central

    Qiu, Zilong; Ghosh, Anirvan

    2009-01-01

    Activity-dependent gene expression plays an important role in mediating the effects of sensory experience on nervous system development and function. While several activity-dependent transcription factors have been identified, the mechanism by which calcium signaling converts a promoter from a silenced to an active state is not well understood. Here we show that a CREST-BRG1 complex plays a critical role in regulating promoter activation by orchestrating a calcium-dependent release of a repressor complex, and a recruitment of an activator complex. In resting neurons, transcription of the c-fos promoter is inhibited by BRG1-dependent recruitment of a phospho-Rb-HDAC repressor complex. Upon calcium influx, Rb becomes dephosphorylated at Serine 795 by Calcineurin, which leads to release of the repressor complex. At the same time there is increased recruitment of CBP to the promoter by a CREST-dependent mechanism, which leads to transcriptional activation. The CREST-BRG1 also binds to the NR2B promoter and activity-dependent induction of NR2B expression involves a release of HDAC1 and recruitment of CBP, suggesting that this mechanism may be generally involved in regulating calcium-dependent transcription of neuronal genes. PMID:19081374

  11. Growth-rate dependent global effects on gene expression in bacteria

    PubMed Central

    Klumpp, Stefan; Zhang, Zhongge; Hwa, Terence

    2010-01-01

    Summary Bacterial gene expression depends not only on specific regulations but also directly on bacterial growth, because important global parameters such as the abundance of RNA polymerases and ribosomes are all growth-rate dependent. Understanding these global effects is necessary for a quantitative understanding of gene regulation and for the robust design of synthetic genetic circuits. The observed growth-rate dependence of constitutive gene expression can be explained by a simple model using the measured growth-rate dependence of the relevant cellular parameters. More complex growth dependences for genetic circuits involving activators, repressors and feedback control were analyzed, and salient features were verified experimentally using synthetic circuits. The results suggest a novel feedback mechanism mediated by general growth-dependent effects and not requiring explicit gene regulation, if the expressed protein affects cell growth. This mechanism can lead to growth bistability and promote the acquisition of important physiological functions such as antibiotic resistance and tolerance (persistence). PMID:20064380

  12. Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM

    PubMed Central

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren; Lorey, Thomas S.; Sahasrabuddhe, Vikrant V.; Lamere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Castle, Philip E.

    2014-01-01

    Objective Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM. Methods A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs). Results For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P< 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3. Conclusion Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management. PMID:23018436

  13. Apolipoprotein E Genotype-Dependent Paradoxical Short-Term Effects of {sup 56}Fe Irradiation on the Brain

    SciTech Connect

    Haley, Gwendolen E.; Villasana, Laura; Dayger, Catherine; Davis, Matthew J.; Raber, Jacob

    2012-11-01

    Purpose: In humans, apolipoprotein E (apoE) is encoded by three major alleles ({epsilon}2, {epsilon}3, and {epsilon}4) and, compared to apoE3, apoE4 increases the risk of developing Alzheimer disease and cognitive impairments following various environmental challenges. Exposure to irradiation, including that of {sup 56}Fe, during space missions poses a significant risk to the central nervous system, and apoE isoform might modulate this risk. Methods and Materials: We investigated whether apoE isoform modulates hippocampus-dependent cognitive performance starting 2 weeks after {sup 56}Fe irradiation. Changes in reactive oxygen species (ROS) can affect cognition and are induced by irradiation. Therefore, after cognitive testing, we assessed hippocampal ROS levels in ex vivo brain slices, using the ROS-sensitive fluorescent probe, dihydroethidium (DHE). Brain levels of 3-nitrotyrosine (3-NT), CuZn superoxide dismutase (CuZnSOD), extracellular SOD, and apoE were assessed using Western blotting analysis. Results: In the water maze, spatial memory retention was impaired by irradiation in apoE2 and apoE4 mice but enhanced by irradiation in apoE3 mice. Irradiation reduced DHE-oxidation levels in the enclosed blade of the dentate gyrus and levels of 3-NT and CuZnSOD in apoE2 but not apoE3 or apoE4 mice. Finally, irradiation increased apoE levels in apoE3 but not apoE2 or apoE4 mice. Conclusions: The short-term effects of {sup 56}Fe irradiation on hippocampal ROS levels and hippocampus-dependent spatial memory retention are apoE isoform-dependent.

  14. Expression of ectonucleotidases in the prosencephalon of melatonin-proficient C3H and melatonin-deficient C57Bl mice: spatial distribution and time-dependent changes.

    PubMed

    Homola, Moran; Pfeffer, Martina; Fischer, Claudia; Zimmermann, Herbert; Robson, Simon C; Korf, Horst-Werner

    2015-10-01

    Extracellular purines (ATP, ADP, AMP and adenosine) are important signaling molecules in the CNS. Levels of extracellular purines are regulated by enzymes located at the cell surface referred to as ectonucleotidases. Time-dependent changes in their expression could profoundly influence the availability of extracellular purines and thereby purinergic signaling. Using radioactive in situ hybridization, we analyzed the mRNA distribution of the enzymes NTPDase1, -2 and -3 and ecto-5'-nucleotidase in the prosencephalon of two mouse strains: melatonin-proficient C3H and melatonin-deficient C57Bl. The mRNAs of these enzymes were localized to specific brain regions, such as hippocampus, striatum, medial habenula and ventromedial hypothalamus. NTPDase3 expression was more widely distributed than previously thought. All ectonucleotidases investigated revealed a prominent time-dependent expression pattern. In C3H, the mRNA expression of all four enzymes gradually increased during the day and peaked during the night. In contrast, in C57Bl, ecto-5'-nucleotidase expression peaked at the beginning of the day and gradually decreased to trough levels at night. Recording of locomotor activity revealed higher daytime activity of C57Bl than of C3H. Our results indicate that the expression of ectonucleotidases varies according to time and genotype and suggest that melatonin exerts modulatory effects associated with different regulations of purinergic signaling in the brain. These findings provide an important basis for further examination of the complexity of the purinergic system in the brain. PMID:25959293

  15. Fish oil induced increase in walking distance, but not ankle brachial pressure index, in peripheral arterial disease is dependent on both body mass index and inflammatory genotype.

    PubMed

    Madden, Jacqueline; Brunner, Andreas; Dastur, Neville D; Tan, Rebecca M; Nash, Gerard B; Rainger, G Ed; Shearman, Cliff P; Calder, Philip C; Grimble, Robert F

    2007-06-01

    Peripheral arterial disease (PAD) is an atherosclerotic disease. Evidence suggests that atherosclerosis is an inflammatory condition and long chain n-3 fatty acids, found in oily fish and fish oils, have been shown to reduce inflammation. Genetic and lifestyle factors such as body mass index (BMI) also influence inflammation. In this study we have examined the effect of fish oil in patients with claudication secondary to PAD. Fish oil supplementation, providing 1g EPA and 0.7 g DHA per day for 12 weeks, increased walking distance on a treadmill set at 3.2 km/h with a 7% incline. Walking distance to first pain increased from 76.2+/-8.5 m before fish oil to 140.6+/-25.5 m after fish oil (mean+/-SEM, p=0.004) and total distance walked increased from 160.0+/-21.5 m before fish oil to 242.1+/-34.5 m after fish oil (p=0.002). Fish oil supplementation also improved ankle brachial pressure index (ABPI) from 0.599+/-0.017 before fish oil to 0.776+/-0.030 after fish oil (p<0.001). The increase in walking distance was dependent on both BMI and genotype for single nucleotide polymorphisms in the genes encoding the pro-inflammatory cytokines tumour necrosis factor-alpha and interleukin (IL)-1beta and the anti-inflammatory cytokine IL-10 (detected using amplification refractory mutation system polymerase chain reaction). Neither BMI nor any of the genotypes examined affected the ability of fish oil to increase ABPI. The mechanisms by which fish oil affects walking distance and ABPI do not appear to be the same. PMID:17600695

  16. Facial expression recognition on a people-dependent personal facial expression space (PFES)

    NASA Astrophysics Data System (ADS)

    Chandrasiri, N. P.; Park, Min Chul; Naemura, Takeshi; Harashima, Hiroshi

    2000-04-01

    In this paper, a person-specific facial expression recognition method which is based on Personal Facial Expression Space (PFES) is presented. The multidimensional scaling maps facial images as points in lower dimensions in PFES. It reflects personality of facial expressions as it is based on the peak instant of facial expression images of a specific person. In constructing PFES for a person, his/her whole normalized facial image is considered as a single pattern without block segmentation and differences of 2-D DCT coefficients from neutral facial image of the same person are used as features. Therefore, in the early part of the paper, separation characteristics of facial expressions in the frequency domain are analyzed using a still facial image database which consists of neutral, smile, anger, surprise and sadness facial images for each of 60 Japanese males (300 facial images). Results show that facial expression categories are well separated in the low frequency domain. PFES is constructed using multidimensional scaling by taking these low frequency domain of differences of 2-D DCT coefficients as features. On the PFES, trajectory of a facial image sequence of a person can be calculated in real time. Based on this trajectory, facial expressions can be recognized. Experimental results show the effectiveness of this method.

  17. Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes

    PubMed Central

    Dey, Avishek; Samanta, Milan Kumar; Gayen, Srimonta; Sen, Soumitra K.; Maiti, Mrinal K.

    2016-01-01

    Drought is one of the major limiting factors for productivity of crops including rice (Oryza sativa L.). Understanding the role of allelic variations of key regulatory genes involved in stress-tolerance is essential for developing an effective strategy to combat drought. The bZIP transcription factors play a crucial role in abiotic-stress adaptation in plants via abscisic acid (ABA) signaling pathway. The present study aimed to search for allelic polymorphism in the OsbZIP23 gene across selected drought-tolerant and drought-sensitive rice genotypes, and to characterize the new allele through overexpression (OE) and gene-silencing (RNAi). Analyses of the coding DNA sequence (CDS) of the cloned OsbZIP23 gene revealed single nucleotide polymorphism at four places and a 15-nucleotide deletion at one place. The single-copy OsbZIP23 gene is expressed at relatively higher level in leaf tissues of drought-tolerant genotypes, and its abundance is more in reproductive stage. Cloning and sequence analyses of the OsbZIP23-promoter from drought-tolerant O. rufipogon and drought-sensitive IR20 cultivar showed variation in the number of stress-responsive cis-elements and a 35-nucleotide deletion at 5’-UTR in IR20. Analysis of the GFP reporter gene function revealed that the promoter activity of O. rufipogon is comparatively higher than that of IR20. The overexpression of any of the two polymorphic forms (1083 bp and 1068 bp CDS) of OsbZIP23 improved drought tolerance and yield-related traits significantly by retaining higher content of cellular water, soluble sugar and proline; and exhibited decrease in membrane lipid peroxidation in comparison to RNAi lines and non-transgenic plants. The OE lines showed higher expression of target genes-OsRab16B, OsRab21 and OsLEA3-1 and increased ABA sensitivity; indicating that OsbZIP23 is a positive transcriptional-regulator of the ABA-signaling pathway. Taken together, the present study concludes that the enhanced gene expression rather

  18. Enhanced Gene Expression Rather than Natural Polymorphism in Coding Sequence of the OsbZIP23 Determines Drought Tolerance and Yield Improvement in Rice Genotypes.

    PubMed

    Dey, Avishek; Samanta, Milan Kumar; Gayen, Srimonta; Sen, Soumitra K; Maiti, Mrinal K

    2016-01-01

    Drought is one of the major limiting factors for productivity of crops including rice (Oryza sativa L.). Understanding the role of allelic variations of key regulatory genes involved in stress-tolerance is essential for developing an effective strategy to combat drought. The bZIP transcription factors play a crucial role in abiotic-stress adaptation in plants via abscisic acid (ABA) signaling pathway. The present study aimed to search for allelic polymorphism in the OsbZIP23 gene across selected drought-tolerant and drought-sensitive rice genotypes, and to characterize the new allele through overexpression (OE) and gene-silencing (RNAi). Analyses of the coding DNA sequence (CDS) of the cloned OsbZIP23 gene revealed single nucleotide polymorphism at four places and a 15-nucleotide deletion at one place. The single-copy OsbZIP23 gene is expressed at relatively higher level in leaf tissues of drought-tolerant genotypes, and its abundance is more in reproductive stage. Cloning and sequence analyses of the OsbZIP23-promoter from drought-tolerant O. rufipogon and drought-sensitive IR20 cultivar showed variation in the number of stress-responsive cis-elements and a 35-nucleotide deletion at 5'-UTR in IR20. Analysis of the GFP reporter gene function revealed that the promoter activity of O. rufipogon is comparatively higher than that of IR20. The overexpression of any of the two polymorphic forms (1083 bp and 1068 bp CDS) of OsbZIP23 improved drought tolerance and yield-related traits significantly by retaining higher content of cellular water, soluble sugar and proline; and exhibited decrease in membrane lipid peroxidation in comparison to RNAi lines and non-transgenic plants. The OE lines showed higher expression of target genes-OsRab16B, OsRab21 and OsLEA3-1 and increased ABA sensitivity; indicating that OsbZIP23 is a positive transcriptional-regulator of the ABA-signaling pathway. Taken together, the present study concludes that the enhanced gene expression rather than

  19. Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4+ T cells, and disease activity

    PubMed Central

    Nagafuchi, Yasuo; Shoda, Hirofumi; Sumitomo, Shuji; Nakachi, Shinichiro; Kato, Rika; Tsuchida, Yumi; Tsuchiya, Haruka; Sakurai, Keiichi; Hanata, Norio; Tateishi, Shoko; Kanda, Hiroko; Ishigaki, Kazuyoshi; Okada, Yukinori; Suzuki, Akari; Kochi, Yuta; Fujio, Keishi; Yamamoto, Kazuhiko

    2016-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4+CD4+ T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4+CD4+ T cells. Moreover, the frequency of memory CXCR4+CD4+ T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4+CD4+ T cells. Clinically, a higher frequency of memory CXCR4+CD4+ T cells predicted a better response to CTLA4-Ig. Memory CXCR4+CD4+ T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA. PMID:27385284

  20. Negative density-dependent dispersal in the American black bear (Ursus americanus) revealed by noninvasive sampling and genotyping

    PubMed Central

    Roy, Justin; Yannic, Glenn; Côté, Steeve D; Bernatchez, Louis

    2012-01-01

    Although the dispersal of animals is influenced by a variety of factors, few studies have used a condition-dependent approach to assess it. The mechanisms underlying dispersal are thus poorly known in many species, especially in large mammals. We used 10 microsatellite loci to examine population density effects on sex-specific dispersal behavior in the American black bear, Ursus americanus. We tested whether dispersal increases with population density in both sexes. Fine-scale genetic structure was investigated in each of four sampling areas using Mantel tests and spatial autocorrelation analyses. Our results revealed male-biased dispersal pattern in low-density areas. As population density increased, females appeared to exhibit philopatry at smaller scales. Fine-scale genetic structure for males at higher densities may indicate reduced dispersal distances and delayed dispersal by subadults. PMID:22822432

  1. Cultivar-dependent root colonization, antifungal metabolite accumulation and gene expression in the wheat-Pseudomonas interaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We explored the role of host genotype in three aspects of the wheat-Pseudomonas biocontrol interaction: rhizosphere population density, accumulation of rhizosphere 2,4-diacetylphloroglucinol (DAPG), and Pseudomonas-mediated changes in root gene expression. Wheat cultivars varied in ability to suppo...

  2. Role of activity-dependent BDNF expression in hippocampal-prefrontal cortical regulation of behavioral perseverance.

    PubMed

    Sakata, Kazuko; Martinowich, Keri; Woo, Newton H; Schloesser, Robert J; Jimenez, Dennisse V; Ji, Yuanyuan; Shen, Liya; Lu, Bai

    2013-09-10

    Activity-dependent gene transcription, including that of the brain-derived neurotrophic factor (Bdnf) gene, has been implicated in various cognitive functions. We previously demonstrated that mutant mice with selective disruption of activity-dependent BDNF expression (BDNF-KIV mice) exhibit deficits in GABA-mediated inhibition in the prefrontal cortex (PFC). Here, we show that disruption of activity-dependent BDNF expression impairs BDNF-dependent late-phase long-term potentiation (L-LTP) in CA1, a site of hippocampal output to the PFC. Interestingly, early-phase LTP and conventional L-LTP induced by strong tetanic stimulation were completely normal in BDNF-KIV mice. In parallel, attenuation of activity-dependent BDNF expression significantly impairs spatial memory reversal and contextual memory extinction, two executive functions that require intact hippocampal-PFC circuitry. In contrast, spatial and contextual memory per se were not affected. Thus, activity-dependent BDNF expression in the hippocampus and PFC may contribute to cognitive and behavioral flexibility. These results suggest distinct roles for different forms of L-LTP and provide a link between activity-dependent BDNF expression and behavioral perseverance, a hallmark of several psychiatric disorders. PMID:23980178

  3. De Novo Polymerase Activity and Oligomerization of Hepatitis C Virus RNA-Dependent RNA-Polymerases from Genotypes 1 to 5

    PubMed Central

    Bellón-Echeverría, Itxaso; Encinar, José Antonio; Martínez-Alfaro, Elisa; Pérez-Flores, Ricardo; Mas, Antonio

    2011-01-01

    Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The Vmax/Km ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in Vmax values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions. PMID:21490973

  4. The serotonin transporter genotype is associated with intermediate brain phenotypes that depend on the context of eliciting stressor

    PubMed Central

    Kalin, NH; Shelton, SE; Fox, AS; Rogers, J; Oakes, TR; Davidson, RJ

    2009-01-01

    A variant allele in the promoter region of the serotonin transporter gene, SLC6A4, the s allele, is associated with increased vulnerability to develop anxiety-related traits and depression. Furthermore, functional magnetic resonance imaging (fMRI) studies reveal that s carriers have increased amygdala reactivity in response to aversive stimuli, which is thought to be an intermediate phenotype mediating the influences of the s allele on emotionality. We used high-resolution microPET [18F]fluoro-2-deoxy-D-glucose (FDG) scanning to assess regional brain metabolic activity in rhesus monkeys to further explore s allele-related intermediate phenotypes. Rhesus monkeys provide an excellent model to understand mechanisms underlying human anxiety, and FDG microPET allows for the assessment of brain activity associated with naturalistic environments outside the scanner. During FDG uptake, monkeys were exposed to different ethologically relevant stressful situations (relocation and threat) as well as to the less stressful familiar environment of their home cage. The s carriers displayed increased orbitofrontal cortex activity in response to both relocation and threat. However, during relocation they displayed increased amygdala reactivity and in response to threat they displayed increased reactivity of the bed nucleus of the stria terminalis. No increase in the activity of any of these regions occurred when the animals were administered FDG in their home cages. These findings demonstrate context-dependent intermediate phenotypes in s carriers that provide a framework for understanding the mechanisms underlying the vulnerabilities of s-allele carriers exposed to different types of stressors. PMID:18414408

  5. Protein expression and genetic structure of the coral Porites lobata in an environmentally extreme Samoan back reef: Does host genotype limit phenotypic plasticity?

    USGS Publications Warehouse

    Barshis, D.J.; Stillman, J.H.; Gates, R.D.; Toonen, R.J.; Smith, L.W.; Birkeland, C.

    2010-01-01

    The degree to which coral reef ecosystems will be impacted by global climate change depends on regional and local differences in corals' susceptibility and resilience to environmental stressors. Here, we present data from a reciprocal transplant experiment using the common reef building coral Porites lobata between a highly fluctuating back reef environment that reaches stressful daily extremes, and a more stable, neighbouring forereef. Protein biomarker analyses assessing physiological contributions to stress resistance showed evidence for both fixed and environmental influence on biomarker response. Fixed influences were strongest for ubiquitin-conjugated proteins with consistently higher levels found in back reef source colonies both pre and post-transplant when compared with their forereef conspecifics. Additionally, genetic comparisons of back reef and forereef populations revealed significant population structure of both the nuclear ribosomal and mitochondrial genomes of the coral host (FST = 0.146 P < 0.0001, FST = 0.335 P < 0.0001 for rDNA and mtDNA, respectively), whereas algal endosymbiont populations were genetically indistinguishable between the two sites. We propose that the genotype of the coral host may drive limitations to the physiological responses of these corals when faced with new environmental conditions. This result is important in understanding genotypic and environmental interactions in the coral algal symbiosis and how corals may respond to future environmental changes. ?? 2010 Blackwell Publishing Ltd.

  6. Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers.

    PubMed

    Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K; Müller, Carsten T; Rosati, Carlo; Rogers, Hilary J

    2012-04-01

    Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar 'Sweet Laura' is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. 'Sweet Laura' with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. 'Sweet Laura' and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. 'Sweet Laura' placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R(28)(R)X(8)W and D(321)DXXD are the putative Mg(2+)-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. 'Sweet Laura' flowers. PMID:22268153

  7. Analysis of SLC16A11 Variants in 12,811 American Indians: Genotype-Obesity Interaction for Type 2 Diabetes and an Association With RNASEK Expression.

    PubMed

    Traurig, Michael; Hanson, Robert L; Marinelarena, Alejandra; Kobes, Sayuko; Piaggi, Paolo; Cole, Shelley; Curran, Joanne E; Blangero, John; Göring, Harald; Kumar, Satish; Nelson, Robert G; Howard, Barbara V; Knowler, William C; Baier, Leslie J; Bogardus, Clifton

    2016-02-01

    Genetic variants in SLC16A11 were recently reported to be associated with type 2 diabetes in Mexican and other Latin American populations. The diabetes risk haplotype had a frequency of 50% in Native Americans from Mexico but was rare in Europeans and Africans. In the current study, we analyzed SLC16A11 in 12,811 North American Indians and found that the diabetes risk haplotype, tagged by the rs75493593 A allele, was nominally associated with type 2 diabetes (P = 0.001, odds ratio 1.11). However, there was a strong interaction with BMI (P = 5.1 × 10(-7)) such that the diabetes association was stronger in leaner individuals. rs75493593 was also strongly associated with BMI in individuals with type 2 diabetes (P = 3.4 × 10(-15)) but not in individuals without diabetes (P = 0.77). Longitudinal analyses suggest that this is due, in part, to an association of the A allele with greater weight loss following diabetes onset (P = 0.02). Analyses of global gene expression data from adipose tissue, skeletal muscle, and whole blood provide evidence that rs75493593 is associated with expression of the nearby RNASEK gene, suggesting that RNASEK expression may mediate the effect of genotype on diabetes. PMID:26487785

  8. Genotype × genotype interactions between the toxic cyanobacterium Microcystis and its grazer, the waterflea Daphnia

    PubMed Central

    Lemaire, Veerle; Brusciotti, Silvia; van Gremberghe, Ineke; Vyverman, Wim; Vanoverbeke, Joost; De Meester, Luc

    2012-01-01

    Toxic algal blooms are an important problem worldwide. The literature on toxic cyanobacteria blooms in inland waters reports widely divergent results on whether zooplankton can control cyanobacteria blooms or cyanobacteria suppress zooplankton by their toxins. Here we test whether this may be due to genotype × genotype interactions, in which interactions between the large-bodied and efficient grazer Daphnia and the widespread cyanobacterium Microcystis are not only dependent on Microcystis strain or Daphnia genotype but are specific to genotype × genotype combinations. We show that genotype × genotype interactions are important in explaining mortality in short-time exposures of Daphnia to Microcystis. These genotype × genotype interactions may result in local coadaptation and a geographic mosaic of coevolution. Genotype × genotype interactions can explain why the literature on zooplankton–cyanobacteria interactions is seemingly inconsistent, and provide hope that zooplankton can contribute to the suppression of cyanobacteria blooms in restoration projects. PMID:25568039

  9. Isolation and Identification of Genes Activating Uas2-Dependent Adh2 Expression in Saccharomyces Cerevisiae

    PubMed Central

    Donoviel, M. S.; Young, E. T.

    1996-01-01

    Two cis-acting elements have been identified that act synergistically to regulate expression of the glucose-repressed alcohol dehydrogenase 2 (ADH2) gene. UAS1 is bound by the trans-activator Adr1p. UAS2 is thought to be the binding site for an unidentified regulatory protein. A genetic selection based on a UAS2-dependent ADH2 reporter was devised to isolate genes capable of activating UAS2-dependent transcription. One set of UAS2-dependent genes contained SPT6/CRE2/SSN20. Multicopy SPT6 caused improper expression of chromosomal ADH2. A second set of UAS2-dependent clones contained a previously uncharacterized open reading frame designated MEU1 (Multicopy Enhancer of UAS2). A frame shift mutation in MEU1 abolished its ability to activate UAS2-dependent gene expression. Multicopy MEU1 expression suppressed the constitutive ADH2 expression caused by cre2-1. Disruption of MEU1 reduced endogenous ADH2 expression about twofold but had no effect on cell viability or growth. No homologues of MEU1 were identified by low-stringency Southern hybridization of yeast genomic DNA, and no significant homologues were found in the sequence data bases. A MEU1/β-gal fusion protein was not localized to a particular region of the cell. MEU1 is linked to PPR1 on chromosome XII. PMID:8807288

  10. Flow-Dependent Epigenetic DNA Methylation in Endothelial Gene Expression and Atherosclerosis.

    PubMed

    Dunn, Jessilyn; Thabet, Salim; Jo, Hanjoong

    2015-07-01

    Epigenetic mechanisms that regulate endothelial cell gene expression are now emerging. DNA methylation is the most stable epigenetic mark that confers persisting changes in gene expression. Not only is DNA methylation important in rendering cell identity by regulating cell type-specific gene expression throughout differentiation, but it is becoming clear that DNA methylation also plays a key role in maintaining endothelial cell homeostasis and in vascular disease development. Disturbed blood flow causes atherosclerosis, whereas stable flow protects against it by differentially regulating gene expression in endothelial cells. Recently, we and others have shown that flow-dependent gene expression and atherosclerosis development are regulated by mechanisms dependent on DNA methyltransferases (1 and 3A). Disturbed blood flow upregulates DNA methyltransferase expression both in vitro and in vivo, which leads to genome-wide DNA methylation alterations and global gene expression changes in a DNA methyltransferase-dependent manner. These studies revealed several mechanosensitive genes, such as HoxA5, Klf3, and Klf4, whose promoters were hypermethylated by disturbed blood flow, but rescued by DNA methyltransferases inhibitors such as 5Aza-2-deoxycytidine. These findings provide new insight into the mechanism by which flow controls epigenomic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and modulates atherosclerosis development. PMID:25953647

  11. Context-dependent expression of the foraging gene in field colonies of ants: the interacting roles of age, environment and task

    PubMed Central

    Gordon, Deborah M.; Greene, Michael; Kahler, John; Peteru, Swetha

    2016-01-01

    Task allocation among social insect workers is an ideal framework for studying the molecular mechanisms underlying behavioural plasticity because workers of similar genotype adopt different behavioural phenotypes. Elegant laboratory studies have pioneered this effort, but field studies involving the genetic regulation of task allocation are rare. Here, we investigate the expression of the foraging gene in harvester ant workers from five age- and task-related groups in a natural population, and we experimentally test how exposure to light affects foraging expression in brood workers and foragers. Results from our field study show that the regulation of the foraging gene in harvester ants occurs at two time scales: levels of foraging mRNA are associated with ontogenetic changes over weeks in worker age, location and task, and there are significant daily oscillations in foraging expression in foragers. The temporal dissection of foraging expression reveals that gene expression changes in foragers occur across a scale of hours and the level of expression is predicted by activity rhythms: foragers have high levels of foraging mRNA during daylight hours when they are most active outside the nests. In the experimental study, we find complex interactions in foraging expression between task behaviour and light exposure. Oscillations occur in foragers following experimental exposure to 13 L : 11 D (LD) conditions, but not in brood workers under similar conditions. No significant differences were seen in foraging expression over time in either task in 24 h dark (DD) conditions. Interestingly, the expression of foraging in both undisturbed field and experimentally treated foragers is also significantly correlated with the expression of the circadian clock gene, cycle. Our results provide evidence that the regulation of this gene is context-dependent and associated with both ontogenetic and daily behavioural plasticity in field colonies of harvester ants. Our results underscore

  12. Context-dependent expression of the foraging gene in field colonies of ants: the interacting roles of age, environment and task.

    PubMed

    Ingram, Krista K; Gordon, Deborah M; Friedman, Daniel A; Greene, Michael; Kahler, John; Peteru, Swetha

    2016-08-31

    Task allocation among social insect workers is an ideal framework for studying the molecular mechanisms underlying behavioural plasticity because workers of similar genotype adopt different behavioural phenotypes. Elegant laboratory studies have pioneered this effort, but field studies involving the genetic regulation of task allocation are rare. Here, we investigate the expression of the foraging gene in harvester ant workers from five age- and task-related groups in a natural population, and we experimentally test how exposure to light affects foraging expression in brood workers and foragers. Results from our field study show that the regulation of the foraging gene in harvester ants occurs at two time scales: levels of foraging mRNA are associated with ontogenetic changes over weeks in worker age, location and task, and there are significant daily oscillations in foraging expression in foragers. The temporal dissection of foraging expression reveals that gene expression changes in foragers occur across a scale of hours and the level of expression is predicted by activity rhythms: foragers have high levels of foraging mRNA during daylight hours when they are most active outside the nests. In the experimental study, we find complex interactions in foraging expression between task behaviour and light exposure. Oscillations occur in foragers following experimental exposure to 13 L : 11 D (LD) conditions, but not in brood workers under similar conditions. No significant differences were seen in foraging expression over time in either task in 24 h dark (DD) conditions. Interestingly, the expression of foraging in both undisturbed field and experimentally treated foragers is also significantly correlated with the expression of the circadian clock gene, cycle Our results provide evidence that the regulation of this gene is context-dependent and associated with both ontogenetic and daily behavioural plasticity in field colonies of harvester ants. Our results underscore

  13. Nicotine dependence measures among adolescents with psychiatric disorders: evaluating symptom expression as a function of dependence severity.

    PubMed

    Strong, David R; Brown, Richard A; Ramsey, Susan E; Myers, Mark G

    2003-10-01

    Using methods based in item response theory, we examined a structured interview assessment of Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) nicotine dependence and the Modified Fagerström Tolerance Questionnaire (mFTQ) symptoms to explore the expression of particular symptoms as a function of level of nicotine involvement in a sample of 191 adolescents with psychiatric disorders. Despite our attempts to capture a broad range of smokers, 64% of teens were daily smokers and 68% met DSM-IV criteria for nicotine dependence. This paper describes the relative severity of DSM-IV and mFTQ items, as well as each item's ability to discriminate among individuals at various levels of nicotine involvement. Comparisons across measures revealed that the mFTQ was not particularly sensitive to individual variation in DSM-IV symptom counts, suggesting the physiological components were not strongly related to the predominantly cognitive and behavioral components of the DSM-IV nicotine dependence syndrome. However, the mFTQ relative to the DSM-IV consistently showed stronger relationships to the immediate consequences of nicotine deprivation (urge, craving), supporting the conceptualization of the mFTQ as measuring nicotine exposure. These analyses provide us with some preliminary understanding of the severity of particular symptoms and the order in which symptoms are likely to be expressed across levels of nicotine dependence. PMID:14577990

  14. High-Fat Diet Changes Hippocampal Apolipoprotein E (ApoE) in a Genotype- and Carbohydrate-Dependent Manner in Mice

    PubMed Central

    Lane-Donovan, Courtney; Herz, Joachim

    2016-01-01

    Alzheimer’s disease is a currently incurable neurodegenerative disease affecting millions of individuals worldwide. Risk factors for Alzheimer’s disease include genetic risk factors, such as possession of ε4 allele of apolipoprotein E (ApoE4) over the risk-neutral ApoE3 allele, and lifestyle risk factors, such as diet and exercise. The intersection of these two sources of disease risk is not well understood. We investigated the impact of diet on ApoE levels by feeding wildtype, ApoE3, and ApoE4 targeted replacement (TR) mice with chow, high-fat, or ketogenic (high-fat, very-low-carbohydrate) diets. We found that high-fat diet affected both plasma and hippocampal levels of ApoE in an isoform-dependent manner, with high-fat diet causing a surprising reduction of hippocampal ApoE levels in ApoE3 TR mice. Conversely, the ketogenic diet had no effect on hippocampal ApoE. Our findings suggest that the use of dietary interventions to slow the progression AD should take ApoE genotype into consideration. PMID:26828652

  15. High-Fat Diet Changes Hippocampal Apolipoprotein E (ApoE) in a Genotype- and Carbohydrate-Dependent Manner in Mice.

    PubMed

    Lane-Donovan, Courtney; Herz, Joachim

    2016-01-01

    Alzheimer's disease is a currently incurable neurodegenerative disease affecting millions of individuals worldwide. Risk factors for Alzheimer's disease include genetic risk factors, such as possession of ε4 allele of apolipoprotein E (ApoE4) over the risk-neutral ApoE3 allele, and lifestyle risk factors, such as diet and exercise. The intersection of these two sources of disease risk is not well understood. We investigated the impact of diet on ApoE levels by feeding wildtype, ApoE3, and ApoE4 targeted replacement (TR) mice with chow, high-fat, or ketogenic (high-fat, very-low-carbohydrate) diets. We found that high-fat diet affected both plasma and hippocampal levels of ApoE in an isoform-dependent manner, with high-fat diet causing a surprising reduction of hippocampal ApoE levels in ApoE3 TR mice. Conversely, the ketogenic diet had no effect on hippocampal ApoE. Our findings suggest that the use of dietary interventions to slow the progression AD should take ApoE genotype into consideration. PMID:26828652

  16. Genotype-environment interaction expressed in the foraging behaviour of dogwhelks, Nucella lapillus (L.), under simulated environmental hazard

    PubMed Central

    Hughes, R. N.; Taylor, M. J.

    1997-01-01

    represent genotype/environment interaction that is apparently adaptive, in part, through its effect on foraging behaviour.

  17. Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation of resistance or susceptibility of corn inbreds to infection by Aspergillus flavus was evaluated by a kernel screening assay. A GFP-expressing strain of A. flavus was used to accomplish this study to measure fungal spread and aflatoxin levels in real time. Among the four inbreds tested, ...

  18. Cezanne regulates E2F1-dependent HIF2α expression.

    PubMed

    Moniz, Sonia; Bandarra, Daniel; Biddlestone, John; Campbell, Kirsteen J; Komander, David; Bremm, Anja; Rocha, Sonia

    2015-08-15

    Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling. PMID:26148512

  19. Cezanne regulates E2F1-dependent HIF2α expression

    PubMed Central

    Moniz, Sonia; Bandarra, Daniel; Biddlestone, John; Campbell, Kirsteen J.; Komander, David; Bremm, Anja; Rocha, Sonia

    2015-01-01

    ABSTRACT Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling. PMID:26148512

  20. De novo transcriptome sequencing of Acer palmatum and comprehensive analysis of differentially expressed genes under salt stress in two contrasting genotypes.

    PubMed

    Rong, Liping; Li, Qianzhong; Li, Shushun; Tang, Ling; Wen, Jing

    2016-04-01

    Maple (Acer palmatum) is an important species for landscape planting worldwide. Salt stress affects the normal growth of the Maple leaf directly, leading to loss of esthetic value. However, the limited availability of Maple genomic information has hindered research on the mechanisms underlying this tolerance. In this study, we performed comprehensive analyses of the salt tolerance in two genotypes of Maple using RNA-seq. Approximately 146.4 million paired-end reads, representing 181,769 unigenes, were obtained. The N50 length of the unigenes was 738 bp, and their total length over 102.66 Mb. 14,090 simple sequence repeats and over 500,000 single nucleotide polymorphisms were identified, which represent useful resources for marker development. Importantly, 181,769 genes were detected in at least one library, and 303 differentially expressed genes (DEGs) were identified between salt-sensitive and salt-tolerant genotypes. Among these DEGs, 125 were upregulated and 178 were downregulated genes. Two MYB-related proteins and one LEA protein were detected among the first 10 most downregulated genes. Moreover, a methyltransferase-related gene was detected among the first 10 most upregulated genes. The three most significantly enriched pathways were plant hormone signal transduction, arginine and proline metabolism, and photosynthesis. The transcriptome analysis provided a rich genetic resource for gene discovery related to salt tolerance in Maple, and in closely related species. The data will serve as an important public information platform to further our understanding of the molecular mechanisms involved in salt tolerance in Maple. PMID:26475609

  1. Maternal high-fat diet interacts with embryonic Cited2 genotype to reduce Pitx2c expression and enhance penetrance of left-right patterning defects.

    PubMed

    Bentham, Jamie; Michell, Anna C; Lockstone, Helen; Andrew, Daniel; Schneider, Jürgen E; Brown, Nigel A; Bhattacharya, Shoumo

    2010-09-01

    Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects and partially penetrant cardiopulmonary laterality defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2-deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume. Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction. We show that a high-fat diet reduces Pitx2c levels >2-fold in Cited2-deficient embryos. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality. They suggest that appropriate modifications of maternal diet may prevent such defects in humans. PMID:20566713

  2. Thyroid Hormone Regulates Hepatic Expression of Fibroblast Growth Factor 21 in a PPARα-dependent Manner*

    PubMed Central

    Adams, Andrew C.; Astapova, Inna; Fisher, ffolliott M.; Badman, Michael K.; Kurgansky, Katherine E.; Flier, Jeffrey S.; Hollenberg, Anthony N.; Maratos-Flier, Eleftheria

    2010-01-01

    Thyroid hormone has profound and diverse effects on liver metabolism. Here we show that tri-iodothyronine (T3) treatment in mice acutely and specifically induces hepatic expression of the metabolic regulator fibroblast growth factor 21 (FGF21). Mice treated with T3 showed a dose-dependent increase in hepatic FGF21 expression with significant induction at doses as low as 100 μg/kg. Time course studies determined that induction is seen as early as 4 h after treatment with a further increase in expression at 6 h after injection. As FGF21 expression is downstream of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), we treated PPARα knock-out mice with T3 and found no increase in expression, indicating that hepatic regulation of FGF21 by T3 in liver is via a PPARα-dependent mechanism. In contrast, in white adipose tissue, FGF21 expression was suppressed by T3 treatment, with other T3 targets unaffected. In cell culture studies with an FGF21 reporter construct, we determined that three transcription factors are required for induction of FGF21 expression: thyroid hormone receptor β (TRβ), retinoid X receptor (RXR), and PPARα. These findings indicate a novel regulatory pathway whereby T3 positively regulates hepatic FGF21 expression, presenting a novel therapeutic target for diseases such as non-alcoholic fatty liver disease. PMID:20236931

  3. GNMT expression increases hepatic folate contents and folate-dependent methionine synthase-mediated homocysteine remethylation.

    PubMed

    Wang, Yi-Cheng; Chen, Yi-Ming; Lin, Yan-Jun; Liu, Shih-Ping; Chiang, En-Pei Isabel

    2011-01-01

    Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT(tg)) and GNMT knockout (GNMT(ko)) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT(tg) had increased hepatic folate significantly, whereas GNMT(ko) had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT(ko), demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway. PMID:21210071

  4. IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies

    PubMed Central

    Duong, Francois H.T.; Trincucci, Gaia; Boldanova, Tujana; Calabrese, Diego; Campana, Benedetta; Krol, Ilona; Durand, Sarah C.; Heydmann, Laura; Zeisel, Mirjam B.; Baumert, Thomas F.

    2014-01-01

    The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α–induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse. PMID:24752298

  5. IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies.

    PubMed

    Duong, Francois H T; Trincucci, Gaia; Boldanova, Tujana; Calabrese, Diego; Campana, Benedetta; Krol, Ilona; Durand, Sarah C; Heydmann, Laura; Zeisel, Mirjam B; Baumert, Thomas F; Heim, Markus H

    2014-05-01

    The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α-induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse. PMID:24752298

  6. Compressed Genotyping

    PubMed Central

    Erlich, Yaniv; Gordon, Assaf; Brand, Michael; Hannon, Gregory J.; Mitra, Partha P.

    2011-01-01

    Over the past three decades we have steadily increased our knowledge on the genetic basis of many severe disorders. Nevertheless, there are still great challenges in applying this knowledge routinely in the clinic, mainly due to the relatively tedious and expensive process of genotyping. Since the genetic variations that underlie the disorders are relatively rare in the population, they can be thought of as a sparse signal. Using methods and ideas from compressed sensing and group testing, we have developed a cost-effective genotyping protocol to detect carriers for severe genetic disorders. In particular, we have adapted our scheme to a recently developed class of high throughput DNA sequencing technologies. The mathematical framework presented here has some important distinctions from the ’traditional’ compressed sensing and group testing frameworks in order to address biological and technical constraints of our setting. PMID:21451737

  7. Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers

    PubMed Central

    Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K.; Müller, Carsten T.; Rosati, Carlo; Rogers, Hilary J.

    2012-01-01

    Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar ‘Sweet Laura’ is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. ‘Sweet Laura’ with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. ‘Sweet Laura’ and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. ‘Sweet Laura’ placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R28(R)X8W and D321DXXD are the putative Mg2+-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. ‘Sweet Laura’ flowers. PMID:22268153

  8. Differentially Expressed Genes in Resistant and Susceptible Common Bean (Phaseolus vulgaris L.) Genotypes in Response to Fusarium oxysporum f. sp. phaseoli

    PubMed Central

    Xue, Renfeng; Wu, Jing; Zhu, Zhendong; Wang, Lanfen; Wang, Xiaoming; Wang, Shumin; Blair, Matthew W.

    2015-01-01

    Fusarium wilt of common bean (Phaseolus vulgaris L.), caused by Fusarium oxysporum Schlechtend.:Fr. f.sp. phaseoli (Fop), is one of the most important diseases of common beans worldwide. Few natural sources of resistance to Fop exist and provide only moderate or partial levels of protection. Despite the economic importance of the disease across multiple crops, only a few of Fop induced genes have been analyzed in legumes. Therefore, our goal was to identify transcriptionally regulated genes during an incompatible interaction between common bean and the Fop pathogen using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. We generated a total of 8,730 transcript-derived fragments (TDFs) with 768 primer pairs based on the comparison of a moderately resistant and a susceptible genotype. In total, 423 TDFs (4.9%) displayed altered expression patterns after inoculation with Fop inoculum. We obtained full amplicon sequences for 122 selected TDFs, of which 98 were identified as annotated known genes in different functional categories based on their putative functions, 10 were predicted but non-annotated genes and 14 were not homologous to any known genes. The 98 TDFs encoding genes of known putative function were classified as related to metabolism (22), signal transduction (21), protein synthesis and processing (20), development and cytoskeletal organization (12), transport of proteins (7), gene expression and RNA metabolism (4), redox reactions (4), defense and stress responses (3), energy metabolism (3), and hormone responses (2). Based on the analyses of homology, 19 TDFs from different functional categories were chosen for expression analysis using quantitative RT-PCR. The genes found to be important here were implicated at various steps of pathogen infection and will allow a better understanding of the mechanisms of defense and resistance to Fop and similar pathogens. The differential response genes discovered here could also be used as molecular

  9. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

    PubMed Central

    Bazovkina, Darya V.; Kondaurova, Elena M.; Naumenko, Vladimir S.; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  10. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity.

    PubMed

    Bazovkina, Darya V; Kondaurova, Elena M; Naumenko, Vladimir S; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  11. Genotype at the PMEL17 locus affects social and explorative behaviour in chickens.

    PubMed

    Karlsson, A-C; Kerje, S; Andersson, L; Jensen, P

    2010-04-01

    1. We studied behaviour and brain gene expression in homozygous PMEL17 genotypes, using chickens originating from an advanced White Leghorn x red junglefowl intercross. The behavioural studies consisted of three social and one explorative behaviour test. There were significant differences between the genotypes in both social and explorative behaviour. 2. Gene expression studies showed no PMEL17 expression in brain, so the genotype differences must depend on extra-neural gene expression or expression during embryonic development. However, linkage or spurious family effects (genetic drift) can not be excluded. 3. The study strongly suggests a correlated effect between plumage colour and behaviour, and we conclude that PMEL17 may have a pleiotropic effect on social and explorative behaviour in chickens. PMID:20461577

  12. Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

    PubMed Central

    2011-01-01

    Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

  13. Glioma cell proliferation controlled by ERK activity-dependent surface expression of PDGFRA.

    PubMed

    Chen, Dongfeng; Zuo, Duo; Luan, Cheng; Liu, Min; Na, Manli; Ran, Liang; Sun, Yingyu; Persson, Annette; Englund, Elisabet; Salford, Leif G; Renström, Erik; Fan, Xiaolong; Zhang, Enming

    2014-01-01

    Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings

  14. The Impact of IL28B Genotype and Liver Fibrosis on the Hepatic Expression of IP10, IFI27, ISG15, and MX1 and Their Association with Treatment Outcomes in Patients with Chronic Hepatitis C

    PubMed Central

    Domagalski, Krzysztof; Pawłowska, Małgorzata; Kozielewicz, Dorota; Dybowska, Dorota; Tretyn, Andrzej; Halota, Waldemar

    2015-01-01

    The strong impact of interleukin 28B (IL28B) polymorphisms on sustained virological response (SVR) after peginterferon and ribavirin treatment in patients with chronic hepatitis C (CHC) is well-known. We investigated IL28B variability and hepatic expression of IP10, IFI27, ISG15, and MX1 in CHC patients, the relation of each with their clinical characteristics, and how they associated with responses to combined therapy. Genotyping and gene expression analysis were conducted in a selected cohort of treatment-naïve patients who underwent interferon and ribavirin treatment. Differential expression of IP10, IFI27, ISG15, and MX1 genes was assessed from pretreatment liver biopsies using quantitative PCR. Histopathological evaluation of liver specimens was performed on the basis of the Scheuer’s modified scale. We showed that hepatic IFI27, ISG15, and MX1 expression was lower in the IL28B CC 12979860 and TT rs8099917 groups than in the CT-TT rs12979860 and TG-GG rs8099917 groups (P < 0.001). We found no differences in IP10 expression between the IL28B genotypes (P > 0.05); in contrast, IP10 expression was significantly affected by the progression of fibrosis (P = 0.007). We showed that the rs12979860 CC genotype was associated with successful treatment when compared to the rs12979860 CT-TT genotype (P = 0.004). Additionally, the expression levels of IP10, IFI27 and ISG15, but not MX1, were significantly higher in non-SVR patients than in SVR patients. The effect of variation in IL28B on the results of IFN-based treatment may be associated with changes in IFI27 and ISG15, but not with IP10. Silencing of IP10 is positive and independent from IL28B prediction of SVR, which is strongly associated with liver fibrosis in CHC patients. PMID:26115415

  15. Different mechanisms of apolipoprotein E isoform–dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia

    PubMed Central

    Li, Xianwu; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2015-01-01

    Several lines of evidence support immune response in brain as a mechanism of injury in Alzheimer disease (AD). Moreover, immune activation is heightened in apolipoprotein E (APOE) ε4 carriers; inhibitors of prostaglandin (PG) synthesis show a partially protective effect on AD risk from APOE ε4; and genetic variants in triggering receptor expressed on myeloid cells 2 (TREM2) are a rare but potent risk for AD. We tested the hypothesis that APOE ε4 inheritance modulates both the PGE2 pathway and TREM2 expression using primary murine microglia from targeted replacement (TR) APOE3/3 and APOE4/4 mice. Microglial cyclooxygenase-2, microsomal PGE synthase, and PGE2 expression were increased 2- to 25-fold in both genotypes by TLR activators; however, this induction was significantly (P < 0.01) greater in TR APOE4/4 microglia with TLR3 and TLR4 activators. Microglial TREM2 expression was reduced approximately 85% by all TLR activators; this reduction was approximately one-third greater in microglia from TR APOE4/4 mice. Importantly, both receptor-associated protein and a nuclear factor κ-light-chain-enhancer inhibitor blocked TR APOE4/4–dependent effects on the PGE2 pathway but not on TREM2 expression. These data demonstrate complementary, but mechanistically distinct, regulation of pro- and anti-inflammatory mediators in TR APOE4/4 murine microglia that yields a more proinflammatory state than with TR APOE3/3.—Li, X., Montine, K. S., Keene, C. D., Montine, T. J. Different mechanisms of apolipoprotein E isoform–dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia. PMID:25593125

  16. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

    PubMed

    Stanga, Serena; Zanou, Nadège; Audouard, Emilie; Tasiaux, Bernadette; Contino, Sabrina; Vandermeulen, Gaëlle; René, Frédérique; Loeffler, Jean-Philippe; Clotman, Frédéric; Gailly, Philippe; Dewachter, Ilse; Octave, Jean-Noël; Kienlen-Campard, Pascal

    2016-05-01

    Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation. PMID:26718890

  17. Engineering the esaR promoter for tunable quorum sensing- dependent gene expression.

    PubMed

    Shong, Jasmine; Collins, Cynthia H

    2013-10-18

    Quorum sensing (QS) systems enable bacteria to coordinate their behavior as a function of local population density and are often used in synthetic systems that require cell−cell communication. We have engineered the esaR promoter, P(esaR), which is repressed by the QS regulator E(saR). E(saR)-dependent gene expression from P(esaR) is induced by 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Here, we report a set of modified P(esaR) promoters that contain a second E(saR) binding site. We observed changes in gene expression levels, regulatory range, 3OC6HSL sensitivity, and the regulatory role of E(saR) that are dependent on the position of the second binding site. Combining the new promoters with endogenous 3OC6HSL production led to QS-dependent systems that exhibit a range of expression levels and timing. These promoters represent a new set of tools for modulating QS-dependent gene expression and may be used to tune the regulation of multiple genes in response to a single QS signal. PMID:23879176

  18. Sex-Dependent Claudin-1 Expression in the Liver of Euthyroid and Hypothyroid Mice

    PubMed Central

    Zwanziger, Denise; Rakov, Helena; Engels, Kathrin; Moeller, Lars C.; Führer, Dagmar

    2015-01-01

    Background In the liver the tight junction protein claudin-1 plays an important role in bile secretion by maintaining the paracellular barrier of bile canaliculi and the bile duct. A diminished bile excretion has been found in hypothyroid patients, and the prevalence of gallstones is increased in hypothyroidism. This association, however, only applies for men and is in contrast to the well-established female preponderance of biliary disease in the general population. Objectives We hypothesized that hypothyroidism could lead to altered claudin-1 expression in the liver, and that this effect may be sex specific. Methods We characterized claudin-1 expression and localization in livers of euthyroid and hypothyroid male and female C57BL/6NTac mice by real-time PCR, Western blot and immunofluorescence. Results Claudin-1 is expressed in canalicular regions and the bile ducts of the murine liver. Livers of female mice showed lower claudin-1 expression than male livers. In hypothyroid livers, female animals showed an elevated claudin-1 expression, whereas reduced claudin-1 expression was found in male animals compared to the euthyroid controls. Conclusion We demonstrate a correlation between claudin-1 expression and hypothyroidism in the murine liver. Furthermore, a sex-dependent alteration of claudin-1 expression was found. PMID:26601075

  19. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  20. Hyaluronan Inhibits Tlr-4-Dependent RANKL Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    PubMed Central

    Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    The Toll-like receptor (TLR) signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA). The receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA) is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS) stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1). Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA. PMID:27054952

  1. Light-dependent expression of flg22-induced defense genes in Arabidopsis

    PubMed Central

    Sano, Satoshi; Aoyama, Mayu; Nakai, Kana; Shimotani, Koji; Yamasaki, Kanako; Sato, Masa H.; Tojo, Daisuke; Suwastika, I. Nengah; Nomura, Hironari; Shiina, Takashi

    2014-01-01

    Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes. PMID:25346742

  2. Wilms' tumour-suppressor protein isoforms have opposite effects on Igf2 expression in primary embryonic cells, independently of p53 genotype.

    PubMed Central

    Duarte, A.; Caricasole, A.; Graham, C. F.; Ward, A.

    1998-01-01

    The p53 protein has been proposed as a modulator of the Wilms' tumour-suppressor protein (WT1) transcriptional regulation activity. To investigate this putative p53 role, the promoter P3 of the mouse insulin-like growth factor II gene (Igf2) was used as a target for WT1 regulation in primary cell cultures derived from p53 wild-type (p53+/+) and knock-out (p53-/-) mouse embryos. In these cells, the WT1 transcriptional activity was observed to be independent of p53 genotype. Furthermore, the two WT1 zinc finger (ZF) isoforms were for the first time found to have opposite effects on gene expression from a single promoter in the same cell type, WT1[-KTS] activating Igf2 P3, whereas WT1[+KTS] repressed its activity. In addition, we have mapped the WT1 binding sites and investigated the effect on WT1 binding activity of individual ZF deletions and Denys-Drash syndrome point mutations to this target. Images Figure 1 Figure 3 Figure 4 PMID:9460996

  3. ATHENA: a tool for meta-dimensional analysis applied to genotypes and gene expression data to predict HDL cholesterol levels.

    PubMed

    Holzinger, Emily R; Dudek, Scott M; Frase, Alex T; Krauss, Ronald M; Medina, Marisa W; Ritchie, Marylyn D

    2013-01-01

    Technology is driving the field of human genetics research with advances in techniques to generate high-throughput data that interrogate various levels of biological regulation. With this massive amount of data comes the important task of using powerful bioinformatics techniques to sift through the noise to find true signals that predict various human traits. A popular analytical method thus far has been the genome-wide association study (GWAS), which assesses the association of single nucleotide polymorphisms (SNPs) with the trait of interest. Unfortunately, GWAS has not been able to explain a substantial proportion of the estimated heritability for most complex traits. Due to the inherently complex nature of biology, this phenomenon could be a factor of the simplistic study design. A more powerful analysis may be a systems biology approach that integrates different types of data, or a meta-dimensional analysis. For this study we used the Analysis Tool for Heritable and Environmental Network Associations (ATHENA) to integrate high-throughput SNPs and gene expression variables (EVs) to predict high-density lipoprotein cholesterol (HDL-C) levels. We generated multivariable models that consisted of SNPs only, EVs only, and SNPs + EVs with testing r-squared values of 0.16, 0.11, and 0.18, respectively. Additionally, using just the SNPs and EVs from the best models, we generated a model with a testing r-squared of 0.32. A linear regression model with the same variables resulted in an adjusted r-squared of 0.23. With this systems biology approach, we were able to integrate different types of high-throughput data to generate meta-dimensional models that are predictive for the HDL-C in our data set. Additionally, our modeling method was able to capture more of the HDL-C variation than a linear regression model that included the same variables. PMID:23424143

  4. Functional expression of double-stranded RNA-dependent protein kinase in rat intestinal epithelial cells.

    PubMed

    Sato, Nagahiro; Morimoto, Hiroyuki; Baba, Ryoko; Nakamata, Junichi; Doi, Yoshiaki; Yamaguchi, Koji

    2010-05-01

    Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs. PMID:20213745

  5. Early MyD88-dependent induction of interleukin-17A expression during Salmonella colitis.

    PubMed

    Keestra, A Marijke; Godinez, Ivan; Xavier, Mariana N; Winter, Maria G; Winter, Sebastian E; Tsolis, Renée M; Bäumler, Andreas J

    2011-08-01

    The development of T helper 17 (T(H)17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1β. IL-23 and IL-1β synergized in inducing expression of IL-17A in splenic T cells in vitro. In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, T(H)17 cells, and CD4(-)CD8(-) T cells. The absence of IL-1β signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis. PMID:21576324

  6. Differentially Expressed Proteins and Associated Histological and Disease Progression Changes in Cotyledon Tissue of a Resistant and Susceptible Genotype of Brassica napus Infected with Sclerotinia sclerotiorum

    PubMed Central

    Garg, Harsh; Li, Hua; Sivasithamparam, Krishnapillai; Barbetti, Martin J.

    2013-01-01

    Sclerotinia rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. To understand the resistance mechanisms in the Brassica napus to S. sclerotiorum, comparative disease progression, histological and proteomic studies were conducted of two B. napus genotypes (resistant cv. Charlton, susceptible cv. RQ001-02M2). At 72 and 96 h post inoculation (hpi), lesion size on cotyledons was significantly (P≤0.001) smaller in the resistant Charlton. Anatomical investigations revealed impeded fungal growth (at 24 hpi and onwards) and hyphal disintegration only on resistant Charlton. Temporal changes (12, 24, 48 and 72 hpi) in protein profile showed certain enzymes up-regulated only in resistant Charlton, such as those related to primary metabolic pathways, antioxidant defence, ethylene biosynthesis, pathogenesis related proteins, protein synthesis and protein folding, play a role in mediating defence responses against S. sclerotiorum. Similarly a eukaryotic translation initiation factor 5A enzyme with increased abundance in susceptible RQ001-02M2 and decreased levels in resistant Charlton has a role in increased susceptibility to this pathogen. This is the first time that the expression of these enzymes has been shown to be associated with mediating the defence response against S. sclerotinia in cotyledon tissue of a resistant cultivar of B. napus at a proteomics level. This study not only provides important new insights into the resistance mechanisms within B. napus against S. sclerotiorum, but opens the way for novel engineering of new B. napus varieties that over-express these key enzymes as a strategy to enhance resistance and better manage this devastating pathogen. PMID:23776450

  7. Hepatitis C virus (HCV) proteins induce NADPH oxidase 4 expression in a transforming growth factor beta-dependent manner: a new contributor to HCV-induced oxidative stress.

    PubMed

    Boudreau, Howard E; Emerson, Suzanne U; Korzeniowska, Agnieszka; Jendrysik, Meghan A; Leto, Thomas L

    2009-12-01

    Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor beta (TGF-beta) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an approximately 2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-beta1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-beta or the expression of dominant negative TGF-beta receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-beta-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease. PMID:19812163

  8. The activity of the TRP-like channel depends on its expression system

    PubMed Central

    Lev, Shaya; Katz, Ben; Minke, Baruch

    2012-01-01

    The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation. PMID:22627924

  9. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  10. Epigenomics and bolting tolerance in sugar beet genotypes.

    PubMed

    Hébrard, Claire; Peterson, Daniel G; Willems, Glenda; Delaunay, Alain; Jesson, Béline; Lefèbvre, Marc; Barnes, Steve; Maury, Stéphane

    2016-01-01

    In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement. PMID:26463996

  11. Epigenomics and bolting tolerance in sugar beet genotypes

    PubMed Central

    Hébrard, Claire; Peterson, Daniel G.; Willems, Glenda; Delaunay, Alain; Jesson, Béline; Lefèbvre, Marc; Barnes, Steve; Maury, Stéphane

    2016-01-01

    In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement. PMID:26463996

  12. Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway.

    PubMed

    Graham, C H; Fitzpatrick, T E; McCrae, K R

    1998-05-01

    Hypoxia underlies a number of biologic processes in which cellular migration and invasion occur. Because earlier studies have shown that the receptor for urokinase-type plasminogen activator (uPAR) may facilitate such events, we studied the effect of hypoxia on the expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo) and human umbilical vein endothelial cells (HUVEC). Compared with control cells cultured under standard conditions (20% O2), HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and [125I]-prourokinase ligand binding analyses. Increased uPAR expression paralleled increases in uPAR mRNA. The involvement of a heme protein in the hypoxia-induced expression of uPAR was suggested by the observations that culture of cells with cobalt chloride, or sodium 4, 5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent, also stimulated uPAR expression, and that the hypoxia-induced uPAR expression was inhibited by adding carbon monoxide to the hypoxic atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that the hypoxia-mediated effect on uPAR expression by these cells did not occur through a VEGF-dependent mechanism. The functional importance of these findings is suggested by the fact that HTR-8/SVneo cells cultured under hypoxia displayed higher levels of cell surface plasminogen activator activity and greater invasion through a reconstituted basement membrane. These results suggest that hypoxia may promote cellular invasion by stimulating the expression of uPAR through a heme protein-dependent pathway. PMID:9558386

  13. An allograft glioma model reveals the dependence of aquaporin-4 expression on the brain microenvironment.

    PubMed

    Noell, Susan; Ritz, Rainer; Wolburg-Buchholz, Karen; Wolburg, Hartwig; Fallier-Becker, Petra

    2012-01-01

    Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don't express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs. PMID:22590566

  14. An Allograft Glioma Model Reveals the Dependence of Aquaporin-4 Expression on the Brain Microenvironment

    PubMed Central

    Noell, Susan; Ritz, Rainer; Wolburg-Buchholz, Karen; Wolburg, Hartwig; Fallier-Becker, Petra

    2012-01-01

    Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don’t express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs. PMID:22590566

  15. GalNAc-T14 promotes metastasis through Wnt dependent HOXB9 expression in lung adenocarcinoma.

    PubMed

    Kwon, Ok-Seon; Oh, Ensel; Park, Jeong-Rak; Lee, Ji-Seon; Bae, Gab-Yong; Koo, Jae-Hyung; Kim, Hyongbum; Choi, Yoon L; Choi, Young Soo; Kim, Jhingook; Cha, Hyuk-Jin

    2015-12-01

    While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the β-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of β-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting β-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC. PMID:26544896

  16. Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo.

    PubMed

    Banerjee, Daliya; Zhao, Linlin; Wu, Lan; Palanichamy, Arumugam; Ergun, Ayla; Peng, Liaomin; Quigley, Catherine; Hamann, Stefan; Dunstan, Robert; Cullen, Patrick; Allaire, Norm; Guertin, Kevin; Wang, Tao; Chao, Jianhua; Loh, Christine; Fontenot, Jason D

    2016-04-01

    Retinoic acid receptor-related orphan nuclear receptor γ (RORγ) orchestrates a pro-inflammatory gene expression programme in multiple lymphocyte lineages including T helper type 17 (Th17) cells, γδ T cells, innate lymphoid cells and lymphoid tissue inducer cells. There is compelling evidence that RORγ-expressing cells are relevant targets for therapeutic intervention in the treatment of autoimmune and inflammatory diseases. Unlike Th17 cells, where RORγ expression is induced under specific pro-inflammatory conditions, γδ T cells and other innate-like immune cells express RORγ in the steady state. Small molecule mediated disruption of RORγ function in cells with pre-existing RORγ transcriptional complexes represents a significant and challenging pharmacological hurdle. We present data demonstrating that a novel, selective and potent small molecule RORγ inhibitor can block the RORγ-dependent gene expression programme in both Th17 cells and RORγ-expressing γδ T cells as well as a disease-relevant subset of human RORγ-expressing memory T cells. Importantly, systemic administration of this inhibitor in vivo limits pathology in an innate lymphocyte-driven mouse model of psoriasis. PMID:26694902

  17. The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression

    PubMed Central

    2012-01-01

    Background Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised. Results Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. Conclusions The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research. PMID:22251276

  18. Blood group genotyping in a population of highly diverse ancestry.

    PubMed

    Pellegrino, J; Castilho, L; Rios, M; De Souza, C A

    2001-01-01

    Accurate phenotyping of red blood cells (RBCs) can be difficult in transfusion-dependent patients such as those with thalassemia and sickle cell anemia because of the presence of previously transfused RBCs in the patient's circulation. Recently, the molecular basis associated with the expression of many blood group antigens was established. This allowed the development of a plethora of polymerase chain reaction (PCR)-based tests for identification of the blood group antigens by testing DNA. The new technologies complement phenotyping and overcome some of the limitations of hemagglutination assays. These molecular assays were developed on the basis of DNA sequences of individuals of Caucasian ancestry. The present study addresses the concern that these genotyping assays may not be applicable to populations of highly diverse ancestry because of variability in intronic regions or because of unrecognized alleles. We determined both phenotype and genotype for RH D, K 1/K 2, JK A/JK B, FY A/ FY B-GATA in 250 normal blood donors using PCR. Phenotype and genotype results agreed in 100% of the cases, indicating that molecular genotyping protocols can be effectively applied to populations with a highly diverse genetic background. However, genotyping for Duffy antigens provided information that could not be obtained by phenotyping. Essentially, 30.5 % of the donors with the FY B gene typed as Fy(b-) because of mutations in the GATA box. This information is very useful for the management of transfusion dependent patients. PMID:11170227

  19. Proteomic Analysis of Terminalia chebula Extract-Dependent Changes in Human Lymphoblastic T Cell Protein Expression

    PubMed Central

    Das, Nando Dulal; Jung, Kyoung Hwa; Park, Ji Hyun; Choi, Mi Ran; Lee, Hyung Tae; Kim, Moo Sung; Lee, Sang Rin

    2012-01-01

    Abstract Terminalia chebula is a native plant from southern Asia to southwestern China that is used in traditional medicine for the treatment of malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. The present study assessed T. chebula extract-dependent protein expression changes in Jurkat cells. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry and Ingenuity Pathways Analysis (IPA) were performed to assess protein expression and networks, respectively. A comparative proteomic profile was determined in T. chebula extract (50 μg/mL)-treated and control cells; the expressions of β-tubulin, ring finger and CHY zinc finger domain containing 1, and insulin-like growth factor 1 receptor kinase were significantly down-regulated in T. chebula extract-treated Jurkat cells. Moreover, the molecular basis for the T. chebula extract-dependent protein expression changes in Jurkat cells was determined by IPA. Treatment with the T. chebula extract significantly inhibited nuclear factor-κB activity and affected the proteomic profile of Jurkat cells. The molecular network signatures and functional proteomics obtained in this study may facilitate the evaluation of potential antitumor therapeutic targets and elucidate the molecular mechanism of T. chebula extract-dependent effects in Jurkat cells. PMID:22471968

  20. The distribution and time-dependent expression of MAGL during skeletal muscle wound healing in rats.

    PubMed

    Jiang, Shu-Kun; Zhang, Miao; Tian, Zhi-Ling; Wang, Lin-Lin; Zhao, Rui; Li, Shan-shan; Liu, Min; Wang, Meng; Guan, Da-Wei

    2015-10-01

    Monoacylglycerol lipase (MAGL) is widely distributed in mammals and largely responsible for metabolizing 2-arachidonoylglycerol (2-AG). Little is known about its expression in skeletal muscles after trauma. A preliminary study on time-dependent expression and distribution of MAGL was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR (qPCR) during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17 and 21 days after contusion, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. Weak immunoreactivity of MAGL was observed in the sarcoplasm of myofibers in control rats. Intensive immunoreactivities of MAGL were observed in polymorphonuclear cells (PMNs), round-shaped mononuclear cells (MNCs), spindle-shaped fibroblastic cells (FBCs) and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as MAGL-positive cells by double immunofluorescent procedure. MAGL expression was remarkably up-regulated after contusion by qPCR and Western blot analysis. The results demonstrate that the expression of MAGL is distributed in certain cell types and time-dependently expressed in skeletal muscles after trauma, suggesting that MAGL may be involved in inflammatory response, fibrogenesis and muscle regeneration during skeletal muscle wound healing. PMID:25921063

  1. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

    PubMed

    Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

    2015-07-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. PMID:26037463

  2. COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons.

    PubMed

    Bovetti, Serena; Bonzano, Sara; Garzotto, Donatella; Giannelli, Serena Gea; Iannielli, Angelo; Armentano, Maria; Studer, Michèle; De Marchis, Silvia

    2013-12-01

    COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons. PMID:24227652

  3. Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

    PubMed Central

    Westfall, Susan; Aguilar-Valles, Argel; Mongrain, Valérie; Luheshi, Giamal N.; Cermakian, Nicolas

    2013-01-01

    Many aspects of the immune system, including circulating cytokine levels as well as counts and function of various immune cell types, present circadian rhythms. Notably, the mortality rate of animals subjected to high doses of lipopolysaccharide is dependent on the time of treatment. In addition, the severity of symptoms of various inflammatory conditions follows a daily rhythmic pattern. The mechanisms behind the crosstalk between the circadian and immune systems remain elusive. Here we demonstrate that localized inflammation induced by turpentine oil (TURP) causes a time-dependent induction of interleukin (IL)-6 and has time-, gene- and tissue-specific effects on clock gene expression. More precisely, TURP blunts the peak of Per1 and Per2 expression in the liver while in other tissues, the expression nadir is elevated. In contrast, Rev-erbα expression remains relatively unaffected by TURP treatment. Co-treatment with the anti-inflammatory agent IL-1 receptor antagonist (IL-1Ra) did not alter the response of Per2 to TURP treatment in liver, despite the reduced induction of fever and IL-6 serum levels. This indicates that the TURP-mediated changes of Per2 in the liver might be due to factors other than systemic IL-6 and fever. Accordingly, IL-6 treatment had no effect on clock gene expression in HepG2 liver carcinoma cells. Altogether, we show that localized inflammation causes significant time-dependent changes in peripheral circadian clock gene expression, via a mechanism likely involving mediators independent from IL-6 and fever. PMID:23527270

  4. Condition-dependent expression of melanin-based coloration in the Eurasian kestrel

    NASA Astrophysics Data System (ADS)

    Piault, Romain; van den Brink, Valentijn; Roulin, Alexandre

    2012-05-01

    Melanin is the most common pigment in animal integuments and is responsible for some of the most striking ornaments. A central tenet of sexual selection theory states that melanin-based traits can signal absolute individual quality in any environment only if their expression is condition-dependent. Significant costs imposed by an ornament would ensure that only the highest quality individuals display the most exaggerated forms of the signal. Firm evidence that melanin-based traits can be condition-dependent is still rare in birds. In an experimental test of this central assumption, we report condition-dependent expression of a melanin-based trait in the Eurasian kestrel ( Falco tinnunculus). We manipulated nestling body condition by reducing or increasing the number of nestlings soon after hatching. A few days before fledging, we measured the width of sub-terminal black bands on the tail feathers. Compared to nestlings from enlarged broods, individuals raised in reduced broods were in better condition and thereby developed larger sub-terminal bands. Furthermore, in 2 years, first-born nestlings also developed larger sub-terminal bands than their younger siblings that are in poorer condition. This demonstrates that expression of melanin-based traits can be condition-dependent.

  5. Cellular mechanisms of activity-dependent BDNF expression in primary sensory neurons.

    PubMed

    Vermehren-Schmaedick, A; Khanjian, R A; Balkowiec, A

    2015-12-01

    Brain-derived neurotrophic factor (BDNF) is abundantly expressed by both developing and adult rat visceral sensory neurons from the nodose ganglion (NG) in vivo and in vitro. We have previously shown that BDNF is released from neonatal NG neurons by activity and regulates dendritic development in their postsynaptic targets in the brainstem. The current study was carried out to examine the cellular and molecular mechanisms of activity-dependent BDNF expression in neonatal rat NG neurons, using our established in vitro model of neuronal activation by electrical field stimulation with patterns that mimic neuronal activity in vivo. We show that BDNF mRNA (transcript 4) increases over threefold in response to a 4-h tonic or bursting pattern delivered at the frequency of 6 Hz, which corresponds to the normal heart rate of a newborn rat. No significant increase in BDNF expression was observed following stimulation at 1 Hz. The latter effect suggests a frequency-dependent mechanism of regulated BDNF expression. In addition to BDNF transcript 4, which is known to be regulated by activity, transcript 1 also showed significant upregulation. The increases in BDNF mRNA were followed by BDNF protein upregulation of a similar magnitude after 24h of stimulation at 6 Hz. Electrical stimulation-evoked BDNF expression was inhibited by pretreating neurons with the blocker of voltage-gated sodium channels tetrodotoxin and by removing extracellular calcium. Moreover, our data show that repetitive stimulation-evoked BDNF expression requires calcium influx through N-, but not L-type, channels. Together, our study reveals novel mechanisms through which electrical activity stimulates de novo synthesis of BDNF in sensory neurons, and points to the role of N-type calcium channels in regulating BDNF expression in sensory neurons in response to repetitive stimulation. PMID:26459016

  6. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    SciTech Connect

    Singh, Raman Deep Schroeder, Andreas S.; Scheffer, Luana; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L. Pagano, Richard E.

    2013-05-10

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  7. Do sexual ornaments demonstrate heightened condition-dependent expression as predicted by the handicap hypothesis?

    PubMed Central

    Cotton, Samuel; Fowler, Kevin; Pomiankowski, Andrew

    2004-01-01

    The handicap hypothesis of sexual selection predicts that sexual ornaments have evolved heightened condition-dependent expression. The prediction has only recently been subject to experimental investigation. Many of the experiments are of limited value as they: (i) fail to compare condition dependence in sexual ornaments with suitable non-sexual trait controls; (ii) do not adequately account for body size variation; and (iii) typically consider no stress and extreme stress manipulations rather than a range of stresses similar to those experienced in nature. There is also a dearth of experimental studies investigating the genetic basis of condition dependence. Despite the common claim that sexual ornaments are condition-dependent, the unexpected conclusion from our literature review is that there is little support from well-designed experiments. PMID:15255094

  8. Transcriptome Analysis of Duck Liver and Identification of Differentially Expressed Transcripts in Response to Duck Hepatitis A Virus Genotype C Infection

    PubMed Central

    Zhang, Huanrong; Ma, Jing; Yue, Hua

    2013-01-01

    Background Duck is an economically important poultry and animal model for human viral hepatitis B. However, the molecular mechanisms underlying host–virus interaction remain unclear because of limited information on the duck genome. This study aims to characterize the duck normal liver transcriptome and to identify the differentially expressed transcripts at 24 h after duck hepatitis A virus genotype C (DHAV-C) infection using Illumina–Solexa sequencing. Results After removal of low-quality sequences and assembly, a total of 52,757 unigenes was obtained from the normal liver group. Further blast analysis showed that 18,918 unigenes successfully matched the known genes in the database. GO analysis revealed that 25,116 unigenes took part in 61 categories of biological processes, cellular components, and molecular functions. Among the 25 clusters of orthologous group categories (COG), the cluster for “General function prediction only” represented the largest group, followed by “Transcription” and “Replication, recombination, and repair.” KEGG analysis showed that 17,628 unigenes were involved in 301 pathways. Through comparison of normal and infected transcriptome data, we identified 20 significantly differentially expressed unigenes, which were further confirmed by real-time polymerase chain reaction. Of the 20 unigenes, nine matched the known genes in the database, including three up-regulated genes (virus replicase polyprotein, LRRC3B, and PCK1) and six down-regulated genes (CRP, AICL-like 2, L1CAM, CYB26A1, CHAC1, and ADAM32). The remaining 11 novel unigenes that did not match any known genes in the database may provide a basis for the discovery of new transcripts associated with infection. Conclusion This study provided a gene expression pattern for normal duck liver and for the previously unrecognized changes in gene transcription that are altered during DHAV-C infection. Our data revealed useful information for future studies on the duck genome

  9. Strain magnitude-dependent calcific marker expression in valvular and vascular cells.

    PubMed

    Ferdous, Zannatul; Jo, Hanjoong; Nerem, Robert M

    2013-01-01

    Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study is that bone morphogenic protein (BMP)-2 and BMP-4 and transforming growth factor-β1 mRNA expression significantly changed in response to the magnitude of applied strain in valvular cells, while the lowest expression was observed for the representative physiological strain. On the other hand, mRNA expression in vascular cells did not vary in response to the magnitude of strain. Regarding BMP-2 and BMP-4 protein expression determined by immunostaining, trends were similar to mRNA expression in vascular and valvular cells, where only valvular cells showed a varied protein expression depending on the magnitude of the strain applied. Our results suggest that cellular differences exist between vascular and valvular cells in their response to altered levels of cyclic strain during calcification. PMID:23548742

  10. Lhx4 Deficiency: Increased Cyclin-Dependent Kinase Inhibitor Expression and Pituitary Hypoplasia

    PubMed Central

    Gergics, Peter; Brinkmeier, Michelle L.

    2015-01-01

    Defects in the Lhx4, Lhx3, and Pitx2 genes can cause combined pituitary hormone deficiency and pituitary hypoplasia in both humans and mice. Not much is known about the mechanism underlying hypoplasia in these mutants beyond generally increased cell death and poorly maintained proliferation. We identified both common and unique abnormalities in developmental regulation of key cell cycle regulator gene expression in each of these three mutants. All three mutants exhibit reduced expression of the proliferative marker Ki67 and the transitional marker p57. We discovered that expression of the cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) is expanded dorsally in the pituitary primordium of both Lhx3 and Lhx4 mutants. Uniquely, Lhx4 mutants exhibit reduced cyclin D1 expression and have auxiliary pouch-like structures. We show evidence for indirect and direct effects of LHX4 on p21 expression in αT3-1 pituitary cells. In summary, Lhx4 is necessary for efficient pituitary progenitor cell proliferation and restriction of p21 expression. PMID:25668206

  11. Sugar-Dependent Gibberellin-Induced Chalcone Synthase Gene Expression in Petunia Corollas.

    PubMed Central

    Moalem-Beno, D.; Tamari, G.; Leitner-Dagan, Y.; Borochov, A.; Weiss, D.

    1997-01-01

    The induction of anthocyanin synthesis and anthocyanin biosynthetic gene expression in detached petunia (Petunia hybrida) corollas by gibberellic acid (GA3) requires sucrose. Neither sucrose nor GA3 alone can induce these processes. We found that GA3 enhances sucrose uptake by 20 to 30%, and we tested whether this is the mechanism by which the hormone induces gene expression. Changing the intracellular level of sucrose with the inhibitors p-chloromercuribenzenesulfonic acid and vanadate did not inhibit the induction of chalcone synthase gene (chs) expression by GA3. Growing detached corollas in various sucrose concentrations did not affect the induction of the gene but did affect its level of expression and the level of anthocyanin accumulated. Only metabolic sugars promoted GA3-induced anthocyanin accumulation. Mannitol and sorbitol had no effect and 3-O-methylglucose only slightly promoted chs expression and anthocyanin accumulation. Our results do not support the suggestion that sugars act as specific signals in the activation of anthocyanin biosynthetic gene expression during petunia corolla development. We suggest that sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent. PMID:12223616

  12. Temperature-dependent reaction-rate expression for oxygen recombination at Shuttle entry conditions

    NASA Technical Reports Server (NTRS)

    Zoby, E. V.; Simmonds, A. L.; Gupta, R. N.

    1984-01-01

    A temperature-dependent oxygen surface reaction-rate coefficient has been determined from experimental STS-2 heating and wall temperature data at altitudes of 77.91 km, 74.98 km, and 71.29 km. The coefficient is presented in an Arrhenius form and is shown to be less temperature dependent than previous results. Finite-rate viscous-shock-layer heating rates based on this present expression have been compared with predicted heating rates using the previous rate coefficients and with experimental heating data obtained over an extensive range of STS-2 and STS-3 entry conditions. A substantial improvement is obtained in comparison of experimental data and predicted heating rates using the present oxygen reaction-rate expression.

  13. Oncogenic KRAS Impairs EGFR Antibodies' Efficiency by C/EBPβ-Dependent Suppression of EGFR Expression12

    PubMed Central

    Derer, Stefanie; Berger, Sven; Schlaeth, Martin; Schneider-Merck, Tanja; Klausz, Katja; Lohse, Stefan; Overdijk, Marije B; Dechant, Michael; Kellner, Christian; Nagelmeier, Iris; Scheel, Andreas H; Lammerts van Bueren, Jeroen J; van de Winkel, Jan GJ; Parren, Paul WHI; Peipp, Matthias; Valerius, Thomas

    2012-01-01

    Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRASG12V) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRASG12V on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRASG12V impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRASG12V also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs—such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRASG12V downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPβ-LIP. Thus, siRNA-mediated knockdown of C/EBPβ led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRASG12V signaling induced C/EBPβ-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents. PMID

  14. Testosterone regulates FGF-2 expression during testis maturation by an IRES-dependent translational mechanism.

    PubMed

    Gonzalez-Herrera, Irma G; Prado-Lourenco, Leonel; Pileur, Frédéric; Conte, Caroline; Morin, Aurélie; Cabon, Florence; Prats, Hervé; Vagner, Stephan; Bayard, Francis; Audigier, Sylvie; Prats, Anne-Catherine

    2006-03-01

    Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5'UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process. PMID:16423876

  15. Condition-dependent expression of pre- and postcopulatory sexual traits in guppies.

    PubMed

    Rahman, Md Moshiur; Kelley, Jennifer L; Evans, Jonathan P

    2013-07-01

    Female choice can impose persistent directional selection on male sexually selected traits, yet such traits often exhibit high levels of phenotypic variation. One explanation for this paradox is that if sexually selected traits are costly, only the fittest males are able to acquire and allocate the resources required for their expression. Furthermore, because male condition is dependent on resource allocation, condition dependence in sexual traits is expected to underlie trade-offs between reproduction and other life-history functions. In this study we test these ideas by experimentally manipulating diet quality (carotenoid levels) and quantity in the guppy (Poecilia reticulata), a livebearing freshwater fish that is an important model for understanding relationships between pre- and post-copulatory sexually selected traits. Specifically, we test for condition dependence in the expression of pre- and postcopulatory sexual traits (behavior, ornamentation, sperm traits) and determine whether diet manipulation mediates relationships among these traits. Consistent with prior work we found a significant effect of diet quantity on the expression of both pre- and postcopulatory male traits; diet-restricted males performed fewer sexual behaviors and exhibited significant reductions in color ornamentation, sperm quality, sperm number, and sperm length than those fed ad libitum. However, contrary to our expectations, we found no significant effect of carotenoid manipulation on the expression of any of these traits, and no evidence for a trade-off in resource allocation between pre- and postcopulatory episodes of sexual selection. Our results further underscore the sensitivity of behavioral, ornamental, and ejaculate traits to dietary stress, and highlight the important role of condition dependence in maintaining the high variability in male sexual traits. PMID:23919162

  16. Condition-dependent expression of pre- and postcopulatory sexual traits in guppies

    PubMed Central

    Rahman, Md Moshiur; Kelley, Jennifer L; Evans, Jonathan P

    2013-01-01

    Female choice can impose persistent directional selection on male sexually selected traits, yet such traits often exhibit high levels of phenotypic variation. One explanation for this paradox is that if sexually selected traits are costly, only the fittest males are able to acquire and allocate the resources required for their expression. Furthermore, because male condition is dependent on resource allocation, condition dependence in sexual traits is expected to underlie trade-offs between reproduction and other life-history functions. In this study we test these ideas by experimentally manipulating diet quality (carotenoid levels) and quantity in the guppy (Poecilia reticulata), a livebearing freshwater fish that is an important model for understanding relationships between pre- and post-copulatory sexually selected traits. Specifically, we test for condition dependence in the expression of pre- and postcopulatory sexual traits (behavior, ornamentation, sperm traits) and determine whether diet manipulation mediates relationships among these traits. Consistent with prior work we found a significant effect of diet quantity on the expression of both pre- and postcopulatory male traits; diet-restricted males performed fewer sexual behaviors and exhibited significant reductions in color ornamentation, sperm quality, sperm number, and sperm length than those fed ad libitum. However, contrary to our expectations, we found no significant effect of carotenoid manipulation on the expression of any of these traits, and no evidence for a trade-off in resource allocation between pre- and postcopulatory episodes of sexual selection. Our results further underscore the sensitivity of behavioral, ornamental, and ejaculate traits to dietary stress, and highlight the important role of condition dependence in maintaining the high variability in male sexual traits. PMID:23919162

  17. Protein kinase C-dependent regulation of human hepatic drug transporter expression.

    PubMed

    Mayati, Abdullah; Le Vee, Marc; Moreau, Amélie; Jouan, Elodie; Bucher, Simon; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2015-12-15

    Hepatic drug transporters are now recognized as major actors of hepatobiliary elimination of drugs. Characterization of their regulatory pathways is therefore an important issue. In this context, the present study was designed to analyze the potential regulation of human hepatic transporter expression by protein kinase C (PKC) activation. Treatment by the reference PKC activator phorbol 12-myristate 13-acetate (PMA) for 48h was shown to decrease mRNA expression of various sinusoidal transporters, including OATP1B1, OATP2B1, NTCP, OCT1 and MRP3, but to increase that of OATP1B3, whereas mRNA expression of canalicular transporters was transiently enhanced (MDR1), decreased (BSEP and MRP2) or unchanged (BCRP) in human hepatoma HepaRG cells. The profile of hepatic transporter mRNA expression changes in PMA-treated HepaRG cells was correlated to that found in PMA-exposed primary human hepatocytes and was similarly observed in response to the PKC-activating marketed drug ingenol mebutate. It was associated with concomitant repression of OATP1B1 and OATP2B1 protein expression and reduction of OATP, OCT1, NTCP and MRP2 activity. The use of chemical PKC inhibitors further suggested a contribution of novel PKCs isoforms to PMA-mediated regulations of transporter mRNA expression. PMA was finally shown to cause epithelial-mesenchymal transition (EMT) in HepaRG cells and exposure to various additional EMT inducers, i.e., hepatocyte growth factor, tumor growth factor-β1 or the HNF4α inhibitor BI6015, led to transporter expression alterations highly correlated to those triggered by PMA. Taken together, these data highlight PKC-dependent regulation of human hepatic drug transporter expression, which may be closely linked to EMT triggered by PKC activation. PMID:26462574

  18. Combination of microRNA expression profiling with genome-wide SNP genotyping to construct a coronary artery disease-related miRNA-miRNA synergistic network.

    PubMed

    Hua, Lin; Xia, Hong; Zhou, Ping; Li, Dongguo; Li, Lin

    2014-12-01

    In recent years, microRNAs (miRNAs) were found to play critical roles in many important biological processes. On the other hand, the rapid development of genome-wide association studies (GWAS) help identify potential genetic variants associated with the disease phenotypic variance. Therefore, we suggested a combined analysis of microRNA expression profiling with genome-wide Single Nucleotide Polymorphism (SNP) genotyping to identify potential disease-related biomarkers. Considering functional SNPs in miRNA genes or target sites might be important signals associated with human complex diseases, we constructed a miRNA-miRNA synergistic network related to coronary artery disease (CAD) by performing a genome-wide scan for SNPs in human miRNA 3' -untranslated regions (UTRs) target sites and computed potential SNP cooperation effects contributing to disease based on potential miRNA-SNP interactions reported recently. Furthermore, we identified some potential CAD-related miRNAs by analyzing the constructed miRNAmiRNA synergistic network. As a result, the predicted miRNA-miRNA network and miRNA clusters were validated by significantly high interaction effects of CAD-related miRNAs. Accurate classification performances were obtained for all of the identified miRNA clusters, and the sensitivity and specificity were all more than 90%. The network topological analysis confirmed some novel CAD-related miRNAs identified recently by experiments. Our method might help to understand miRNA function and CAD disease, as well as to explore the novel mechanisms involved. PMID:25641175

  19. Cigarette smoke condensate induces aryl hydrocarbon receptor-dependent changes in gene expression in spermatocytes.

    PubMed

    Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Moley, Kelle H

    2012-12-01

    Cigarette smoke contains numerous compounds that cause oxidative stress and alter gene expression in many tissues, and cigarette smoking is correlated with male infertility. To identify mechanisms by which this occurs, we evaluated expression of antioxidant genes in mouse spermatocytes in response to cigarette smoke condensate (CSC). CSC exposure led to oxidative stress and dose-dependent up-regulation of Hsp90aa1, Ahr, Arnt, Sod1, Sod2, and Cyp1a1 expression in a mouse spermatocyte cell line. An antagonist of the aryl hydrocarbon receptor (AHR) abrogated several CSC-mediated changes in mRNA and protein levels. Consistent with these results, spermatocytes isolated by laser-capture microdissection from CSC-treated mice showed increased expression of several antioxidant genes. In vivo exposure to CSC was genotoxic to spermatocytes, resulting in apoptosis and disruptions to the seminiferous tubules. Our in vivo and in vitro data indicate that CSC-mediated damage to murine spermatocytes is AHR-dependent and is mediated by oxidative stress. PMID:23069111

  20. Plasticity of chemoreceptor gene expression: Sensory and circuit inputs modulate state-dependent chemoreceptors.

    PubMed

    Gruner, Matthew; van der Linden, Alexander M

    2015-01-01

    Animals dramatically modify their chemosensory behaviors when starved, which could allow them to alter and optimize their food-search strategies. Dynamic changes in the gene expression of chemoreceptors may be a general mechanism underlying food and state-dependent changes in chemosensory behaviors. In our recent study,(1) we identified chemoreceptors in the ADL sensory neuron type of C. elegans that are modulated by feeding state and food availability. Here, we highllight our recent findings by which sensory inputs into ADL, neuronal outputs from ADL, and circuit inputs from the RMG interneuron, which is electrically connected to ADL, are required to regulate an ADL-expressed chemoreceptor. This sensory and circuit-mediated regulation of chemoreceptor gene expression is dependent on cell-autonomous pathways acting in ADL, e.g. KIN-29, DAF-2, OCR-2 and calcium signaling, and circuit inputs from RMG mediated by NPR-1. Based on these findings, we propose an intriguing but speculative feedback modulatory circuit mechanism by which sensory perception of food and internal state signals may be coupled to regulate ADL-expressed chemoreceptors, which may allow animals to precisely regulate and fine-tune their chemosensory neuron responses as a function of feeding state. PMID:26430563

  1. Serum-dependent expression of promyelocytic leukemia protein suppresses propagation of influenza virus

    SciTech Connect

    Iki, Shigeo; Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Nagata, Kyosuke; Fujii, Nobuhiro . E-mail: fujii@sapmed.ac.jp

    2005-12-05

    The rate of propagation of influenza virus in human adenocarcinoma Caco-2 cells was found to negatively correlate with the concentration of fetal bovine serum (FBS) in the culture medium. Virus replicated more rapidly at lower FBS concentrations (0 or 2%) than at higher concentrations (10 or 20%) during an early stage of infection. Basal and interferon (IFN)-induced levels of typical IFN-inducible anti-viral proteins, such as 2',5'-oligoadenylate synthetase, dsRNA-activated protein kinase and MxA, were unaffected by variation in FBS concentrations. But promyelocytic leukemia protein (PML) was expressed in a serum-dependent manner. In particular, the 65 to 70 kDa isoform of PML was markedly upregulated following the addition of serum. In contrast, other isoforms were induced by IFN treatment, and weakly induced by FBS concentrations. Immunofluorescence microscopy indicated that PML was mainly formed nuclear bodies in Caco-2 cells at various FBS concentrations, and the levels of the PML-nuclear bodies were upregulated by FBS. Overexpression of PML isoform consisting of 560 or 633 amino acid residues by transfection of expression plasmid results in significantly delayed viral replication rate in Caco-2 cells. On the other hand, downregulation of PML expression by RNAi enhanced viral replication. These results indicate that PML isoforms which are expressed in a serum-dependent manner suppress the propagation of influenza virus at an early stage of infection.

  2. Involvement of UTR-dependent gene expression in the maintenance of cancer stem cell like phenotypes

    PubMed Central

    YASUDA, MOTOAKI; HATANAKA, TOMOYUKI; SHIRATO, HIROKI; NISHIOKA, TAKESHI

    2015-01-01

    The present study demonstrated the acquisition of additional malignant characteristics in irradiated mouse fibrosarcoma cells compared with the parent cells. Several reporter assays indicated that hypoxia-inducible factor (HIF)-1α, activator protein-1 and Ets-dependent transcription were activated in irradiated cells. The cis-elements in the 5′-untranslated region (UTR) of these transcription factors plays a major role in their expression in surviving irradiated cancer cells. By contrast, there were no evident differences between the 3′-UTR-dependent repression demonstrated by parent cells and irradiated cells. A small population of parental fibrosarcoma cells was also found to exhibit the same enhanced 5′-UTR-dependent HIF-1α expression as that demonstrated by irradiated cells. These observations may indicate that high-dose X-ray irradiation affects the majority of proliferating cancer cells, but not the cancer stem cells (CSCs), and an increased CSC population may explain the progressive phenotypes of the irradiated cells. It appears likely that the transcription factors that maintain stemness are regulated by the same 5′-UTR-dependent mechanism. PMID:26722307

  3. Fighting experience alters brain androgen receptor expression dependent on testosterone status

    PubMed Central

    Li, Cheng-Yu; Earley, Ryan L.; Huang, Shu-Ping; Hsu, Yuying

    2014-01-01

    Contest decisions are influenced by the outcomes of recent fights (winner–loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner–loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner–loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/β (ERα/β), glucocorticoid (GR) and serotonin (5-HT1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner–loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ERα/β, 5-HT1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly. PMID:25320171

  4. Expression of activity-dependent neuroprotective protein in the brain of adult rats.

    PubMed

    Gennet, N; Herden, C; Bubb, V J; Quinn, J P; Kipar, A

    2008-03-01

    Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple functions. We investigated the pattern of ADNP expression by immunohistology in normal rat brain, in order to generate a framework for future studies examining changes in ADNP expression in response to noxious stimuli or in models of disease. We found widespread ADNP-like immunoreactivity in neurons throughout the rat brain, with the highest expression in the cerebellum, and strong expression in the thalamus, mesencephalon, pons and medulla oblongata. ADNP-like immunoreactivity was mainly observed in the cytoplasm of neurons, and fibre tracts were often strongly positive as well. In addition, positive neuronal nuclei were occasionally observed. ADNP-like immunoreactivity was lost in degenerating "dark" neurons, whereas it appeared to locate to the nucleus in some of the morphologically unaltered adjacent cells. Occasional astrocyte and microglial cells were also positive. We suggest that the widespread expression of ADNP may correlate with the wide-ranging protective effects of NAP, and that the cytoplasmic and axonal localisation of ADNP-like immunoreactivity suggests additional, non-transcriptional functions of ADNP. PMID:18072088

  5. [Expression of cyclin-dependent kinase 2-associated protein 1 in chicken embryos of different sexes].

    PubMed

    Yang, Yu; Feng, Yan-Ping; Gong, Ping; Huang, Pan; Li, Shi-Jun; Peng, Xiu-Li; Gong, Yan-Zhang

    2009-09-01

    To investigate the expression and functions of cyclin-dependent kinase 2-associated protein 1 (cdk2ap1) screened by suppression subtractive hybridization in chicken embryo development, a pair of primers was designed to amplify the cdk2ap1 fragment by RT-PCR and subsequently the fragment obtained was cloned into the plasmid pGEM-T. Sense and antisense probes labeled with digoxigenin were generated using SP6 and T7 RNA polymerases, respectively, and used to examine cdk2ap1 expression in chicken embryos of both sexes by whole-mount in situ hybridization. In both sexes, cdk2ap1 was expressed in the head mesenchyme, rhombencephalon, optic vesicles, spinal neural tube, and forelimb of 4.0-day-old embryos and the expression in males was significantly higher than that in females. In addition, in the genital ridge and hindlimb of the 4.0-day-old chicken embryo, cdk2ap1 was obviously expressed in the males but not in females. It is supposed that cdk2ap1 may play a role in the sexual differentiation and development of gonad of chicken embryo. PMID:19819846

  6. Evidence of Autoinducer-Dependent and -Independent Heterogeneous Gene Expression in Sinorhizobium fredii NGR234

    PubMed Central

    Grote, Jessica; Krysciak, Dagmar; Schorn, Andrea; Dahlke, Renate I.; Soonvald, Liina; Müller, Johannes; Hense, Burkhard A.; Schwarzfischer, Michael; Sauter, Margret; Schmeisser, Christel

    2014-01-01

    Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression. PMID:25002427

  7. Evidence of autoinducer-dependent and -independent heterogeneous gene expression in Sinorhizobium fredii NGR234.

    PubMed

    Grote, Jessica; Krysciak, Dagmar; Schorn, Andrea; Dahlke, Renate I; Soonvald, Liina; Müller, Johannes; Hense, Burkhard A; Schwarzfischer, Michael; Sauter, Margret; Schmeisser, Christel; Streit, Wolfgang R

    2014-09-01

    Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression. PMID:25002427

  8. Rosiglitazone Promotes PPARγ-Dependent and -Independent Alterations in Gene Expression in Mouse Islets

    PubMed Central

    El Ouaamari, Abdelfattah; Kawamori, Dan; Meyer, John; Hu, Jiang; Smith, David M.; Kulkarni, Rohit N.

    2012-01-01

    The glitazone class of insulin-sensitizing agents act, in part, by the activation of peroxisome proliferator-activated receptor (PPAR)-γ in adipocytes. However, it is unclear whether the expression of PPARγ in the islets is essential for their potential β-cell-sparing properties. To investigate the in vivo effects of rosiglitazone on β-cell biology, we used an inducible, pancreatic and duodenal homeobox-1 enhancer element-driven, Cre recombinase to knockout PPARγ expression specifically in adult β-cells (PPARgKO). Subjecting the PPARgKO mice to a chow diet led to virtually undetectable changes in glucose or insulin sensitivity, which was paralleled by minimal changes in islet gene expression. Similarly, challenging the mutant mice with a high-fat diet and treatment with rosiglitazone did not alter insulin sensitivity, glucose-stimulated insulin secretion, islet size, or proliferation in the knockout mice despite PPARγ-dependent and -independent changes in islet gene expression. These data suggest that PPARγ expression in the β-cells is unlikely to be directly essential for normal β-cell function or the insulin-sensitizing actions of rosiglitazone. PMID:22807489

  9. Age- and task-dependent foraging gene expression in the bumblebee Bombus terrestris.

    PubMed

    Tobback, Julie; Mommaerts, Veerle; Vandersmissen, Hans Peter; Smagghe, Guy; Huybrechts, Roger

    2011-01-01

    In eusocial insects, the division of labor within a colony, based on either age or size, is correlated with a differential foraging (for) gene expression and PKG activity. This article presents in the first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the open reading frame allowed phylogenetic tracing, which showed conservation of PKGs among social insects. Our results confirm the proposed role for PKGs in division of labor. Btfor gene expression is significantly higher in the larger foragers compared with the smaller sized nurses. More importantly, we discovered an age-related decrease in Btfor expression in both nursing and foraging bumblebees. We therefore speculate that the presence of BtFOR is required for correct adaptation to new external stimuli and rapid learning for foraging. In a second series of experiments, worker bumblebees of B. terrestris were treated with two insecticides imidacloprid and kinoprene, which have shown to cause impaired foraging behavior. Compared with controls, only the latter treatment resulted in a decreased Btfor expression, which concurs with a stimulation of ovarian growth and a shift in labor toward nest-related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides. PMID:21136525

  10. UGT1A1 genotype-dependent dose adjustment of belinostat in patients with advanced cancers using population pharmacokinetic modeling and simulation.

    PubMed

    Peer, Cody J; Goey, Andrew K L; Sissung, Tristan M; Erlich, Sheryl; Lee, Min-Jung; Tomita, Yusuke; Trepel, Jane B; Piekarz, Richard; Balasubramaniam, Sanjeeve; Bates, Susan E; Figg, William D

    2016-04-01

    Belinostat is a second-generation zinc-binding histone deacetylase inhibitor that is approved for peripheral T-cell lymphoma and is currently being studied in small cell lung cancer and other advanced carcinomas as a 48-hour continuous intravenous infusion. Belinostat is predominantly metabolized by UGT1A1, which is polymorphic. Preliminary analyses revealed a difference in belinostat clearance based on UGT1A1 genotype. A 2-compartment population pharmacokinetic (PK) model was developed and validated that incorporated the UGT1A1 genotype, albumin, and creatinine clearance on the clearance parameter; body weight was a significant covariate on volume. Simulated doses of 600 and 400 mg/m(2) /24 h given to patients considered extensive or impaired metabolizers, respectively, provided equivalent AUCs. This model and subsequent simulations supported additional PK/toxicity and pharmacogenomics/toxicity analyses to suggest a UGT1A1 genotype-based dose adjustment to normalize belinostat exposure and allow for more tolerable therapy. In addition, global protein lysine acetylation was modeled with PK and demonstrated a reversible belinostat exposure/response relationship, consistent with previous reports. PMID:26637161

  11. Wavelength-dependent dynamics of heat shock protein 70 expression in free electron laser wounds

    NASA Astrophysics Data System (ADS)

    Wilmink, Gerald J.; Beckham, Joshua T.; Mackanos, Mark; Contag, Christopher H.; Davidson, Jeffrey M.; Jansen, E. D.

    2007-02-01

    Many medical laser procedures require selecting laser operating parameters that minimize undesirable tissue damage. In this study, heat shock protein 70(hsp70) gene expression was used as a sensitive marker for laser-induced thermal damage. Wound repair and hsp70 expression were compared after surgery with the free electron laser(FEL) as a function of wavelength(λ) and radiant exposure(H). Damage was assessed at λ = 6.45, 6.10, and 2.94 μm using 8-20 J/cm2. The FEL beam (\\Vpgr r=200 μm,30Hz,τ p =5μs) was delivered to produce a 6.5 mm square wound. hsp70 expression was assessed using a transgenic mouse strain with the hsp70 promoter driving luciferase and eGFP expression. Bioluminescent imaging (BLI) was monitored non-invasively and in real time. Hsp70 protein was visualized with laser confocal imaging, blood velocity was measured with 2D-laser doppler, and depth of tissue damage was measured using histological methods. BLI verified the model's sensitivity and peak hsp70 expression was bi-phasic, with maxima occurring 12 and 24 hours after FEL irradiation. hsp70 expression exhibited wavelength-dependence, and it increased with radiant exposure. Histology indicated that tissue damage at 6.45 µm was ~2x deeper than 6.10 μm. Quantitative BLI with the Hsp70-luc transgene can be used to non-invasively measure gene expression in laser-tissue interaction studies.

  12. Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism

    PubMed Central

    Frampton, Gabriel; Invernizzi, Pietro; Bernuzzi, Francesca; Pae, Hae Yong; Quinn, Matthew; Horvat, Darijana; Galindo, Cheryl; Huang, Li; McMillin, Matthew; Cooper, Brandon; Rimassa, Lorenza; DeMorrow, Sharon

    2015-01-01

    Background and objectives Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. The growth factor, progranulin, is overexpressed in a number of tumours. The study aims were to assess the expression of progranulin in cholangiocarcinoma and to determine its effects on tumour growth. Methods The expression and secretion of progranulin were evaluated in multiple cholangiocarcinoma cell lines and in clinical samples from patients with cholangiocarcinoma. The role of interleukin 6 (IL-6)-mediated signalling in the expression of progranulin was assessed using a combination of specific inhibitors and shRNA knockdown techniques. The effect of progranulin on proliferation and Akt activation and subsequent effects of FOXO1 phosphorylation were assessed in vitro. Progranulin knockdown cell lines were established, and the effects on cholangiocarcinoma growth were determined. Results Progranulin expression and secretion were upregulated in cholangiocarcinoma cell lines and tissue, which were in part via IL-6-mediated activation of the ERK1/2/RSK1/C/EBPβ pathway. Blocking any of these signalling molecules, by either pharmacological inhibitors or shRNA, prevented the IL-6-dependent activation of progranulin expression. Treatment of cholangiocarcinoma cells with recombinant progranulin increased cell proliferation in vitro by a mechanism involving Akt phosphorylation leading to phosphorylation and nuclear extrusion of FOXO1. Knockdown of progranulin expression in cholangiocarcinoma cells decreased the expression of proliferating cellular nuclear antigen, a marker of proliferative capacity, and slowed tumour growth in vivo. Conclusions Evidence is presented for a role for progranulin as a novel growth factor regulating cholangiocarcinoma growth. Specific targeting of progranulin may represent an alternative for the development of therapeutic strategies. PMID:22068162

  13. Tissue- and age-dependent expression of the bovine DEFB103 gene and protein.

    PubMed

    Mirabzadeh-Ardakani, Ali; Solie, Jay; Gonzalez-Cano, Patricia; Schmutz, Sheila M; Griebel, Philip J

    2016-02-01

    Beta-defensin 103 (DEFB103) shares little homology with 8 other members of the bovine beta-defensin family and in other species DEFB103 protein has diverse functions, including antimicrobial activity, a chemoattractant for dendritic cells, enhancing epithelial wound repair and regulating hair colour. Expression of the bovine DEFB103 gene was surveyed in 27 tissues and transcript was most abundant in tissues with stratified squamous epithelium. Oral cavity epithelial tissues and nictitating membrane consistently expressed high levels of DEFB103 gene transcript. An age-dependent decrease (P < 0.05) in DEFB103 gene expression was only observed for buccal epithelium when comparing healthy 10- to 14-day-old and 10- to 12-month-old calves. A bovine herpesvirus-1 respiratory infection did, however, significantly (P < 0.05) up-regulate DEFB103 gene expression in the buccal epithelium of 6- to 8-month-old calves. Finally, DEFB103 transcript was low in lymph nodes draining the skin and at the limit of detection in other internal organs such as lung, intestine and kidney. Affinity-purified rabbit antisera to bovine DEFB103 was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells prior to birth. Beta-defensins have been identified as regulators of dendritic cell (DC) chemokine responses and we observed a close association between DCs and epithelial cells expressing DEFB103 in both the fetus and newborn calf. In conclusion, bovine DEFB103 gene expression is most abundant in stratified squamous epithelium with DEFB103 protein localised to basal epithelial cells. These observations are consistent with proposed roles for DEFB103 in DC recruitment and repair of stratified squamous epithelium. PMID:26299200

  14. Cryptochrome expression in the eye of migratory birds depends on their migratory status.

    PubMed

    Fusani, Leonida; Bertolucci, Cristiano; Frigato, Elena; Foà, Augusto

    2014-03-15

    Most passerine birds are nocturnal migrants. When kept in captivity during the migratory periods, these species show a migratory restlessness, or Zugunruhe. Recent studies on Sylvia warblers have shown that Zugunruhe is an excellent proxy of migratory disposition. Passerine birds can use the Earth's geomagnetic field as a compass to keep their course during their migratory flight. Among the candidate magnetoreceptive mechanisms are the cryptochromes, flavoproteins located in the retina that are supposed to perceive the magnetic field through a light-mediated process. Previous work has suggested that expression of Cryptochrome 1 (Cry1) is increased in migratory birds compared with non-migratory species. Here we tested the hypothesis that Cry1 expression depends on migratory status. Blackcaps Sylvia atricapilla were caught before fall migration and held in registration cages. When the birds were showing robust Zugunruhe, we applied a food deprivation protocol that simulates a long migratory flight. When the birds were refed after 2 days, their Zugunruhe decreased substantially, as is expected from birds that would interrupt migration for a refuelling stopover. We found that Cry1 expression was higher at night than during daytime in birds showing Zugunruhe, whereas in birds that underwent the fasting-and-refeeding protocol and reduced their levels of Zugunruhe, night Cry1 expression decreased to daytime levels. Our work shows that Cry1 expression is dependent on the presence of Zugunruhe and not on species-specific or seasonal factors, or on the birds being active versus inactive. These results support the hypothesis that cryptochromes underlie magnetoreceptive mechanisms in birds. PMID:24622895

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

    PubMed Central

    Nawandar, Dhananjay M.; Wang, Anqi; Makielski, Kathleen; Lee, Denis; Ma, Shidong; Barlow, Elizabeth; Reusch, Jessica; Jiang, Ru; Wille, Coral K.; Greenspan, Deborah; Greenspan, John S.; Mertz, Janet E.; Hutt-Fletcher, Lindsey; Johannsen, Eric C.; Lambert, Paul F.; Kenney, Shannon C.

    2015-01-01

    Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells. PMID:26431332

  16. Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius

    NASA Technical Reports Server (NTRS)

    Woodman, C. R.; Schrage, W. G.; Rush, J. W.; Ray, C. A.; Price, E. M.; Hasser, E. M.; Laughlin, M. H.

    2001-01-01

    We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.

  17. Breast cancer risk associated with gene expression and genotype polymorphisms of the folate-metabolizing MTHFR gene: a case-control study in a high altitude Ecuadorian mestizo population.

    PubMed

    López-Cortés, Andrés; Echeverría, Carolina; Oña-Cisneros, Fabián; Sánchez, María Eugenia; Herrera, Camilo; Cabrera-Andrade, Alejandro; Rosales, Felipe; Ortiz, Malena; Paz-Y-Miño, César

    2015-08-01

    Breast cancer (BC) is the leading cause of cancer-related death among women in 2014. Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and MTR reductase (MTRR) are enzymes that play an important role in folate metabolism. The single nucleotide polymorphisms, MTHFR C677T, A1298C, MTR A2756G, and MTRR A66G, alter plasmatic folate and homocysteine concentrations, causing problems during the repairment, synthesis, and methylation of the genetic material. Therefore, it is essential to know how BC risk is associated with histopathological and immunohistochemical characteristics, genotype polymorphisms, and gene expression in a high altitude Ecuadorian mestizo population. DNA was extracted from 195 healthy and 114 affected women. Genotypes were determined by restriction enzymes and genomic sequencing. mRNA was extracted from 26 glandular breast tissue samples, both from cancerous tissue and healthy tissue adjacent to the tumor. Relative gene expression was determined with the comparative Livak method (2(-ΔΔCT)). We found significant association between the rs1801133 (A222V) genotypes and an increased risk of BC development: C/T (odds ratio [OR] = 1.8; 95 % confidence interval [CI] = 1.1-3.2; P = 0.039), T/T (OR = 2.9; 95 % CI = 1.2-7.2; P = 0.025), and C/T + T/T (OR = 1.9; 95 % CI = 1.1-3.3; P = 0.019). Regarding relative gene expression, we found significant mRNA subexpression between the combined genotypes C/T + T/T (rs1801133) and triple negative breast cancer (TNBC) (P = 0.034). In brief, the MTHFR gene and its protein could act as potential predictive biomarkers of BC, especially TNBC among the high altitude Ecuadorian mestizo population. PMID:25801246

  18. Strain-dependent variations in visceral sensitivity: relationship to stress, anxiety and spinal glutamate transporter expression.

    PubMed

    Moloney, R D; Dinan, T G; Cryan, J F

    2015-04-01

    Responses to painful stimuli differ between populations, ethnic groups, sexes and even among individuals of a family. However, data regarding visceral pain are still lacking. Thus, we investigated differences in visceral nociception across inbred and outbred mouse strains using colorectal distension. Anxiety and depression-like behaviour were assessed using the open field and forced swim test as well as the corticosterone stress response. Possible mechanistic targets [excitatory amino acid transporter (EAAT-1), brain-derived neurotrophic factor (BDNF) and 5HT1A receptor] were also assessed using quantitative real-time polymerase chain reaction. Adult, male, inbred and outbred mouse strains were used in all assays (inbred strains; CBA/J Hsd, C3H/HeNHsd, BALB/c OlaHsd, C57 BL/6JOlaHsd, DBA/2J RccHsd, CAST/EiJ, SM/J, A/J OlaHsd, 129P2/OlaHsd, FVB/NHan Hsd and outbred strains: Swiss Webster, CD-1). mRNA expression levels of EAAT-1, BDNF and 5HT1A receptor (HTR1A) were quantified in the lumbosacral spinal cord, amygdala and hippocampus. A significant effect of strain was found in visceral sensitivity, anxiety and depressive-like behaviours. Strain differences were also seen in both baseline and stress-induced corticosterone levels. CBA/J mice consistently exhibited heightened visceral sensitivity, anxiety behaviour and depression-like behaviour which were associated with decreased spinal EAAT-1 and hippocampal BDNF and HTR1A. Our results show the CBA/J mouse strain as a novel mouse model to unravel the complex mechanisms of brain-gut axis disorders such as irritable bowel syndrome, in particular the underlying mechanisms of visceral hypersensitivity, for which there is great need. Furthermore, this study highlights the importance of genotype and the consequences for future development of transgenic strains in pain research. PMID:25851919

  19. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

    PubMed

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

    2016-04-20

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  20. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms

    PubMed Central

    Mabb, Angela M.; Simon, Jeremy M.; King, Ian F.; Lee, Hyeong-Min; An, Lin-Kun; Philpot, Benjamin D.; Zylka, Mark J.

    2016-01-01

    Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression. PMID:27231886

  1. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  2. MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae.

    PubMed

    Hu, Z; Nehlin, J O; Ronne, H; Michels, C A

    1995-08-01

    Glucose repression is a global regulatory system in Saccharomyces cerevisiae controlling carbon-source utilization, mitochondrial biogenesis, gluconeogenesis and other metabolic pathways. Mig1p, a zinc-finger class of DNA-binding protein, is a transcriptional repressor regulating GAL and SUC gene expression in response to glucose. This report demonstrates that Mig1 protein represses transcription of the MAL61 and MAL62 structural genes and also the MAL63 gene, which encodes the Mal-activator. Mig1p DNA-binding sites were identified upstream of all three MAL genes. Both of the Mig1p-binding sites found in the bidirectional MAL61-MAL62 promoter were shown to function in the Mig1p-dependent glucose repression. Studies using constitutive Mal-activator alleles suggest that glucose regulation of inducer availability is a second major contributing factor in glucose repression of MAL gene expression and is even stronger than the Mig1p-dependent component of repression. Moreover, our results also suggest the contribution of other minor mechanisms in glucose regulation of MAL gene expression. PMID:8529272

  3. Temperature-Dependent Expression of NodC and Community Structure of Soybean-Nodulating Bradyrhizobia

    PubMed Central

    Shiro, Sokichi; Kuranaga, Chika; Yamamoto, Akihiro; Sameshima-Saito, Reiko; Saeki, Yuichi

    2016-01-01

    In order to assess the physiological responses of bradyrhizobia and competition for the nodulation of soybean at different temperatures, we investigated the expression of the nodC gene at 20, 25, and 30°C and the abilities of bacteria to nodulate soybean in microcosms at day/night cultivation temperatures of 23/18°C, 28/23°C, and 33/28°C for 16/8 h. We tested five Bradyrhizobium USDA strains: B. diazoefficiens USDA 110T and 122, B. japonicum USDA 123, and B. elkanii USDA 31 and 76T. The expression of nodC was up-regulated by increasing culture temperatures in USDA 110T, 122, 31, and 76T, but was down-regulated in USDA 123. The proportions of USDA 110T and 122 within the community were the greatest at 28/23°C. The population of USDA 31 increased, whereas that of USDA 123 decreased with increasing cultivation temperatures. On the other hand, infection by USDA 76T was not detected, and low numbers of USDA 76T nodules confirmed its poor nodulation ability. These results indicate that the competitiveness of and infection by USDA 110T, 122, 123, and 31 for soybean nodulation depend on cultivation temperatures, and suggest that the temperature dependence of nodC expression affects the bradyrhizobial community structure. PMID:26877137

  4. Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes.

    PubMed

    Beilstein, Kim; Wittmann, Alexander; Grez, Manuel; Suess, Beatrix

    2015-05-15

    Robust synthetic devices are requisite for the construction of synthetic genetic circuits and important scientific and technological tools to control cellular processes. We developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. A tetracycline aptamer was grafted onto the hammerhead ribozyme in such a way that ligand binding to the aptamers destroys a loop-loop interaction within the ribozyme thereby inhibiting ribozyme cleavage and allowing gene expression. The advantage of the presented regulatory system is its independence of any regulatory proteins. The stable integration of the ribozyme into the genome of HeLa cells indicates a low background activity in the absence of ligand. Furthermore, the ligand concentration required to robustly flip the switch does not affect cell viability and therefore allows a long-term application of the system. These properties turn the tetracycline-dependent ribozymes into a very promising tool for conditional gene expression in mammalian cells. PMID:25265236

  5. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms.

    PubMed

    Mabb, Angela M; Simon, Jeremy M; King, Ian F; Lee, Hyeong-Min; An, Lin-Kun; Philpot, Benjamin D; Zylka, Mark J

    2016-01-01

    Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc's) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc's, and a TOP1 mutation (T718A) that stabilizes TOP1cc's. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression. PMID:27231886

  6. Expression of apoptosis-related genes in liver-specific growth hormone receptor gene-disrupted mice is sex dependent.

    PubMed

    Gesing, Adam; Wang, Feiya; List, Edward O; Berryman, Darlene E; Masternak, Michal M; Lewinski, Andrzej; Karbownik-Lewinska, Malgorzata; Kopchick, John J; Bartke, Andrzej

    2015-01-01

    Apoptosis is a process that affects life span and health. Mice with liver-specific disruption of the growth hormone receptor (GHR) gene (ie, Ghr gene) liver-specific growth hormone receptor knockout [LiGHRKO] mice), as opposed to mice with global deletion of the Ghr gene (GHRKO; Ghr-/-), are characterized by severe hepatic steatosis and lack of improved insulin sensitivity. We have previously shown that levels of proapoptotic factors are decreased in long-lived and insulin-sensitive GHRKO mice. In the current study, expression of specific apoptosis-related genes was assessed in brains, kidneys, and livers of male and female LiGHRKO and wild-type mice using real-time PCR. In the brain, expression of Caspase 3, Caspase 9, Smac/DIABLO, and p53 was decreased in females compared with males. Renal expression of Caspase 3 and Noxa also decreased in female mice. In the liver, no differences were seen between males and females. Also, no significant genotype effects were detected in the examined organs. Lack of significant genotype effect in kidneys contrasts with previous observations in GHRKO mice. Apparently, global GHR deletion induces beneficial changes in apoptotic factors, whereas liver-specific GHR disruption does not. Furthermore, sexual dimorphism may play an important role in regulating apoptosis during liver-specific suppression of the somatotrophic signaling. PMID:24550353

  7. Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression

    PubMed Central

    Muzikar, Katy A.; Nickols, Nicholas G.; Dervan, Peter B.

    2009-01-01

    The glucocorticoid receptor (GR) affects the transcription of genes involved in diverse processes, including energy metabolism and the immune response, through DNA-binding dependent and independent mechanisms. The DNA-binding dependent mechanism occurs by direct binding of GR to glucocorticoid response elements (GREs) at regulatory regions of target genes. The DNA-binding independent mechanism involves binding of GR to transcription factors and coactivators that, in turn, contact DNA. A small molecule that competes with GR for binding to GREs could be expected to affect the DNA-dependent pathway selectively by interfering with the protein-DNA interface. We show that a DNA-binding polyamide that targets the consensus GRE sequence binds the glucocorticoid-induced zipper (GILZ) GRE, inhibits expression of GILZ and several other known GR target genes, and reduces GR occupancy at the GILZ promoter. Genome-wide expression analysis of the effects of this polyamide on a set of glucocorticoid-induced and -repressed genes could help to elucidate the mechanism of GR regulation for these genes. PMID:19805343

  8. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase.

    PubMed

    Failer, Bernd U; Braun, Norbert; Zimmermann, Herbert

    2002-10-01

    We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi. The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa. The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance. The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates. Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen. It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >CDP but not ADP. Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm. Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum. PMID:12167635

  9. T Lymphocytes Induce Endothelial Cell Matrix Metalloproteinase Expression by a CD40L-Dependent Mechanism

    PubMed Central

    Mach, François; Schönbeck, Uwe; Fabunmi, Rosalind P.; Murphy, Curran; Atkinson, Elizabeth; Bonnefoy, Jean-Yves; Graber, Pierre; Libby, Peter

    1999-01-01

    Neovascularization frequently accompanies chronic immune responses characterized by T cell infiltration and activation. Angiogenesis requires endothelial cells (ECs) to penetrate extracellular matrix, a process that involves matrix metalloproteinases (MMPs). We report here that activated human T cells mediate contact-dependent expression of MMPs in ECs through CD40/CD40 ligand signaling. Ligation of CD40 on ECs induced de novo expression of gelatinase B (MMP-9), increased interstitial collagenase (MMP-1) and stromelysin (MMP-3), and activated gelatinase A (MMP-2). Recombinant human CD40L induced expression of MMPs by human vascular ECs to a greater extent than did maximally effective concentrations of interleukin-1β or tumor necrosis factor-α. Moreover, activation of human vascular ECs through CD40 induced tube formation in a three-dimensional fibrin matrix gel assay, an effect antagonized by a MMP inhibitor. These results demonstrated that activation of ECs by interaction with T cells induced synthesis and release of MMPs and promoted an angiogenic function of ECs via CD40L-CD40 signaling. As vascular cells at the sites of chronic inflammation, such as atherosclerotic plaques, express CD40 and its ligand, our findings suggest that ligation of CD40 on ECs can mediate aspects of vascular remodeling and neovessel formation during atherogenesis and other chronic immune reactions. PMID:9916937

  10. Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis

    PubMed Central

    Kanthi, Yogendra; Hyman, Matthew C.; Liao, Hui; Baek, Amy E.; Visovatti, Scott H.; Sutton, Nadia R.; Goonewardena, Sascha N.; Neral, Mithun K.; Jo, Hanjoong; Pinsky, David J.

    2015-01-01

    The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E–deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39+/–Apoe–/– mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe–/– mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress. PMID:26121751

  11. p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells

    PubMed Central

    Mitkin, Nikita A.; Hook, Christina D.; Schwartz, Anton M.; Biswas, Subir; Kochetkov, Dmitry V.; Muratova, Alisa M.; Afanasyeva, Marina A.; Kravchenko, Julia E.; Bhattacharyya, Arindam; Kuprash, Dmitry V.

    2015-01-01

    Elevated expression of chemokine receptors in tumors has been reported in many instances and is related to a number of survival advantages for tumor cells including abnormal activation of prosurvival intracellular pathways. In this work we demonstrated an inverse correlation between expression levels of p53 tumor suppressor and CXCR5 chemokine receptor in MCF-7 human breast cancer cell line. Lentiviral transduction of MCF-7 cells with p53 shRNA led to elevated CXCR5 at both mRNA and protein levels. Functional activity of CXCR5 in p53-knockdown MCF-7 cells was also increased as shown by activation of target gene expression and chemotaxis in response to B-lymphocyte chemoattractant CXCL13. Using deletion analysis and site-directed mutagenesis of the cxcr5 gene promoter and enhancer elements, we demonstrated that p53 appears to act upon cxcr5 promoter indirectly, by repressing the activity of NFκB transcription factors. Using chromatin immunoprecipitation and reporter gene analysis, we further demonstrated that p65/RelA was able to bind the cxcr5 promoter in p53-dependent manner and to directly transactivate it when overexpressed. Through the described mechanism, elevated CXCR5 expression may contribute to abnormal cell survival and migration in breast tumors that lack functional p53. PMID:25786345

  12. Calorie restriction regulates circadian clock gene expression through BMAL1 dependent and independent mechanisms

    PubMed Central

    Patel, Sonal A.; Velingkaar, Nikkhil; Makwana, Kuldeep; Chaudhari, Amol; Kondratov, Roman

    2016-01-01

    Feeding behavior, metabolism and circadian clocks are interlinked. Calorie restriction (CR) is a feeding paradigm known to extend longevity. We found that CR significantly affected the rhythms in the expression of circadian clock genes in mice on the mRNA and protein levels, suggesting that CR reprograms the clocks both transcriptionally and post-transcriptionally. The effect of CR on gene expression was distinct from the effects of time-restricted feeding or fasting. Furthermore, CR affected the circadian output through up- or down-regulation of the expression of several clock-controlled transcriptional factors and the longevity candidate genes. CR-dependent effects on some clock gene expression were impaired in the liver of mice deficient for BMAL1, suggesting importance of this transcriptional factor for the transcriptional reprogramming of the clock, however, BMAL1- independent mechanisms also exist. We propose that CR recruits biological clocks as a natural mechanism of metabolic optimization under conditions of limited energy resources. PMID:27170536

  13. Calorie restriction regulates circadian clock gene expression through BMAL1 dependent and independent mechanisms.

    PubMed

    Patel, Sonal A; Velingkaar, Nikkhil; Makwana, Kuldeep; Chaudhari, Amol; Kondratov, Roman

    2016-01-01

    Feeding behavior, metabolism and circadian clocks are interlinked. Calorie restriction (CR) is a feeding paradigm known to extend longevity. We found that CR significantly affected the rhythms in the expression of circadian clock genes in mice on the mRNA and protein levels, suggesting that CR reprograms the clocks both transcriptionally and post-transcriptionally. The effect of CR on gene expression was distinct from the effects of time-restricted feeding or fasting. Furthermore, CR affected the circadian output through up- or down-regulation of the expression of several clock-controlled transcriptional factors and the longevity candidate genes. CR-dependent effects on some clock gene expression were impaired in the liver of mice deficient for BMAL1, suggesting importance of this transcriptional factor for the transcriptional reprogramming of the clock, however, BMAL1- independent mechanisms also exist. We propose that CR recruits biological clocks as a natural mechanism of metabolic optimization under conditions of limited energy resources. PMID:27170536

  14. Electroacupuncture Upregulates SIRT1-Dependent PGC-1α Expression in SAMP8 Mice

    PubMed Central

    Dong, Weiguo; Guo, Wanqing; Wang, Feng; Li, Changzheng; Xie, Yongcai; Zheng, Xueha; Shi, Hong

    2015-01-01

    Background Abnormalities of brain energy metabolism are involved in Alzheimer disease (AD). Sirtuin 1 (SIRT1) is a class III histone deacetylase and activates peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which enhances mitochondrial biogenesis and energy homeostasis. Electroacupuncture (EA) has been reported to improve brain energy metabolism in AD. However, the effect of EA on SIRT1 and PGC-1α in AD remains unclear. Material/Methods ATP levels were measured using assay kits in the hippocampus and frontal cortex of senescence-accelerated mouse prone 8 (SAMP8) mice. Western blotting analysis and quantitative real-time RT-PCR were performed to measure the expression of SIRT1 and PGC-1α in the hippocampus of SAMP8 mice. PGC-1α acetylation was analyzed using immunoprecipitation. Results Compared with senescence-accelerated resistant mice 1 (SAMR1) mice, SAMP8 mice had a decline in ATP levels and the expression of SIRT1 and PGC-1α. EA treatment improved ATP levels, upregulated the expression of SIRT1 and PGC-1α, and decreased PGC-1α acetylation. Conclusions These data suggest that EA improved brain energy metabolism, potentially associated with the upregulation of SIRT1-dependent PGC-1α expression. PMID:26530101

  15. Maternal High Fat Diet Affects Offspring’s Vitamin K-Dependent Proteins Expression Levels

    PubMed Central

    Lanham, Stuart; Cagampang, Felino R.; Oreffo, Richard O. C.

    2015-01-01

    Studies suggest bone growth & development and susceptibility to vascular disease in later life are influenced by maternal nutrition, during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs) including Osteocalcin, Matrix-gla protein, Periostin, and Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF) diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure. PMID:26381752

  16. VEGFR-2 Expression in Glioblastoma Multiforme Depends on Inflammatory Tumor Microenvironment

    PubMed Central

    Jaal, Jana; Kase, Marju; Minajeva, Ave; Saretok, Mikk; Adamson, Aidi; Junninen, Jelizaveta; Metsaots, Tõnis; Jõgi, Tõnu; Joonsalu, Madis; Vardja, Markus; Asser, Toomas

    2015-01-01

    Glioblastoma multiforme (GBM) is one of the most angiogenic tumors. However, antiangiogenic therapy has not shown significant clinical efficacy. The aim of our study was to evaluate the impact of inflammatory tumor microenvironment on the expression of vascular endothelial growth factor receptor 2 (VEGFR-2). Surgically excised primary GBM tissues were histologically examined for overall extent of inflammation (score 1–3). After immunohistochemistry, the tissue expression of ICAM-1 (optical density), the number of VEGFR-2 positive (VEGFR-2+) blood vessels (per microscopic field), and the endothelial staining intensity of VEGFR-2 (score 0–3) were determined. In GBM, the extent of inflammation was 1.9 ± 0.7 (group mean ± SD). Mean optical density of inflammatory mediator ICAM-1 was 57.0 ± 27.1 (pixel values). The number of VEGFR-2+ blood vessels and endothelial VEGFR-2 staining intensity were 6.2 ± 2.4 and 1.2 ± 0.8, respectively. A positive association was found between endothelial VEGFR-2 staining intensity and the extent of inflammation (p = 0.005). Moreover, VEGFR-2 staining intensity correlated with the expression level of ICAM-1 (p = 0.026). The expression of VEGFR-2, one of the main targets of antiangiogenic therapy, depends on GBM microenvironment. Higher endothelial VEGFR-2 levels were seen in the presence of more pronounced inflammation. Target dependence on inflammatory tumor microenvironment has to be taken into consideration when treatment approaches that block VEGFR-2 signaling are designed. PMID:26798546

  17. Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization.

    PubMed

    Kneuer, Carsten; Honscha, Kerstin U; Honscha, Walther

    2004-03-01

    Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a K(m) of 41 microM and a V(max) of 337 pmol.mg protein(-1).min(-1) was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model. PMID:14612385

  18. Genotypic differences in behavioural entropy: unpredictable genotypes are composed of unpredictable individuals

    PubMed Central

    Stamps, Judy A.; Saltz, Julia B.; Krishnan, V.V.

    2013-01-01

    Intra-genotypic variability (IGV) occurs when individuals with the same genotype, raised in the same environment and then tested under the same conditions, express different trait values. Game theoretical and bet-hedging models have suggested two ways that a single genotype might generate variable behaviour when behavioural variation is discrete rather than continuous: behavioural polyphenism (a genotype produces different types of individuals, each of which consistently expresses a different type of behaviour) or stochastic variability (a genotype produces one type of individual who randomly expresses different types of behaviour over time). We first demonstrated significant differences across 14 natural genotypes of male Drosophila melanogaster in the variability (as measured by entropy) of their microhabitat choice, in an experiment in which each fly was allowed free access to four different types of habitat. We then tested four hypotheses about ways that within-individual variability might contribute to differences across genotypes in the variability of microhabitat choice. There was no empirical support for three hypotheses (behavioural polymorphism, consistent choice, or time-based choice), nor could our results be attributed to genotypic differences in activity levels. The stochastic variability hypothesis accurately predicted the slope and the intercept of the relationship across genotypes between entropy at the individual level and entropy at the genotype level. However, our initial version of the stochastic model slightly but significantly overestimated the values of individual entropy for each genotype, pointing to specific assumptions of this model that might need to be adjusted in future studies of the IGV of microhabitat choice. This is among a handful of recent studies to document genotypic differences in behavioural IGV, and the first to explore ways that genotypic differences in within-individual variability might contribute to differences among

  19. Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms

    PubMed Central

    King, Elizabeth M.; Chivers, Joanna E.; Rider, Christopher F.; Minnich, Anne; Giembycz, Mark A.; Newton, Robert

    2013-01-01

    Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the de novo synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent

  20. Sympathetic Activation Induces Skeletal Fgf23 Expression in a Circadian Rhythm-dependent Manner*

    PubMed Central

    Kawai, Masanobu; Kinoshita, Saori; Shimba, Shigeki; Ozono, Keiichi; Michigami, Toshimi

    2014-01-01

    The circadian clock network is well known to link food intake and metabolic outputs. Phosphorus is a pivotal nutritional factor involved in energy and skeletal metabolisms and possesses a circadian profile in the circulation; however, the precise mechanisms whereby phosphate metabolism is regulated by the circadian clock network remain largely unknown. Because sympathetic tone, which displays a circadian profile, is activated by food intake, we tested the hypothesis that phosphate metabolism was regulated by the circadian clock network through the modification of food intake-associated sympathetic activation. Skeletal Fgf23 expression showed higher expression during the dark phase (DP) associated with elevated circulating FGF23 levels and enhanced phosphate excretion in the urine. The peaks in skeletal Fgf23 expression and urine epinephrine levels, a marker for sympathetic tone, shifted from DP to the light phase (LP) when mice were fed during LP. Interestingly, β-adrenergic agonist, isoproterenol (ISO), induced skeletal Fgf23 expression when administered at ZT12, but this was not observed in Bmal1-deficient mice. In vitro reporter assays revealed that ISO trans-activated Fgf23 promoter through a cAMP responsive element in osteoblastic UMR-106 cells. The mechanism of circadian regulation of Fgf23 induction by ISO in vivo was partly explained by the suppressive effect of Cryptochrome1 (Cry1) on ISO signaling. These results indicate that the regulation of skeletal Fgf23 expression by sympathetic activity is dependent on the circadian clock system and may shed light on new regulatory networks of FGF23 that could be important for understanding the physiology of phosphate metabolism. PMID:24302726

  1. Potential antioxidant response to coffee - A matter of genotype?

    PubMed

    Hassmann, Ute; Haupt, Larisa M; Smith, Robert A; Winkler, Swantje; Bytof, Gerhard; Lantz, Ingo; Griffiths, Lyn R; Marko, Doris

    2014-12-01

    In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the - 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the - 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential. PMID:25606436

  2. Potential antioxidant response to coffee — A matter of genotype?

    PubMed Central

    Hassmann, Ute; Haupt, Larisa M.; Smith, Robert A.; Winkler, Swantje; Bytof, Gerhard; Lantz, Ingo; Griffiths, Lyn R.; Marko, Doris

    2014-01-01

    In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the − 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the − 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential. PMID:25606436

  3. Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

    PubMed

    Pozo, Karine; Hillmann, Antje; Augustyn, Alexander; Plattner, Florian; Hai, Tao; Singh, Tanvir; Ramezani, Saleh; Sun, Xiankai; Pfragner, Roswitha; Minna, John D; Cote, Gilbert J; Chen, Herbert; Bibb, James A; Nwariaku, Fiemu E

    2015-05-20

    Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis. PMID:25900242

  4. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes

    PubMed Central

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R.; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1–5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1–5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response. PMID:23327879

  5. Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro.

    PubMed Central

    Indest, K J; Ramamoorthy, R; Solé, M; Gilmore, R D; Johnson, B J; Philipp, M T

    1997-01-01

    Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi. PMID:9119447

  6. Calcium activates the light-dependent conductance in melanopsin-expressing photoreceptors of amphioxus

    PubMed Central

    Peinado, Gabriel; Osorno, Tomás; Gomez, María del Pilar; Nasi, Enrico

    2015-01-01

    Melanopsin, the photopigment of the “circadian” receptors that regulate the biological clock and the pupillary reflex in mammals, is homologous to invertebrate rhodopsins. Evidence supporting the involvement of phosphoinositides in light-signaling has been garnered, but the downstream effectors that control the light-dependent conductance remain unknown. Microvillar photoreceptors of the primitive chordate amphioxus also express melanopsin and transduce light via phospholipase-C, apparently not acting through diacylglycerol. We therefore examined the role of calcium in activating the photoconductance, using simultaneous, high time-resolution measurements of membrane current and Ca2+ fluorescence. The light-induced calcium rise precedes the onset of the photocurrent, making it a candidate in the activation chain. Moreover, photolysis of caged Ca elicits an inward current of similar size, time course and pharmacology as the physiological photoresponse, but with a much shorter latency. Internally released calcium thus emerges as a key messenger to trigger the opening of light-dependent channels in melanopsin-expressing microvillar photoreceptors of early chordates. PMID:26056310

  7. Calcium activates the light-dependent conductance in melanopsin-expressing photoreceptors of amphioxus.

    PubMed

    Peinado, Gabriel; Osorno, Tomás; Gomez, María del Pilar; Nasi, Enrico

    2015-06-23

    Melanopsin, the photopigment of the "circadian" receptors that regulate the biological clock and the pupillary reflex in mammals, is homologous to invertebrate rhodopsins. Evidence supporting the involvement of phosphoinositides in light-signaling has been garnered, but the downstream effectors that control the light-dependent conductance remain unknown. Microvillar photoreceptors of the primitive chordate amphioxus also express melanopsin and transduce light via phospholipase-C, apparently not acting through diacylglycerol. We therefore examined the role of calcium in activating the photoconductance, using simultaneous, high time-resolution measurements of membrane current and Ca(2+) fluorescence. The light-induced calcium rise precedes the onset of the photocurrent, making it a candidate in the activation chain. Moreover, photolysis of caged Ca elicits an inward current of similar size, time course and pharmacology as the physiological photoresponse, but with a much shorter latency. Internally released calcium thus emerges as a key messenger to trigger the opening of light-dependent channels in melanopsin-expressing microvillar photoreceptors of early chordates. PMID:26056310

  8. Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways.

    PubMed

    Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

    2015-01-01

    Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

  9. Strain-dependent response to Cu(2+) in the expression of laccase in Pycnoporus coccineus.

    PubMed

    Park, Ju-Wan; Kang, Hyeon-Woo; Ha, Byung-Suk; Kim, Sin-Il; Kim, Soonok; Ro, Hyeon-Su

    2015-05-01

    The effects of Cu(2+) on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu(2+) was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO4 and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu(2+) significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu(2+), despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu(2+). Taken together, these results indicate that Cu(2+) regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu(2+)-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus. PMID:25677944

  10. p53-dependent SIRT6 expression protects Aβ42-induced DNA damage

    PubMed Central

    Jung, Eun Sun; Choi, Hyunjung; Song, Hyundong; Hwang, Yu Jin; Kim, Ahbin; Ryu, Hoon; Mook-Jung, Inhee

    2016-01-01

    Alzheimer’s disease (AD) is the most common type of dementia and age-related neurodegenerative disease. Elucidating the cellular changes that occur during ageing is an important step towards understanding the pathogenesis and progression of neurodegenerative disorders. SIRT6 is a member of the mammalian sirtuin family of anti-aging genes. However, the relationship between SIRT6 and AD has not yet been elucidated. Here, we report that SIRT6 protein expression levels are reduced in the brains of both the 5XFAD AD mouse model and AD patients. Aβ42, a major component of senile plaques, decreases SIRT6 expression, and Aβ42-induced DNA damage is prevented by the overexpression of SIRT6 in HT22 mouse hippocampal neurons. Also, there is a strong negative correlation between Aβ42-induced DNA damage and p53 levels, a protein involved in DNA repair and apoptosis. In addition, upregulation of p53 protein by Nutlin-3 prevents SIRT6 reduction and DNA damage induced by Aβ42. Taken together, this study reveals that p53-dependent SIRT6 expression protects cells from Aβ42-induced DNA damage, making SIRT6 a promising new therapeutic target for the treatment of AD. PMID:27156849

  11. Pathology-Dependent Effects Linked to Small Heat Shock Proteins Expression: An Update

    PubMed Central

    Arrigo, A.-P.

    2012-01-01

    Small heat shock proteins (small Hsps) are stress-induced molecular chaperones that act as holdases towards polypeptides that have lost their folding in stress conditions or consequently of mutations in their coding sequence. A cellular protection against the deleterious effects mediated by damaged proteins is thus provided to cells. These chaperones are also highly expressed in response to protein conformational and inflammatory diseases and cancer pathologies. Through specific and reversible modifications in their phospho-oligomeric organization, small Hsps can chaperone appropriate client proteins in order to provide cells with resistance to different types of injuries or pathological conditions. By helping cells to better cope with their pathological status, their expression can be either beneficial, such as in diseases characterized by pathological cell degeneration, or deleterious when they are required for tumor cell survival. Moreover, small Hsps are actively released by cells and can act as immunogenic molecules that have dual effects depending on the pathology. The cellular consequences linked to their expression levels and relationships with other Hsps as well as therapeutic strategies are discussed in view of their dynamic structural organization required to interact with specific client polypeptides. PMID:24278676

  12. Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression.

    PubMed

    Ambite, Ines; Lutay, Nataliya; Stork, Christoph; Dobrindt, Ulrich; Wullt, Björn; Svanborg, Catharina

    2016-01-01

    Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease-associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that

  13. The 14-3-3 gene expression specificity in response to stress is promoter-dependent.

    PubMed

    Aksamit, Anna; Korobczak, Alina; Skala, Jacek; Lukaszewicz, Marcin; Szopa, Jan

    2005-10-01

    Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms. PMID:16081528

  14. Sleep-Dependent Gene Expression in the Hippocampus and Prefrontal Cortex Following Long-Term Potentiation

    PubMed Central

    Romcy-Pereira, Rodrigo N.; Erraji-Benchekroun, Loubna; Smyrniotopoulos, Peggy; Ogawa, Sonoko; Mello, Claudio V.; Sibille, Etienne; Pavlides, Constantine

    2009-01-01

    The activity-dependent transcription factor zif268 is re-activated in sleep following hippocampal long-term potentiation (LTP). However, the activation of secondary genes, possibly involved in modifying local synaptic strengths and ultimately stabilizing memory traces during sleep, has not yet been studied. Here, we investigated changes in hippocampal and cortical gene expression at a time point subsequent to the previously reported initial zif268 re-activation during sleep. Rats underwent unilateral hippocampal LTP and were assigned to SLEEP or AWAKE groups. Eighty minutes after a long rapid-eye-movement sleep (REMS) episode (or an equivalent amount of time for awake group) animals had their hippocampi dissected and processed for gene microarray hybridization. Prefrontal and parietal cortices were also collected for qRT-PCR analysis. The microarray analysis identified 28 up-regulated genes in the hippocampus: 11 genes were enhanced in the LTPed hemisphere of sleep animals; 13 genes were enhanced after sleep, regardless of hemisphere; and 4 genes were enhanced in LTPed hemisphere, regardless of behavioral state. qRT-PCR analysis confirmed the upregulation of aif-1 and sc-65 during sleep. Moreover, we observed a down-regulation of the purinergic receptor, P2Y4R in the LTP hemisphere of awake animals and a trend for the protein kinase, CaMKI to be up-regulated in the LTP hemisphere of sleep animals. In the prefrontal cortex, we showed a significant LTP-dependent down-regulation of gluR1 and spinophilin specifically during sleep. Zif268 was downregulated in sleep regardless of the hemisphere. No changes in gene expression were observed in the parietal cortex. Our findings indicate that a set of synaptic plasticity-related genes have their expression modulated during sleep following LTP, which can reflect biochemical events associated with reshaping of synaptic connections in sleep following learning. PMID:19389414

  15. Age-Dependent Brain Gene Expression and Copy Number Anomalies in Autism Suggest Distinct Pathological Processes at Young Versus Mature Ages

    PubMed Central

    Winn, Mary E.; Barnes, Cynthia Carter; Li, Hai-Ri; Weiss, Lauren; Fan, Jian-Bing; Murray, Sarah; April, Craig; Belinson, Haim; Fu, Xiang-Dong; Wynshaw-Boris, Anthony; Schork, Nicholas J.; Courchesne, Eric

    2012-01-01

    Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs) in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess prefrontal neurons

  16. Experience-Dependent Changes in Excitatory and Inhibitory Receptor Subunit Expression in Visual Cortex

    PubMed Central

    Beston, Brett R.; Jones, David G.; Murphy, Kathryn M.

    2010-01-01

    Experience-dependent development of visual cortex depends on the balance between excitatory and inhibitory activity. This activity is regulated by key excitatory (NMDA, AMPA) and inhibitory (GABAA) receptors. The composition of these receptors changes developmentally, affecting the excitatory–inhibitory (E/I) balance and synaptic plasticity. Until now, it has been unclear how abnormal visual experience affects this balance. To examine this question, we measured developmental changes in excitatory and inhibitory receptor subunits in visual cortex following normal visual experience and monocular deprivation. We used Western blot analysis to quantify expression of excitatory (NR1, NR2A, NR2B, GluR2) and inhibitory (GABAAα1, GABAAα3) receptor subunits. Monocular deprivation promoted a complex pattern of changes in receptor subunit expression that varied with age and was most severe in the region of visual cortex representing the central visual field. To characterize the multidimensional pattern of experience-dependent change in these synaptic mechanisms, we applied a neuroinformatics approach using principal component analysis. We found that monocular deprivation (i) causes a large portion of the normal developmental trajectory to be bypassed, (ii) shifts the E/I balance in favor of more inhibition, and (iii) accelerates the maturation of receptor subunits. Taken together, these results show that monocularly deprived animals have an abnormal balance of the synaptic machinery needed for functional maturation of cortical circuits and for developmental plasticity. This raises the possibility that interventions intended to treat amblyopia may need to address multiple synaptic mechanisms to produce optimal recovery. PMID:21423524

  17. Where Is the Extended Phenotype in the Wild? The Community Composition of Arthropods on Mature Oak Trees Does Not Depend on the Oak Genotype

    PubMed Central

    Gossner, Martin M.; Brändle, Martin; Brandl, Roland; Bail, Johannes; Müller, Jörg; Opgenoorth, Lars

    2015-01-01

    Through a series of common garden experiments, it has been shown that heritable phenotypic differences between individual trees can affect arthropod communities. However, field studies under heterogeneous environmental conditions remain rare. In the present study, we investigated the genetic constitution of 121 mature oak host trees at different trophic levels from 10 sites across Bavaria, southern Germany and their associated insect communities. A total of 23,576 individuals representing 395 species of beetles and true bugs were evaluated. In particular, we determined whether the composition of arthropod communities is related to the oak genotype and whether the strength of the relationships decreases from lower to higher trophic levels, such as for phytophagous, xylophagous, zoophagous, and mycetophagous species. The genetic differentiation of oaks was assessed using eight microsatellite markers. We found no significant influence of the oak genotype on neither the full beetle and true bug community nor on any of the analyzed trophic guilds. In contrast, the community composition of the insects was highly related to the space and climate, such that the community similarity decreased with increases in spatial distance and climatic differences. The relationship with space and climate was much stronger in beetles than in true bugs, particularly in mycetophagous species. Our results suggest that spatial processes override the genetic effects of the host plant in structuring arthropod communities on oak trees. Because we used neutral markers, we cannot exclude the possibility that trait-specific markers may reveal a genetic imprint of the foundation tree species on the composition of the arthropod community. However, based on the strength of the spatial patterns in our data set, we assume that genetic differences among oaks are less important in the structuring of arthropod communities. Future whole-genome studies are required to draw a final conclusion. PMID:25635387

  18. Water Channels Aquaporin 4 and -1 Expression in Subependymoma Depends on the Localization of the Tumors

    PubMed Central

    Mack, Andreas F.; Hoffmeister, Maike; Beschorner, Rudi; Ritz, Rainer

    2015-01-01

    Background We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior. Methods and Results Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels. Conclusions Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1. PMID:26115524

  19. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression.

    PubMed

    Wissmann, Melanie; Yin, Na; Müller, Judith M; Greschik, Holger; Fodor, Barna D; Jenuwein, Thomas; Vogler, Christine; Schneider, Robert; Günther, Thomas; Buettner, Reinhard; Metzger, Eric; Schüle, Roland

    2007-03-01

    Posttranslational modifications of histones, such as methylation, regulate chromatin structure and gene expression. Recently, lysine-specific demethylase 1 (LSD1), the first histone demethylase, was identified. LSD1 interacts with the androgen receptor and promotes androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9) only. Here, we identify the Jumonji C (JMJC) domain-containing protein JMJD2C as the first histone tridemethylase regulating androgen receptor function. JMJD2C interacts with androgen receptor in vitro and in vivo. Assembly of ligand-bound androgen receptor and JMJD2C on androgen receptor-target genes results in demethylation of trimethyl H3K9 and in stimulation of androgen receptor-dependent transcription. Conversely, knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumour cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities. PMID:17277772

  20. Induction of carcinoma cell migration on vitronectin by NF-kappa B-dependent gene expression.

    PubMed Central

    Yebra, M; Filardo, E J; Bayna, E M; Kawahara, E; Becker, J C; Cheresh, D A

    1995-01-01

    Integrin alpha v beta 5 promotes FG carcinoma cell adhesion to vitronectin yet requires protein kinase C (PKC) activation for migration on this ligand. Here we report that this PKC-dependent cell motility event requires NF-kappaB-dependent transcription. Specifically, a component within nuclear extracts prepared from PKC-stimulated FG cells exhibited a significant increase in binding activity to a synthetic oligonucleotide containing a consensus kappa B sequence. These nuclear DNA-binding complexes were shown to be comprised of p65 and p50 NF-kappaB/rel family members and appeared functionally active because they promoted transcription of a reporter construct containing a kappa B site. The NF-kappa B activation event was directly linked to the alpha v beta 5 motility response because the NF-kappa B-binding oligonucleotide, when introduced into FG cells, inhibited cell migration on vitronectin but not on collagen and had no effect on cell adhesion to either ligand. These results suggest that the detected DNA-binding complexes interact with kappa B transcriptional elements to regulate gene expression required for alpha v beta 5-dependent cell motility on vitronectin. Images PMID:7579698

  1. Age-dependent chloride channel expression in skeletal muscle fibres of normal and HSALR myotonic mice

    PubMed Central

    DiFranco, Marino; Yu, Carl; Quiñonez, Marbella; Vergara, Julio L

    2013-01-01

    We combine electrophysiological and optical techniques to investigate the role that the expression of chloride channels (ClC-1) plays on the age-dependent electrical properties of mammalian muscle fibres. To this end, we comparatively evaluate the magnitude and voltage dependence of chloride currents (ICl), as well as the resting resistance, in fibres isolated from control and human skeletal actin (HSA)LR mice (a model of myotonic dystrophy) of various ages. In control mice, the maximal peak chloride current ([peak-ICl]max) increases from −583 ± 126 to −956 ± 260 μA cm−2 (mean ± SD) between 3 and 6 weeks old. Instead, in 3-week-old HSALR mice, ICl are significantly smaller (−153 ± 33 μA cm−2) than in control mice, but after a long period of ∼14 weeks they reach statistically comparable values. Thus, the severe ClC-1 channelopathy in young HSALR animals is slowly reversed with aging. Frequency histograms of the maximal chloride conductance (gCl,max) in fibres of young HSALR animals are narrow and centred in low values; alternatively, those from older animals show broad distributions, centred at larger gCl,max values, compatible with mosaic expressions of ClC-1 channels. In fibres of both animal strains, optical data confirm the age-dependent increase in gCl, and additionally suggest that ClC-1 channels are evenly distributed between the sarcolemma and transverse tubular system membranes. Although gCl is significantly depressed in fibres of young HSALR mice, the resting membrane resistance (Rm) at −90 mV is only slightly larger than in control mice due to upregulation of a Rb-sensitive resting conductance (gK,IR). In adult animals, differences in Rm are negligible between fibres of both strains, and the contributions of gCl and gK,IR are less altered in HSALR animals. We surmise that while hyperexcitability in young HSALR mice can be readily explained on the basis of reduced gCl, myotonia in adult HSALR animals may be explained on the basis of a

  2. DNA vaccine cocktail expressing genotype A and C HBV surface and consensus core antigens generates robust cytotoxic and antibody responses in mice and Rhesus macaques.

    PubMed

    Obeng-Adjei, N; Hutnick, N A; Yan, J; Chu, J S; Myles, D J F; Morrow, M P; Sardesai, N Y; Weiner, D B

    2013-12-01

    There are well over a quarter of a billion chronic hepatitis B virus (HBV) carriers across the globe. Most carriers are at high risk for development of liver cirrhosis and subsequent progression to hepatocellular carcinoma. It is therefore imperative to develop new approaches for immunotherapy against this infection. Antibodies and cytotoxic T cells to different HBV antigens are believed to be important for reducing viral load and clearing HBV-infected cells from the liver. Some of the major challenges facing current vaccine candidates have been their inability to induce both humoral and cellular immunity to multiple antigenic targets and the induction of potent immune responses against the major genotypes of HBV. In this study, highly optimized synthetic DNA plasmids against the HBV consensus core (HBc) and surface (HBs) antigens genotypes A and C were developed and evaluated for their immune potential. These plasmids, which encode the most prevalent genotypes of the virus, were observed to individually induce binding antibodies to HBs antigens and drove robust cell-mediated immunity in animal models. Similar responses to both HBc and HBs antigens were observed when mice and non-human primates were inoculated with the HBc-HBs cocktails. In addition to the cytotoxic T lymphocyte activities exhibited by the immunized mice, the vaccine-induced responses were broadly distributed across multiple antigenic epitopes. These elements are believed to be important to develop an effective therapeutic vaccine. These data support further evaluation of multivalent synthetic plasmids as therapeutic HBV vaccines. PMID:24310062

  3. Nonlinear Network Reconstruction from Gene Expression Data Using Marginal Dependencies Measured by DCOL

    PubMed Central

    Zhu, Mengyao; Wang, Xiaofei; Lu, Jianwei; Yu, Tianwei

    2016-01-01

    Reconstruction of networks from high-throughput expression data is an important tool to identify new regulatory relations. Given that nonlinear and complex relations exist between biological units, methods that can utilize nonlinear dependencies may yield insights that are not provided by methods using linear associations alone. We have previously developed a distance to measure predictive nonlinear relations, the Distance based on Conditional Ordered List (DCOL), which is sensitive and computationally efficient on large matrices. In this study, we explore the utility of DCOL in the reconstruction of networks, by combining it with local false discovery rate (lfdr)–based inference. We demonstrate in simulations that the new method named nlnet is effective in recovering hidden nonlinear modules. We also demonstrate its utility using a single cell RNA seq dataset. The method is available as an R package at https://cran.r-project.org/web/packages/nlnet. PMID:27380516

  4. HIF-1α restricts NF-κB-dependent gene expression to control innate immunity signals

    PubMed Central

    Bandarra, Daniel; Biddlestone, John; Mudie, Sharon; Müller, H.-Arno J.; Rocha, Sonia

    2015-01-01

    Hypoxia and inflammation are intimately linked. It is known that nuclear factor κB (NF-κB) regulates the hypoxia-inducible factor (HIF) system, but little is known about how HIF regulates NF-κB. Here, we show that HIF-1α represses NF-κB-dependent gene expression. HIF-1α depletion results in increased NF-κB transcriptional activity both in mammalian cells and in the model organism Drosophila melanogaster. HIF-1α depletion enhances the NF-κB response, and this required not only the TAK-IKK complex, but also CDK6. Loss of HIF-1α results in an increased angiogenic response in mammalian cancer cells and increased mortality in Drosophila following infection. These results indicate that HIF-1α is required to restrain the NF-κB response, and thus prevents excessive and damaging pro-inflammatory responses. PMID:25510503

  5. Antioxidant Houttuynia cordata extract upregulates filaggrin expression in an aryl hydrocarbon-dependent manner.

    PubMed

    Doi, Kazuko; Mitoma, Chikage; Nakahara, Takeshi; Uchi, Hiroshi; Hashimoto-Hachiya, Akiko; Takahara, Masakazu; Tsuji, Gaku; Nakahara, Makiko; Furue, Masutaka

    2014-11-01

    The plant Houttuynia cordata, which is called "dokudami" in Japanese, is known as a potent antioxidant herb that has been traditionally consumed as a folk medicine for various ailments, such as diabetes, obesity, cough, fever and skin diseases, in Asia. However, its antioxidant mechanism remains largely unknown. In the present study, we investigated the effects of Houttuynia cordata extract (HCE) on human keratinocytes. HCE activated aryl hydrocarbon receptor (AHR) and nuclear factor E2-related factor 2, with subsequent induction of the antioxidative enzyme NAD (P)H: quinone oxidoreductase 1 gene. HCE inhibited the generation of reactive oxygen species (ROS) in keratinocytes stimulated with tumor necrosis factor α or benzo(α)pyrene. Moreover, HCE upregulated the gene expression of filaggrin, an essential skin barrier protein, in an AHR-dependent manner. HCE may be beneficial for treating ROS-related photoaging and barrier-disrupted skin conditions. PMID:25816564

  6. Chemotherapy-induced Dkk-1 expression by primary human mesenchymal stem cells is p53 dependent.

    PubMed

    Hare, Ian; Evans, Rebecca; Fortney, James; Moses, Blake; Piktel, Debbie; Slone, William; Gibson, Laura F

    2016-10-01

    Mesenchymal stem cells (MSCs) are abundant throughout the body and regulate signaling within tumor microenvironments. Wnt signaling is an extrinsically regulated pathway that has been shown to regulate tumorigenesis in many types of cancer. After evaluating a panel of Wnt activating and inhibiting molecules, we show that primary human MSCs increase the expression of Dkk-1, an inhibitor of Wnt signaling, into the extracellular environment following chemotherapy exposure in a p53-dependent manner. Dkk-1 has been shown to promote tumor growth in several models of malignancy, suggesting that MSC-derived Dkk-1 could counteract the intent of cytotoxic chemotherapy, and that pharmacologic inhibition of Dkk-1 in patients receiving chemotherapy treatment for certain malignancies may be warranted. PMID:27586146

  7. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

    SciTech Connect

    Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha; Massey, Thomas E.

    2014-03-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in

  8. Impaired Regulation of ALDH2 Protein Expression Revealing a Yet Unknown Epigenetic Impact of rs886205 on Specific Methylation of a Negative Regulatory Promoter Region in Alcohol-Dependent Patients.

    PubMed

    Haschemi Nassab, Mani; Rhein, Mathias; Hagemeier, Lars; Kaeser, Marius; Muschler, Marc; Glahn, Alexander; Pich, Andreas; Heberlein, Annemarie; Kornhuber, Johannes; Bleich, Stefan; Frieling, Helge; Hillemacher, Thomas

    2016-01-01

    Acetaldehyde, the carcinogenic metabolite of ethanol known to provoke aversive symptoms of alcohol consumption, is predominantly eliminated by aldehyde dehydrogenase 2 (ALDH2). Reduced ALDH2 activity correlates with low alcohol tolerance and low risk for alcohol dependence. The ALDH2 promoter polymorphism rs886205 (A>G) is associated with decreased promoter activity, but a molecular mechanism and allele-dependent ALDH2 protein expression has not been described yet. On the basis of allele-dependent epigenetic effects, we analyzed the rs886205 genotype, methylation rates of cytosine-phosphatidyl-guanine (CpG)-sites within a regulatory promoter region and ALDH2 protein levels in 82 alcohol-dependent patients during a 2-week withdrawal and compared them to 34 matched controls. Patients without the G-allele of rs886205 showed higher methylation of the promoter region than controls and readily adapted epigenetically as well as on protein level during withdrawal, while patients with the G-allele displayed retarded methylation readjustment and no change in ALDH2 protein levels. Our data provide novel insights into an unknown genetic-epigenetic interaction, revealing impaired ALDH2 protein expression in patients with the G-allele of rs886205. Additionally, we checked for an association between rs886205 and protection against alcohol dependence and found a trend association between the G-allele and protection against alcohol dependence that needs replication in a larger Caucasian cohort. PMID:26339786

  9. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    SciTech Connect

    Dalgaard, Louise T.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. Black-Right-Pointing-Pointer UCP2 mRNA up-regulation by glucose is dependent on glucokinase. Black-Right-Pointing-Pointer Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. Black-Right-Pointing-Pointer This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic {beta}-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/- islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2-/- and GK+/- islets compared with GK+/- islets and UCP2 deficiency improved glucose tolerance of GK+/- mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/- mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  10. Dendritic remodeling of hippocampal neurons is associated with altered NMDA receptor expression in alcohol dependent rats

    PubMed Central

    Staples, Miranda C.; Kim, Airee; Mandyam, Chitra D.

    2015-01-01

    Prolonged alcohol exposure has been previously shown to impair the structure and function of the hippocampus, although the underlying structural and biochemical alterations contributing to these deleterious effects are unclear. Also unclear is whether these changes persist into prolonged periods of abstinence. Previous work from our lab utilizing a clinically relevant rodent model of alcohol consumption demonstrated that alcohol dependence (induced by chronic intermittent ethanol vapor exposure or CIE) decreases proliferation and survival of neural stem cells in the hippocampal subgranular zone and hippocampal neurogenesis in the dentate gyrus, implicating this region of the cortex as particularly sensitive to the toxic effects of prolonged ethanol exposure. For this study, we investigated seven weeks of CIE-induced morphological changes (dendritic complexity and dendritic spine density) of dentate gyrus (DG) granule cell neurons, CA3, and CA1 pyramidal neurons and the associated alterations in biochemical markers of synaptic plasticity and toxicity (NMDA receptors and PSD-95) in the hippocampus in ethanol-experienced Wistar rats 3h (CIE) and 21 days (protracted abstinence) after the last ethanol vapor exposure. CIE reduced dendritic arborization of DG neurons and this effect persisted into protracted abstinence. CIE enhanced dendritic arborization of pyramidal neurons and this effect did not persist into protracted abstinence. The architectural changes in dendrites did not correlate with alterations in dendritic spine density, however, they were associated with increases in the expression of pNR2B, total NR2B, and total NR2A immediately following CIE with expression levels returning to control levels in prolonged abstinence. Overall, these data provide the evidence that CIE produces profound changes in hippocampal structural plasticity and in molecular tools that maintain hippocampal structural plasticity, and these alterations may underlie cognitive dysfunction

  11. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements.

    PubMed

    van der Velden, Yme U; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. PMID:26615334

  12. Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat.

    PubMed Central

    Tseng, C C; Jarboe, L A; Landau, S B; Williams, E K; Wolfe, M M

    1993-01-01

    Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose. Approximately 7.8 x 10(5) recombinant clones of a neonatal rat intestinal cDNA library were screened by using plaque hybridization, and three clones were identified and sequenced with the dideoxynucleotide chain-termination method. The translated amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that rat GIP was derived by proteolytic processing of a 144-amino acid precursor polypeptide. The mature peptide is flanked by a 43-amino acid NH2-terminal peptide that contains a 21-amino acid signal peptide and by a 59-amino acid COOH-terminal peptide. Analysis of the nucleotide and amino acid sequence of rat GIP revealed only two substitutions from the known human GIP peptide. The use of high-stringency RNA blot-hybridization analysis of total RNA extracted from various organs demonstrated expression of the GIP gene in the duodenum and jejunum and, to a lesser extent, in the ileum. In addition, expression of the GIP gene was observed in the submandibular salivary gland both by RNA analysis and RIA. In response to duodenal perfusion of a 20% Lipomul meal for 60 min, duodenal mucosal GIP mRNA concentrations increased by 42.8% and 48.2% at 30 and 60 min, respectively. Images Fig. 1 Fig. 4 PMID:8446620

  13. PPARgamma-Dependent Control of Renin Expression: Molecular Mechanisms and Pathophysiological Relevance

    PubMed Central

    Todorov, Vladimir T.

    2013-01-01

    During the last years accumulating evidence demonstrated that the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) regulates the expression of renin gene and thus the overall renin production. This review summarizes the current knowledge of the transcriptional control of the renin gene by PPARgamma received from variety of models ranging from cell culture to transgenic animals. The molecular mechanisms of the PPARgamma action on renin are particularly interesting because they are featured by two newly described characteristics: one of them is the recently identified PPARgamma target sequence Pal3 which is specific for the human renin gene and mediates exceptionally high sensitivity to transactivation; the other is the potentiating effect of PPARgamma on the cAMP signaling in the renin-producing cells. Furthermore, I discuss the need for generating of additional transgenic animal models which are more appropriate with regard to the role of the PPARgamma-dependent regulation of the renin gene expression in human diseases such as arterial hypertension and metabolic syndrome. PMID:24288524

  14. Expression and Stress-Dependent Induction of Potassium Channel Transcripts in the Common Ice Plant1

    PubMed Central

    Su, Hua; Golldack, Dortje; Katsuhara, Maki; Zhao, Chengsong; Bohnert, Hans J.

    2001-01-01

    We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mm NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant. PMID:11161018

  15. Unexpected reduction of skin tumorigenesis on expression of cyclin-dependent kinase 6 in mouse epidermis.

    PubMed

    Wang, Xian; Sistrunk, Christopher; Rodriguez-Puebla, Marcelo L

    2011-01-01

    Cyclin-dependent kinases (CDKs) 4 and 6 are important regulators of the G(1) phase of the cell cycle, share 71% amino acid identity, and are expressed ubiquitously. As a result, it was assumed that each of these kinases plays a redundant role regulating normal and neoplastic proliferation. In previous reports, we have described the effects of CDK4 expression in transgenic mice, including the development of epidermal hyperplasia and increased malignant progression to squamous cell carcinoma. To study the role of CDK6 in epithelial growth and tumorigenesis, we generated transgenic mice carrying the CDK6 gene under the keratin 5 promoter (K5CDK6). Similar to K5CDK4 mice, epidermal proliferation increased substantially in K5CDK6 mice; however, no hyperplasia was observed. CDK6 overexpression also triggered keratinocyte apoptosis in interfollicular and follicular epidermis as a compensatory mechanism to override aberrant proliferation. Unexpectedly, CDK6 overexpression results in decreased skin tumor development compared with wild-type siblings. The inhibition in skin tumorigenesis was similar to that previously reported in K5-cyclin D3 mice. Furthermore, biochemical analysis of the K5CDK6 epidermis showed preferential complex formation between CDK6 and cyclin D3, suggesting that this particular complex plays an important role in tumor restraint. These studies provide in vivo evidence that CDK4 and CDK6 play a similar role as a mediator of keratinocyte proliferation but differ in apoptosis activation and skin tumor development. PMID:21224071

  16. Structure and expression of mouse mitochondrial voltage dependent anion channel genes

    SciTech Connect

    Craigen, W.J.; Lovell, R.S.; Sampson, M.J.

    1994-09-01

    Voltage dependent anion channels (VDACs) are small abundant proteins of the outer mitochondrial membrane that interact with the adenine nucleotide translocater and bind glycerol kinase and hexokinase. Kinase binding is developmentally regulated, tissue specific, and increased in various tumor cell lines. VDACs are also components of the peripheral benzodiazepine receptor and GABA{sub A} receptor. Two human VDAC cDNAs have previously been reported, and expression of these isoforms appears ubiquitous. Genomic Southern analysis suggests the presence of other as yet uncharacterised VDAC genes. To study VDAC function in a mammal more amenable to experimental manipulation, we have isolated three mouse VDAC genes by cDNA cloning from a mouse brain cDNA library. DNA sequencing of the cDNAs shows that they share 65-75% amino acid identity. Northern analysis indicates that MVDAC1 is expressed most highly in kidney, heart, and brain. Using an MVDAC3 3{prime} untranslated exon as a probe, three distinct transcripts can be detected. The gene structure for MVDAC3 and MVDAC2 has been completed and suggests that the VDAC isoforms did not arise by gene duplication and divergence. The intron/exon boundaries are not conserved between MVDAC1 and MVDAC3, and MVDAC2 appears to be encoded by a single intronless gene.

  17. Dynamic Emotional and Neural Responses to Music Depend on Performance Expression and Listener Experience

    PubMed Central

    Chapin, Heather; Jantzen, Kelly; Scott Kelso, J. A.; Steinberg, Fred; Large, Edward

    2010-01-01

    Apart from its natural relevance to cognition, music provides a window into the intimate relationships between production, perception, experience, and emotion. Here, emotional responses and neural activity were observed as they evolved together with stimulus parameters over several minutes. Participants listened to a skilled music performance that included the natural fluctuations in timing and sound intensity that musicians use to evoke emotional responses. A mechanical performance of the same piece served as a control. Before and after fMRI scanning, participants reported real-time emotional responses on a 2-dimensional rating scale (arousal and valence) as they listened to each performance. During fMRI scanning, participants listened without reporting emotional responses. Limbic and paralimbic brain areas responded to the expressive dynamics of human music performance, and both emotion and reward related activations during music listening were dependent upon musical training. Moreover, dynamic changes in timing predicted ratings of emotional arousal, as well as real-time changes in neural activity. BOLD signal changes correlated with expressive timing fluctuations in cortical and subcortical motor areas consistent with pulse perception, and in a network consistent with the human mirror neuron system. These findings show that expressive music performance evokes emotion and reward related neural activations, and that music's affective impact on the brains of listeners is altered by musical training. Our observations are consistent with the idea that music performance evokes an emotional response through a form of empathy that is based, at least in part, on the perception of movement and on violations of pulse-based temporal expectancies. PMID:21179549

  18. Dynamic emotional and neural responses to music depend on performance expression and listener experience.

    PubMed

    Chapin, Heather; Jantzen, Kelly; Kelso, J A Scott; Steinberg, Fred; Large, Edward

    2010-01-01

    Apart from its natural relevance to cognition, music provides a window into the intimate relationships between production, perception, experience, and emotion. Here, emotional responses and neural activity were observed as they evolved together with stimulus parameters over several minutes. Participants listened to a skilled music performance that included the natural fluctuations in timing and sound intensity that musicians use to evoke emotional responses. A mechanical performance of the same piece served as a control. Before and after fMRI scanning, participants reported real-time emotional responses on a 2-dimensional rating scale (arousal and valence) as they listened to each performance. During fMRI scanning, participants listened without reporting emotional responses. Limbic and paralimbic brain areas responded to the expressive dynamics of human music performance, and both emotion and reward related activations during music listening were dependent upon musical training. Moreover, dynamic changes in timing predicted ratings of emotional arousal, as well as real-time changes in neural activity. BOLD signal changes correlated with expressive timing fluctuations in cortical and subcortical motor areas consistent with pulse perception, and in a network consistent with the human mirror neuron system. These findings show that expressive music performance evokes emotion and reward related neural activations, and that music's affective impact on the brains of listeners is altered by musical training. Our observations are consistent with the idea that music performance evokes an emotional response through a form of empathy that is based, at least in part, on the perception of movement and on violations of pulse-based temporal expectancies. PMID:21179549

  19. NFATc1 Mediates HDAC-Dependent Transcriptional Repression of Osteocalcin Expression During Osteoblast Differentiation

    PubMed Central

    Choo, Min-Kyung; Yeo, Hyeonju; Zayzafoon, Majd

    2009-01-01

    We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter possibly in an HDAC-dependent manner. PMID:19463978

  20. Dose-dependent microRNA expression in human fibroblasts after LET irradiation

    NASA Astrophysics Data System (ADS)

    Maes, Olivier Charles; An, Jin; Wu, Honglu; Wang, Eugenia; Sarojini, Harshini

    Humans are exposed to various levels of radiation during spaceflight voyages. In cells, exposure to linear energy transfer (LET) radiation causes cellular damage and triggers responses controlled by unique gene-directed signaling pathways. MicroRNAs (miRNAs) are small ( 22- nucleotide) non-coding RNAs, which regulate gene expression generally by either degrading the messager RNA or inhibiting translation. Their implication in specific cellular response pathways is largely unknown. Here, we investigated the role of radiation-dependent changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray exposure in human fibroblasts, and correlated their predicted targets with the cells' genomics and proteomics profiles. A differential miRNA expression pattern was observed between low and high doses of irradiation, with early (0.5 and 2 hrs) significant changes mostly after a high dose and, late (6 and 24 hrs) significant changes after both low and high doses of irradiation. The results suggest that miRNAs may act as ‘hub' regulators of signaling pathways initially to derepress their target genes for cellular responses such as DNA repair, followed by up-regulation to suppress apoptosis, and finally down-regulation to reestablish cellular normalcy. Functional attributions are made to key microRNAs, potentially regulating known radiation biomarkers as well as radiation-responsive mechanisms of cell cycle checkpoint, proliferation and apoptosis. In summary, radiation-responsive miRNAs may have functional roles in the regulation of cell death or survival, and may become biodosimeters for radiation dose exposure. Specific microRNAs may exert a hormetic effect after low-dose radiation, and prove useful in future applications for radiation adaptive therapy and in the prevention and treatment of radiation-induced damage. The confirmation of specific miRNAs as biodosimetry markers with therapeutic applications will be necessary in future functional

  1. Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent.

    PubMed

    Glukhova, M A; Frid, M G; Shekhonin, B V; Vasilevskaya, T D; Grunwald, J; Saginati, M; Koteliansky, V E

    1989-07-01

    Different fibronectin (FN) variants arise from the single gene transcript alternatively spliced in a tissue-specific manner (Hynes, R. O. 1985. Annu. Rev. Cell Biol. 1:67-90; Owens, R. J., A. R. Kornblihtt, and F. E. Baralle. 1986. Oxf. Surv. Eurcaryotic Genes. 3:141-160). We used mAb IST-9, specific for extra domain A (ED-A) FN sequence, and cDNA probe to ED-A exon to determine whether ED-A is present in FN synthesized by vascular smooth muscle cells (SMCs) and, if so, whether expression of ED-A is SMC phenotype dependent. ED-A-containing FN (A-FN) was not revealed in tunica media of human arteries and normal rat aorta by immunofluorescence and immunoblotting techniques. A cDNA probe to ED-A exon did not hybridize with RNA isolated from human aortic media. A positive reaction with IST-9 was observed in (a) diffuse intimal thickening and atherosclerotic plaque from human arteries; (b) experimentally induced intimal thickening in rat aorta; and (c) cultured vascular SMCs. A-FN mRNA was present in the RNA preparation from human aortic intima as judged by hybridization with cDNA probe to ED-A. On the other hand, an mAb interacting with an epitope common for all FN variants revealed FN in both intima and media of human arteries and in the normal rat aorta. A cDNA probe to a sequence shared by all FN variants hybridized with RNA from both intima and media of human aorta, though the level of expression was higher in intima. The data suggest that ED-A exon is omitted during splicing of the FN mRNA precursor in medial SMCs while the expression of A-FN is characteristic of "modulated" SMCs--those of intimal thickenings, of atherosclerotic lesions, and growing in culture. PMID:2663879

  2. Genotypic Context and Epistasis in Individuals and Populations.

    PubMed

    Sackton, Timothy B; Hartl, Daniel L

    2016-07-14

    Genes encode components of coevolved and interconnected networks. The effect of genotype on phenotype therefore depends on genotypic context through gene interactions known as epistasis. Epistasis is important in predicting phenotype from genotype for an individual. It is also examined in population studies to identify genetic risk factors in complex traits and to predict evolution under selection. Paradoxically, the effects of genotypic context in individuals and populations are distinct and sometimes contradictory. We argue that predicting genotype from phenotype for individuals based on population studies is difficult and, especially in human genetics, likely to result in underestimating the effects of genotypic context. PMID:27419868

  3. Short communication: The effect of genotyping cows to improve the reliability of genomic predictions for selection candidates.

    PubMed

    Edel, C; Pimentel, E C G; Plieschke, L; Emmerling, R; Götz, K-U

    2016-03-01

    In this study we investigate the potential of enlarging the reference population for genomic prediction in dairy cattle by routinely genotyping a random sample of the first-crop daughters of every AI bull in the breeding program. We analyzed small nuclear pedigrees, each consisting of a genotyped selection candidate and 3 generations of genotyped male ancestors. Genotypes were taken from the genomic routine evaluation of Fleckvieh cattle in Germany and Austria. The phenotypic information of a daughter of any one male in each of these pedigrees was either considered to be part of the daughter yield deviation of the corresponding sire, or was assumed to be an individually observed genotyped daughter of this sire. Daughter genotypes in this case were simulated from phased haplotypes of their sires and random maternal gametes drawn from a haplotype library. We measured the gain from genotyping daughters as the increase in model-based theoretical reliability of the genomic prediction for a putative selection candidate. We expressed the improvements as a marginal increase, corresponding to an increase in reliability at a reliability baseline level of zero, to simplify comparisons. Results were encouraging with 2 to 40% of marginal reliability increase for selection candidates depending on the assumed heritability of the trait and the number of daughters modeled to be genotyped in the design. PMID:26723131

  4. Effect of temperature on biomass allocation in seedlings of two contrasting genotypes of the oilseed crop Ricinus communis.

    PubMed

    Ribeiro, Paulo R; Zanotti, Rafael F; Deflers, Carole; Fernandez, Luzimar G; Castro, Renato D de; Ligterink, Wilco; Hilhorst, Henk W M

    2015-08-01

    Ricinus communis is becoming an important crop for oil production, and studying the physiological and biochemical aspects of seedling development may aid in the improvement of crop quality and yield. The objective of this study was to assess the effect of temperature on biomass allocation in two R. communis genotypes. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Root length of each seedling was measured. Biomass allocation was strongly affected by temperature. Seedlings grown at 25°C and 35°C showed greater biomass than seedlings grown at 20°C. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behavior. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Based on metabolite and gene expression profiles, genotype MPA11 increases its level of osmoprotectant molecules and transcripts of genes encoding for antioxidant enzymes and heat shock proteins to a higher extent than genotype IAC80. This might be causal for the ability to maintain homeostasis and support root growth at elevated temperatures in genotype MPA11. PMID:26276402

  5. ERK Oscillation-Dependent Gene Expression Patterns and Deregulation by Stress-Response

    SciTech Connect

    Waters, Katrina M.; Cummings, Brian S.; Shankaran, Harish; Scholpa, Natalie E.; Weber, Thomas J.

    2014-09-15

    Studies were undertaken to determine whether ERK oscillations regulate a unique subset of genes in human keratinocytes and subsequently, whether the p38 stress response inhibits ERK oscillations. A DNA microarray identified many genes that were unique to ERK oscillations, and network reconstruction predicted an important role for the mediator complex subunit 1 (MED1) node in mediating ERK oscillation-dependent gene expression. Increased ERK-dependent phosphorylation of MED1 was observed in oscillating cells compared to non-oscillating counterparts as validation. Treatment of keratinocytes with a p38 inhibitor (SB203580) increased ERK oscillation amplitudes and MED1 and phospho-MED1 protein levels. Bromate is a probable human carcinogen that activates p38. Bromate inhibited ERK oscillations in human keratinocytes and JB6 cells and induced an increase in phospho-p38 and decrease in phospho-MED1 protein levels. Treatment of normal rat kidney cells and primary salivary gland epithelial cells with bromate decreased phospho-MED1 levels in a reversible fashion upon treatment with p38 inhibitors (SB202190; SB203580). Our results indicate that oscillatory behavior in the ERK pathway alters homeostatic gene regulation patterns and that the cellular response to perturbation may manifest differently in oscillating vs non-oscillating cells.

  6. DNA polymerase β-dependent cell survival independent of XRCC1 expression.

    PubMed

    Horton, Julie K; Gassman, Natalie R; Dunigan, Brittany D; Stefanick, Donna F; Wilson, Samuel H

    2015-02-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  7. DNA polymerase β-dependent cell survival independent of XRCC1 expression

    PubMed Central

    Horton, Julie K.; Gassman, Natalie R.; Dunigan, Brittany B.; Stefanick, Donna F.; Wilson, Samuel H.

    2014-01-01

    Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1+/+ and Xrcc1−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β- dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants. PMID:25541391

  8. Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a.

    PubMed

    Jung, Bo-Kyoung; Kim, Hye-Ran; Park, Gyu-Nam; Luo, Guangxiang; Chang, Kyung-Soo

    2016-06-01

    Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype-dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3-specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents. PMID:27225463

  9. Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes.

    PubMed

    Nakajo, Koichi; Kubo, Yoshihiro

    2005-11-15

    It is well established that stimulation of G(q)-coupled receptors such as the M1 muscarinic acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that this muscarinic inhibition is mainly caused by the breakdown of PIP(2), the role of the subsequent activation of protein kinase C (PKC) is not well understood. By reconstituting M currents in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 microm oxotremorine M (oxo-M) induces a positive shift (4-30 mV, depending on which KCNQ channels are expressed) in the conductance-voltage relationship (G-V) of KCNQ channels. When we applied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large positive shift (17.8 +/- 1.6 mV) in the G-V curve for KCNQ2, while chelerythrine, a PKC inhibitor, attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP(2) had little effect on the G-V curve for KCNQ2 channels; although pretreating cells with 10 mum wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G-V curve was shifted only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T cells, the G-V curve seemed already to be shifted in a positive direction, even before activation of PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipette shifted the G-V curve in a negative direction suggests KCNQ2/3 channels are constitutively phosphorylated in HEK 293T cells. PMID:16179364

  10. IL7Rα expression and upregulation by IFNβ in dendritic cell subsets is haplotype-dependent.

    PubMed

    McKay, Fiona C; Hoe, Edwin; Parnell, Grant; Gatt, Prudence; Schibeci, Stephen D; Stewart, Graeme J; Booth, David R

    2013-01-01

    The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response. PMID:24147013

  11. Agonist ligand discrimination by the two orexin receptors depends on the expression system.

    PubMed

    Putula, Jaana; Turunen, Pauli M; Jäntti, Maria H; Ekholm, Marie E; Kukkonen, Jyrki P

    2011-04-20

    Despite the recent successes in producing orexin receptor subtype-selective antagonists, these are not commonly available, and therefore, agonist ligands are regularly used to ascribe cell and tissue responses to OX(1) or OX(2) receptors. In the current study, we have compared the native "subtype-selective" agonist, orexin-B, and its reputedly enhanced synthetic variant, Ala(11), d-Leu(15)-orexin-B, in two different recombinant cell lines. Ca2+ elevation was used as readout, and the two "selective" ligands were compared to the subtype-non-selective orexin-A, as is customary with these ligands. In transiently transfected HEK-293 cells, orexin-B showed 9-fold selectivity for the OX(2) receptor and Ala(11), d-Leu(15)-orexin-B 23-fold selectivity, when the potency ratios of ligands were compared between OX(1) and OX(2). In stable CHO-K1 cells, the corresponding values were only 2.6- and 14-fold, respectively. In addition to being low, the selectivity of the ligands was also variable, as indicated by the comparison of the two cell lines. For instance, the relative potency of Ala(11), d-Leu(15)-orexin-B at OX(2) in CHO cells was only 2.3-fold higher than its relative potency at OX(1) in HEK-293 cells; this indicates that Ala(11), d-Leu(15)-orexin-B does not show high enough selectivity for OX(2) to be useful for determination of receptor subtype expression. Comparison of the potencies of orexin-A and -B with respect to a number of published responses in OX(1)-expressing CHO cells, demonstrates that these show great variation: i.e., orexin-A is 1.6-18-fold more potent than orexin-B, depending on the response assessed. These data together suggest that orexin receptor ligands show signal trafficking, which makes agonist-based pharmacology unreliable. PMID:21362456

  12. Effects of Diisodecyl Phthalate on PPAR:RXR-Dependent Gene Expression Pathways in Sea Bream Hepatocytes.

    PubMed

    Cocci, Paolo; Mosconi, Gilberto; Arukwe, Augustine; Mozzicafreddo, Matteo; Angeletti, Mauro; Aretusi, Graziano; Palermo, Francesco Alessandro

    2015-05-18

    Evidence that endocrine-disrupting chemicals (EDCs) may target metabolic disturbances, beyond interference with the functions of the endocrine systems has recently accumulated. Among EDCs, phthalate plasticizers like the diisodecyl phthalate (DiDP) are commonly found contaminants of aquatic environments and have been suggested to function as obesogens by activating peroxisome proliferator activated receptors (PPARs), a subset of nuclear receptors (NRs) that act as metabolic sensors, playing pivotal roles in lipid homeostasis. However, little is known about the modulation of PPAR signaling pathways by DiDP in fish. In this study, we have first investigated the ligand binding efficiency of DiDP to the ligand binding domains of PPARs and retinoid-X-receptor-α (RXRα) proteins in fish using a molecular docking approach. Furthermore, in silico predictions were integrated by in vitro experiments to show possible dose-relationship effects of DiDP on PPAR:RXR-dependent gene expression pathways using sea bream hepatocytes. We observed that DiDP shows high binding efficiency with piscine PPARs demonstrating a greater preference for RXRα. Our studies also demonstrated the coordinate increased expression of PPARs and RXRα, as well as their downstream target genes in vitro. Principal component analysis (PCA) showed the strength of relationship between transcription of most genes involved in fatty acid metabolism and PPAR mRNA levels. In particular, fatty acid binding protein (FABP) was highly correlated to all PPARs. The results of this study suggest that DiDP can be considered an environmental stressor that activates PPAR:RXR signaling to promote long-term changes in lipid homeostasis leading to potential deleterious physiological consequences in teleost fish. PMID:25825955

  13. Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors

    PubMed Central

    Luchkina, Natalia V.; Huupponen, Johanna; Clarke, Vernon R. J.; Coleman, Sarah K.; Keinänen, Kari; Taira, Tomi; Lauri, Sari E.

    2014-01-01

    The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2+/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development. PMID:24599589

  14. Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

    SciTech Connect

    Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

    2011-03-01

    The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 {mu}M) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

  15. Effects of a Rhodiola rosea L. extract on acquisition and expression of morphine tolerance and dependence in mice.

    PubMed

    Mattioli, Laura; Perfumi, Marina

    2011-03-01

    This study investigated the effect of Rhodiola rosea L. extract on acquisition and expression of morphine tolerance and dependence in mice. Therefore animals were injected with repeated administration of morphine (10 mg/kg, subcutaneous) twice daily for five or six days, in order to make them tolerant or dependent. Rhodiola rosea L. extract (0, 10, 15 and 20 mg/kg) was administered by the intragastric route 60 min prior to each morphine injection (for acquisition) or prior the last injection of morphine or naloxone on test day (for tolerance or dependence expression, respectively). Morphine tolerance was evaluated by testing its analgesic effect in the tail flick test at the 1st and 5th days. Morphine dependence was evaluated by counting the number of withdrawal signs (jumping, rearing, forepaw tremor, teeth chatter) after naloxone injection (5 mg/kg; intraperitoneal) on the test day (day 6). Results showed that Rhodiola rosea L. extract significantly reduced the expression of morphine tolerance, while it was ineffective in modulating its acquisition. Conversely, Rhodiola rosea L. extract significantly and dose-dependently attenuated both development and expression of morphine dependence after chronic or acute administration. These data suggest that Rhodiola rosea L. may have human therapeutic potential for treatment of opioid addiction. PMID:20142299

  16. Protein Kinase CK2 Expression Predicts Relapse Survival in ERα Dependent Breast Cancer, and Modulates ERα Expression in Vitro

    PubMed Central

    Williams, Marlon D.; Nguyen, Thu; Carriere, Patrick P.; Tilghman, Syreeta L.; Williams, Christopher

    2015-01-01

    The heterotetrameric protein kinase CK2 has been associated with oncogenic transformation, and our previous studies have shown that it may affect estrogenic signaling. Here, we investigate the role of the protein kinase CK2 in regulating ERα (estrogen receptor α) signaling in breast cancer. We determined the correlation of CK2α expression with relapse free breast cancer patient survival utilizing Kaplan Meier Plotter (kmplot.com/analysis/) to mine breast cancer microarrays repositories. Patients were stratified according to ERα status, histological grade, and hormonal therapy. Luciferase reporter assays and flow cytometry were implemented to determine the impact of CK2 inhibition on ERE-mediated gene expression and expression of ERα protein. CK2α expression is associated with shorter relapse free survival among ERα (+) patients with grade 1 or 2 tumors, as well as among those patients receiving hormonal therapy. Biochemical inhibition of CK2 activity results in increased ER-transactivation as well as increased expression among ERα (+) and ERα (−) breast cancer cell lines. These findings suggest that CK2 may contribute to estrogen-independent cell proliferation and breast tumor progression, and may potentially serve as a biomarker and pharmacological target in breast cancer. PMID:26703694

  17. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression

    PubMed Central

    Liu, S-C; Chuang, S-M; Hsu, C-J; Tsai, C-H; Wang, S-W; Tang, C-H

    2014-01-01

    Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts. PMID:25341039

  18. TIM-3/Gal-9 interaction induces IFNγ-dependent IDO1 expression in acute myeloid leukemia blast cells.

    PubMed

    Folgiero, Valentina; Cifaldi, Loredana; Li Pira, Giuseppina; Goffredo, Bianca Maria; Vinti, Luciana; Locatelli, Franco

    2015-01-01

    NK cells expressing TIM-3 show a marked increase in IFNγ production in response to acute myeloid leukemia (AML) blast cells that endogenously express Gal-9. Herein, we demonstrate that NK cell-mediated production of IFNγ, induced by TIM-3/Gal-9 interaction and released in bone marrow microenvironment, is responsible for IDO1 expression in AML blasts. IDO1-expressing AML blasts consequently down-regulate NK cell degranulation activity, by sustaining leukemia immune escape. Furthermore, the blocking of TIM-3/Gal-9 interaction strongly down-regulates IFNγ-dependent IDO1 activity. Thus, the inhibition of TIM-3/Gal-9 immune check point, which affects NK cell-dependent IFNγ production and the consequent IDO1 activation, could usefully integrate current chemotherapeutic approaches. PMID:25886742

  19. Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans.

    PubMed

    Wei, Xuqin; Meng, Xiaolei; Chen, Yunlai; Wei, Yutuo; Du, Liqin; Huang, Ribo

    2014-01-01

    The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase. PMID:24078133

  20. Solar zenith angle-dependent asymmetries in Venusian bow shock location revealed by Venus Express

    NASA Astrophysics Data System (ADS)

    Chai, Lihui; Wan, Weixing; Fraenz, Markus; Zhang, Tielong; Dubinin, Eduard; Wei, Yong; Li, Yi; Rong, Zhaojin; Zhong, Jun; Han, Xiuhong; Futaana, Yoshifumi

    2015-06-01

    It has been long known that the Venusian bow shock (BS) location is asymmetric from the observations of the long-lived Pioneer Venus Orbiter mission. The Venus Express (VEX) mission crossed BS near perpendicularly not only in the terminator region but also in the near-subsolar and tail regions. Taking the advantage of VEX orbit geometry, we examined a large data set of BS crossings observed during the long-lasting solar minimum between solar cycles 23 and 24 and found that the Venusian BS asymmetries exhibit dependence of solar zenith angle. In the terminator and tail regions, both the magnetic pole-equator and north-south asymmetries are observed in Venusian BS location, which is similar to the Pioneer Venus Orbiter (PVO) observation near terminator. However, in the near-subsolar region, only the magnetic north-south is observed; i.e., the BS shape is indented inward over magnetic south pole and bulged outward over magnetic north pole. The absence of the magnetic pole-equator asymmetry in the near-subsolar region suggests that the magnetic pole-equator asymmetry is mainly caused by the asymmetric wave propagation rather than the ion pickup process. The evident magnetic north-south asymmetry in solar minimum, which is not observed by PVO, suggests that even during the low long-lasting solar minimum, the ion pickup process is very important in Venusian space environment.

  1. Expression of vasopressin receptors in hamster hypothalamus is sexually dimorphic and dependent upon photoperiod.

    PubMed Central

    Dubois-Dauphin, M; Theler, J M; Zaganidis, N; Dominik, W; Tribollet, E; Pévet, P; Charpak, G; Dreifuss, J J

    1991-01-01

    The distribution of vasopressin receptors was studied in the brain of a photoperiodic animal, the Siberian hamster. Attention was focused on [3H]vasopressin binding sites located in the hypothalamic ventromedial nucleus, medial tuberal nucleus, and ventral premammillary nucleus in males or females kept in long or short photoperiod conditions. Displacement experiments with structural analogs suggested that vasopressin receptors in the hamster hypothalamus are of the vasopressor (V1) type. Quantitative data obtained with a gaseous detector of beta-particles indicated that in the ventromedial nucleus and in the ventral premammillary nucleus of animals in long photoperiod, the number of beta-particles emitted per unit area was significantly greater in males than in females. In the ventromedial hypothalamic nucleus, in both males and females, the number of beta-particles emitted was significantly lower in short than in long photoperiod conditions. In the ventral premammillary nucleus, shortening of the photoperiod had a significant effect in reducing the amount of [3H]vasopressin bound in females, but not in males. These data suggest that, in the hamster, the control of the expression of vasopressin receptors differs among various hypothalamic nuclei and may depend on the sex and/or on the level of circulating gonadal steroids. Images PMID:1837144

  2. Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice.

    PubMed

    Tanaka, Shinji; Miyamoto, Osamu; Janjua, Najma A; Miyazaki, Tetsuji; Takahashi, Fumio; Konishi, Ryoji; Itano, Toshifumi

    2005-01-01

    Amygdala kindling is regarded as a model of temporal lobe epilepsy in humans because of many points of similarity. In amygdala kindling, bilateralization of epileptic seizures follows from the accumulation of stimulation and commissural fibers may play a role in this process. However, new progenies of cells following amygdala kindling have not been reported and the precise nature of how bilateralization occurs is not clear. In the present study, we aim to clarify the emergence of radial glia during the progress of amygdala kindling in mouse brain. For this purpose, immunohistochemical staining for 3CB2, which is a specific marker of radial glia, was employed. Immunoreactivity for 3CB2 was observed in the forceps minor, radiation of trunk and forceps major regions at Clonus 3 and more strongly at Clonus 5. In the forceps major, the cingulate gyrus showed immunopositive staining at Clonus 3, but the corpus callosum and alveus hippocampi showed staining only at Clonus 5. In the fimbria hippocampus, the anterior commissure posterior showed staining at Clonus 5. However, the anterior commissure anterior was not stained at the stage progressed to Clonus 5. These findings indicate stage and region dependent expression of progenitor cells in commissural fibers and suggest that these changes may accompany the formation of new circuits in seizure progression during amygdala kindling. PMID:16202564

  3. Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates

    PubMed Central

    Kosugi, Akihiko; Murashima, Koichiro; Doi, Roy H.

    2001-01-01

    Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High α-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate. PMID:11717260

  4. VHL-dependent regulation of a β-dystroglycan glycoform and glycogene expression in renal cancer

    PubMed Central

    AGGELIS, VASSILIS; CRAVEN, RACHEL A.; PENG, JIANHE; HARNDEN, PATRICIA; SCHAFFER, LANA; HERNANDEZ, GILBERTO E.; HEAD, STEVEN R.; MAHER, EAMONN R.; TONGE, ROBERT; SELBY, PETER J.; BANKS, ROSAMONDE E.

    2013-01-01

    Identification of novel biomarkers and targets in renal cell carcinoma (RCC) remains a priority and one cellular compartment that is a rich potential source of such molecules is the plasma membrane. A shotgun proteomic analysis of cell surface proteins enriched by cell surface biotinylation and avidin affinity chromatography was explored using the UMRC2- renal cancer cell line, which lacks von Hippel-Lindau (VHL) tumour suppressor gene function, to determine whether proteins of interest could be detected. Of the 814 proteins identified ∼22% were plasma membrane or membrane-associated, including several with known associations with cancer. This included β-dystroglycan, the transmembrane subunit of the DAG1 gene product. VHL-dependent changes in the form of β-dystroglycan were detected in UMRC2−/+VHL transfectants. Deglycosylation experiments showed that this was due to differential sialylation. Analysis of normal kidney cortex and conventional RCC tissues showed that a similar change also occurred in vivo. Investigation of the expression of genes involved in glycosylation in UMRC2−/+VHL cells using a focussed microarray highlighted a number of enzymes involved in sialylation; upregulation of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) was validated in UMRC2− cells compared with their +VHL counterparts and also found in conventional RCC tissue. These results implicate VHL in the regulation of glycosylation and raise interesting questions regarding the extent and importance of such changes in RCC. PMID:23970118

  5. Load Regulates Bone Formation and Sclerostin Expression through a TGFβ-Dependent Mechanism

    PubMed Central

    Nguyen, Daniel; Alliston, Tamara

    2013-01-01

    Bone continually adapts to meet changing physical and biological demands. Osteoblasts, osteoclasts, and osteocytes cooperate to integrate these physical and biochemical cues to maintain bone homeostasis. Although TGFβ acts on all three of these cell types to maintain bone homeostasis, the extent to which it participates in the adaptation of bone to mechanical load is unknown. Here, we investigated the role of the TGFβ pathway in load-induced bone formation and the regulation of Sclerostin, a mechanosensitive antagonist of bone anabolism. We found that mechanical load rapidly represses the net activity of the TGFβ pathway in osteocytes, resulting in reduced phosphorylation and activity of key downstream effectors, Smad2 and Smad3. Loss of TGFβ sensitivity compromises the anabolic response of bone to mechanical load, demonstrating that the mechanosensitive regulation of TGFβ signaling is essential for load-induced bone formation. Furthermore, sensitivity to TGFβ is required for the mechanosensitive regulation of Sclerostin, which is induced by TGFβ in a Smad3-dependent manner. Together, our results show that physical cues maintain bone homeostasis through the TGFβ pathway to regulate Sclerostin expression and the deposition of new bone. PMID:23308287

  6. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence.

    PubMed

    Mamdani, Mohammed; Williamson, Vernell; McMichael, Gowon O; Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; van der Vaart, Andrew D; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S; Miles, Michael F; Dick, Danielle; Riley, Brien P; Dumur, Catherine; Vladimirov, Vladimir I

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  7. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

    PubMed Central

    Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  8. Molecular hydrogen regulates gene expression by modifying the free radical chain reaction-dependent generation of oxidized phospholipid mediators

    PubMed Central

    Iuchi, Katsuya; Imoto, Akemi; Kamimura, Naomi; Nishimaki, Kiyomi; Ichimiya, Harumi; Yokota, Takashi; Ohta, Shigeo

    2016-01-01

    We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress. Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic and preventive medicine. Moreover, H2 regulates various signal transduction pathways and the expression of many genes. However, the primary targets of H2 in the signal transduction pathways are unknown. Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2 gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to the H2-dependently autoxidized phospholipid species reduced Ca2+ signal transduction and mediated the expression of various genes as revealed by comprehensive microarray analysis. In the cultured cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased cellular Ca2+, resulting in the regulation of Ca2+-dependent gene expression. Thus, H2 might regulate gene expression via the Ca2+ signal transduction pathway by modifying the free radical-dependent generation of oxidized phospholipid mediators. PMID:26739257

  9. Describing the Frequency of Marijuana Use: Fuzziness and Context-Dependent Interpretation of Frequency Expressions.

    ERIC Educational Resources Information Center

    Matt, Georg E.; Wilson, Sandra Jo

    1994-01-01

    A fuzzy set model is offered for interpreting vague frequency expressions, such as "rarely" and "sometimes." Results with 152 undergraduates reporting marijuana use reflect different frequency expressions for the same level of use and suggest that self-report validity may be enhanced by analyzing frequency expressions as fuzzy sets. (SLD)

  10. The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression).

    PubMed

    Sax, Joanna K; Dash, Bipin C; Hong, Rui; Dicker, David T; El-Deiry, Wafik S

    2002-01-01

    Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints. PMID:12429914

  11. Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide.

    PubMed

    Tiruppathi, Chinnaswamy; Shimizu, Jun; Miyawaki-Shimizu, Kayo; Vogel, Stephen M; Bair, Angela M; Minshall, Richard D; Predescu, Dan; Malik, Asrar B

    2008-02-15

    We investigated the role of NF-kappaB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-kappaB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-kappaB activation by inhibiting the interaction of IKKgamma with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-kappaB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2-4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-kappaB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability. PMID:18077459

  12. pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

    PubMed Central

    Yohannes, Elizabeth; Barnhart, D. Michael; Slonczewski, Joan L.

    2004-01-01

    During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis

  13. Type 1 diabetes risk for human leukocyte antigen (HLA)-DR3 haplotypes depends on genotypic context: association of DPB1 and HLA class I loci among DR3- and DR4-matched Italian patients and controls.

    PubMed

    Noble, Janelle A; Martin, Adelle; Valdes, Ana M; Lane, Julie A; Galgani, Andrea; Petrone, Antonio; Lorini, Renata; Pozzilli, Paolo; Buzzetti, Raffaella; Erlich, Henry A

    2008-01-01

    Patients with high-risk human leukocyte antigen (HLA)-DR-DQ genotypes for type 1 diabetes (T1D) were compared with HLA-matched controls to evaluate T1D risk for other HLA loci, including HLA-A, -B, -Cw, and DPB1. Patients (n = 133) with high-risk genotypes (DR3/DR3, DR3/DR4, DR4/DR4) were selected from the Lazio (Rome) region of Italy. Screening of more than 9000 patients from the Lazio region and northern Italy yielded 162 controls with high-T1D-risk haplotypes. Although the overall distributions did not differ significantly, allele frequency differences were discovered between the controls from Lazio and controls from northern Italy for some alleles previously determined to affect T1D risk, such as A*3002, DPB1*0301, and DPB1*0402. Therefore, Lazio patient data were compared both with the Lazio subset of controls (n = 53) and with the entire group of controls for association analyses. Significant allele frequency differences between patients and DR-DQ-matched controls existed for specific alleles at all loci. Data for the DR3/DR3 subset of patients and controls demonstrated an increase of Cw*0702 in patients. Compared with controls, reduced patient frequencies were seen for several alleles, including A*0101, B*0801, and Cw*0701, all on the highly conserved, extended DR3 haplotype known as 8.1 in DR3/DR3, but not DR3/DR4, subgroup. DPB1*0101, often reported on 8.1 haplotypes, was also less frequent in DR3/DR3 patients than controls. Analysis of family-based data from the HBDI repository was consistent with the observed results from the Italian patients, indicating the presence of a T1D-protective locus at or near A*0101 and a second T1D-protective locus at or near DPB1*0101. These data indicate that T1D risk conferred by the 8.1 haplotype is genotype dependent. PMID:18486765

  14. MicroRNA dependent regulation of DNMT-1 and tumor suppressor gene expression by Interleukin-6 in human malignant cholangiocytes

    PubMed Central

    Braconi, Chiara; Huang, Nianyuan; Patel, Tushar

    2014-01-01

    Although the inflammation-associated cytokine Interleukin-6 (IL-6) has been implicated in cholangiocarcinoma growth, the relationship between IL-6 and oncogenic changes is unknown. IL-6 can increase expression of DNA methyltransferase 1 (DNMT-1) and epigenetically regulate the expression of several genes, including microRNAs (miRNAs). DNMT-1 up-regulation occurs in hepatobiliary cancers and is associated with a poor prognosis. To understand the potential regulation of DNMT-1 by IL-6 dependent miRNAs, we examined the expression of a group of miRNAs which have sequence complementarity to the 3′-UTR of DNMT-1, namely miR-148a, miR-152 and miR-301. The expression of these miRNAs was decreased in cholangiocarcinoma cells. Moreover, the expression of all three miRNAs was decreased in IL-6 over-expressing malignant cholangiocytes in vitro and in tumor cell xenografts. There was a concomitant decrease in expression of the methylation-sensitive tumor suppressor genes Rassf1a, and p16INK4a. Using luciferase reporter constructs, DNMT-1 was verified as a target for miR-148a and miR-152. Precursors to miR-148a and miR-152 decreased DNMT-1 protein expression, increased Rassf1a and p16INK4a expression and reduced cell proliferation. Conclusion These data indicate that IL-6 can regulate the activity of DNMT-1 and expression of methylation-dependent tumor suppressor genes by modulation of miR-148a and miR-152, and provide a link between this inflammation-associated cytokines and oncogenesis in cholangiocarcinoma. PMID:20146264

  15. The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation

    PubMed Central

    Henning, Amanda N.; Haag, Jill D.; Smits, Bart M. G.; Gould, Michael N.

    2016-01-01

    In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman’s life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage. PMID:27537370

  16. Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

    PubMed

    Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

    2016-07-01

    Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry. PMID:27298373

  17. The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation.

    PubMed

    Henning, Amanda N; Haag, Jill D; Smits, Bart M G; Gould, Michael N

    2016-08-01

    In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman's life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage. PMID:27537370

  18. Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

    PubMed Central

    Graham, M E; Gerke, V; Burgoyne, R D

    1997-01-01

    The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells. Images PMID:9188096

  19. Do Voltage-Dependent K^+ Channels Require Ca2+? A Critical Test Employing a Heterologous Expression System

    NASA Astrophysics Data System (ADS)

    Armstrong, Clay M.; Miller, Christopher

    1990-10-01

    Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K^+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K^+ channels in an altered, debased conformation. Voltage-dependent K^+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K^+ channels. These expressed channels respond to low Ca2+ identically to voltage-dependent K^+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K^+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltage-dependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K^+ channels and is required to stabilize the native conformations of these membrane proteins.

  20. Activity-dependent regulation of prestin expression in mouse outer hair cells

    PubMed Central

    Song, Yohan; Xia, Anping; Lee, Hee Yoon; Wang, Rosalie; Ricci, Anthony J.

    2015-01-01

    Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal hearing. Its regulatory mechanisms are unknown. Several mouse models of hearing loss demonstrate increased prestin, inspiring us to investigate how hearing loss might feedback onto OHCs. To test whether centrally mediated feedback regulates prestin, we developed a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) into adult CBA mice produced significant loss of inner hair cells without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to receive normal auditory mechanical stimulation and having normal function. Patch-clamp experiments demonstrated no change in OHC prestin, indicating that loss of information transfer centrally did not alter prestin expression. To test whether local mechanical feedback regulates prestin, we used TectaC1509G mice, where the tectorial membrane is malformed and only some OHCs are stimulated. OHCs connected to the tectorial membrane had normal prestin levels, whereas OHCs not connected to the tectorial membrane had elevated prestin levels, supporting an activity-dependent model. To test whether the endocochlear potential was necessary for prestin regulation, we studied TectaC1509G mice at different developmental ages. OHCs not connected to the tectorial membrane had lower than normal prestin levels before the onset of the endocochlear potential and higher than normal prestin levels after the onset of the endocochlear potential. Taken together, these data indicate that OHC prestin levels are regulated through local feedback that requires mechanoelectrical transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment. PMID:25810486

  1. Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca.

    PubMed

    Tobimatsu, T; Sakai, T; Hashida, Y; Mizoguchi, N; Miyoshi, S; Toraya, T

    1997-11-01

    Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65%. The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma). Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the beta subunit and alpha2gamma2 complex, respectively. Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S). Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme. PMID:9344474

  2. Oral benzo[a]pyrene-induced cancer: two distinct types in different target organs depend on the mouse Cyp1 genotype

    PubMed Central

    Shi, Zhanquan; Dragin, Nadine; Miller, Marian L.; Stringer, Keith F.; Johansson, Elisabet; Chen, Jing; Uno, Shigeyuki; Gonzalez, Frank J.; Rubio, Carlos A.; Nebert, Daniel W.

    2010-01-01

    Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) can both detoxify PAHs and activate them to cancer-causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ~28 days, whereas Cyp1(+/+) wild-type mice remain healthy for >12 months on this regimen. In the present study, male Cyp1(+/+) wild-type, Cyp1a1(−/−) and Cyp1b1(−/−) single-knockout, and Cyp1a1/1b1(−/−) double-knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs––including proximal small intestine (PSI), liver, preputial gland duct (PGD)––were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(−/−) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(−/−) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(−/−) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking up-regulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y-chromosome; the Rab30 oncogene was up-regulated; the Nr0b2 tumor suppressor gene was down-regulated; paradoxical over-expression of numerous immunoglobulin kappa and heavy chain variable genes was found––although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of “gene-environment interactions” in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just one or two globally absent genes. PMID:20127859

  3. Lymphomas are sensitive to perforin-dependent cytotoxic pathways despite expression of PI-9 and overexpression of bcl-2.

    PubMed

    Godal, Robert; Keilholz, Ulrich; Uharek, Lutz; Letsch, Anne; Asemissen, Anne Marie; Busse, Antonia; Na, Il-Kang; Thiel, Eckhard; Scheibenbogen, Carmen

    2006-04-15

    There is considerable interest in immunotherapeutic approaches for lymphoma. The expression of proteinase inhibitor 9 (PI-9), a molecule that inactivates granzyme B, is considered an immune escape mechanism in lymphoma. Further, lymphomas frequently overexpress the antiapoptotic molecule bcl-2, which is able to inhibit perforin-dependent cytotoxic pathways. In this study, the impact of PI-9 and bcl-2 expression on the sensitivity of lymphomas to T- and natural killer (NK) cell-mediated cytotoxicity was analyzed. We found PI-9 expression in 10 of 18 lymphoma cell lines and in 9 of 14 primary lymphomas. Overexpression of bcl-2 was found in 8 of 18 cell lines and in 12 of 14 primary lymphomas. All lymphoma cells were sensitive to cytolysis by specific T cells and cytokine-activated NK cells, and no difference in sensitivity was observed with respect to PI-9 or bcl-2 expression. Cytolysis was mediated predominantly through perforin-dependent pathways despite expression of PI-9 and bcl-2. Interestingly, the majority of lymphoma cells were resistant to cytolysis by resting allogeneic NK cells. This was due to the failure of lymphomas to induce degranulation of resting NK cells. These results show that resistance to perforin-dependent pathways is not a relevant immune escape mechanism in lymphoma and therefore is unlikely to impair clinical outcome of immunotherapeutic approaches. PMID:16373664

  4. Genes and Small RNA Transcripts Exhibit Dosage-Dependent Expression Pattern in Maize Copy-Number Alterations.

    PubMed

    Zuo, Tao; Zhang, Jianbo; Lithio, Andrew; Dash, Sudhansu; Weber, David F; Wise, Roger; Nettleton, Dan; Peterson, Thomas

    2016-07-01

    Copy-number alterations are widespread in animal and plant genomes, but their immediate impact on gene expression is still unclear. In animals, copy-number alterations usually exhibit dosage effects, except for sex chromosomes which tend to be dosage compensated. In plants, genes within small duplications (<100 kb) often exhibit dosage-dependent expression, whereas large duplications (>50 Mb) are more often dosage compensated. However, little or nothing is known about expression in moderately-sized (1-50 Mb) segmental duplications, and about the response of small RNAs to dosage change. Here, we compared maize (Zea mays) plants with two, three, and four doses of a 14.6-Mb segment of chromosome 1 that contains ∼300 genes. Plants containing the duplicated segment exhibit dosage-dependent effects on ear length and flowering time. Transcriptome analyses using GeneChip and RNA-sequencing methods indicate that most expressed genes and unique small RNAs within the duplicated segments exhibit dosage-dependent transcript levels. We conclude that dosage effect is the predominant regulatory response for both genes and unique small RNA transcripts in the segmental dosage series we tested. To our knowledge this is the first analysis of small RNA expression in plant gene dosage variants. Because segmental duplications comprise a significant proportion of eukaryotic genomes, these findings provide important new insight into the regulation of genes and small RNAs in response to dosage changes. PMID:27129738

  5. Interleukin-1-induced gene expression requires the membrane-raft-dependent internalization of the interleukin-1 receptor.

    PubMed

    Windheim, Mark

    2016-10-01

    Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression. PMID:27327966

  6. TIME-DEPENDENT EFFECTS ON GENE EXPRESSION IN RAT SEMINAL VESICLE DEVELOPMENTALLY ALTERED BY IN UTERO EXPOSURE TO TCDD

    EPA Science Inventory

    TIME-DEPENDENT EFFECTS ON GENE EXPRESSION IN RAT SEMINAL VESICLE DEVELOPMENTALLY ALTERED BY IN UTERO EXPOSURE TO TCDD. V M Richardson', J T Hamm2, and L S Birnbaum1. 'USEPA, ORD/NHEERL/ETD, Research Triangle Park, NC, USA, 'Curriculum in Toxicology, University of North Carolina, ...

  7. Following the time course of face gender and expression processing: a task-dependent ERP study.

    PubMed

    Valdés-Conroy, Berenice; Aguado, Luis; Fernández-Cahill, María; Romero-Ferreiro, Verónica; Diéguez-Risco, Teresa

    2014-05-01

    The effects of task demands and the interaction between gender and expression in face perception were studied using event-related potentials (ERPs). Participants performed three different tasks with male and female faces that were emotionally inexpressive or that showed happy or angry expressions. In two of the tasks (gender and expression categorization) facial properties were task-relevant while in a third task (symbol discrimination) facial information was irrelevant. Effects of expression were observed on the visual P100 component under all task conditions, suggesting the operation of an automatic process that is not influenced by task demands. The earliest interaction between expression and gender was observed later in the face-sensitive N170 component. This component showed differential modulations by specific combinations of gender and expression (e.g., angry male vs. angry female faces). Main effects of expression and task were observed in a later occipito-temporal component peaking around 230 ms post-stimulus onset (EPN or early posterior negativity). Less positive amplitudes in the presence of angry faces and during performance of the gender and expression tasks were observed. Finally, task demands also modulated a positive component peaking around 400 ms (LPC, or late positive complex) that showed enhanced amplitude for the gender task. The pattern of results obtained here adds new evidence about the sequence of operations involved in face processing and the interaction of facial properties (gender and expression) in response to different task demands. PMID:24594443

  8. Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

    PubMed Central

    McDonald, R A; Matthews, R P; Idzerda, R L; McKnight, G S

    1995-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types. Images Fig. 1 Fig. 3 PMID:7543684

  9. The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation.

    PubMed

    Simon, C; Simon, M; Vucelic, G; Hicks, M J; Plinkert, P K; Koitschev, A; Zenner, H P

    2001-12-10

    The invasive phenotype of cancers critically depends on the expression of proteases such as the M(R) 92,000 type IV collagenase (MMP-9). Several growth factors and oncogenes were found to increase promoter activity and as a consequence protease expression. This frequently requires the activation of the transcription factor AP-1 by signal transduction cascades such as the ERK and JNK pathways. We have previously demonstrated that the tumor promoter TPA can induce MMP-9 expression via a third signaling cascade, the p38 pathway. Considering that TPA is a potent activator of AP-1, we hypothesized that this transcription factor might also be required for p38 pathway-dependent MMP-9 regulation. While dominant negative p38 and MKK-6 mutants reduced MMP-9 promoter activity in CAT assays, a construct encoding an activating mutation in the MKK-6 protein potently stimulated it. This was mediated via 144 bp of the 5'flanking region of the wild-type promoter, which contains an AP-1 site at -79. Both point mutations in this motif and the expression of a c-jun protein lacking its transactivation domain and therefore acting as a dominant negative AP-1 mutant abrogated MKK-6-dependent promoter stimulation. Finally SB 203580, a specific p38 pathway inhibitor, reduced MMP-9 expression/secretion and in vitro invasion of cancer cells. Thus, our results provide evidence that also the third SAPK/MAPK signaling cascade, the p38 signal transduction pathway, stimulates MMP-9 expression in an AP-1-dependent fashion. PMID:11716547

  10. Insulin dependent diabetes mellitus epidemiology: HLA genotype study in 12 north eastern Italian families with two siblings affected by type I diabetes.

    PubMed

    Pinelli, L; Drei, F; Gonfiantini, E; Visentin, A; Roata, C; Ciaffoni, S; Maffeis, C

    1989-12-01

    The purpose of our study was to evaluate the relationship between the histocompatibility antigens and type I diabetes mellitus in families living in the north-eastern part of Italy. In each family two siblings were affected by diabetes. HLA-antigens were determined with the lymphocytotoxicity test, utilizing antisera of the series A-B-C-DR. The phenotypic frequencies were compared with those observed in controls. We showed that diabetes has a strong association with HLA DR 3 and/or DR 4 antigens. In particular we registered high frequency of compound heterozygous DR 3 - DR 4 subjects, and this fact supports the hypothesis of the existence of two different genes for diabetes associated with these HLA antigens. Moreover we observed a particular haplotype segregation with a very high percentage of HLA identity between patients belonging to the same family, confirming the association between HLA and genetic susceptibility to insulin dependent diabetes. These results confirm data in the literature and, completed by other data from other patients' families living in our area, will be useful in providing reliable genetic counselling. PMID:2606174

  11. Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

    PubMed Central

    Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

    2016-01-01

    Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype. PMID:26960370

  12. Diastrophic dysplasia and atelosteogenesis type II as expression of compound heterozygosis: first report of a Mexican patient and genotype-phenotype correlation.

    PubMed

    Macías-Gómez, Nelly Margarita; Mégarbané, André; Leal-Ugarte, Evelia; Rodríguez-Rojas, Lisa Ximena; Barros-Núñez, Patricio

    2004-08-30

    The osteochondrodysplasias represent a heterogeneous group of cartilage and bone diseases. Among these, achondrogenesis 1B, atelosteogenesis type II, diastrophic dysplasia, and autosomal recessive multiple epiphyseal dysplasia are caused by mutations in the solute carrier family 26 (sulfate transporter), member 2 gene (SLC26A2). This group of osteochondrodysplasias shows a continuous spectrum of clinical variability and shares many features in common. Usually, it is difficult to distinguish clinically among these patients. To date, several efforts have been made to correlate mutations in the SLC26A2 gene with phenotypic severity in the patients. We report on a Mexican girl with diastrophic dysplasia presenting some unusual clinical and radiographic features that are usually observed in atelosteogenesis type II. Molecular analysis of the SLC26A2 gene in this patient showed compound heterozygosity for the R178X and R279W mutations. In this patient, the combination of a mild and a severe mutation has apparently led to an intermediate or transitional clinical picture, showing an apparent genotype-phenotype correlation. PMID:15316973

  13. Early MyD88-Dependent Induction of Interleukin-17A Expression during Salmonella Colitis ▿ †

    PubMed Central

    Keestra, A. Marijke; Godinez, Ivan; Xavier, Mariana N.; Winter, Maria G.; Winter, Sebastian E.; Tsolis, Renée M.; Bäumler, Andreas J.

    2011-01-01

    The development of T helper 17 (TH17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1β. IL-23 and IL-1β synergized in inducing expression of IL-17A in splenic T cells in vitro. In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, TH17 cells, and CD4−CD8− T cells. The absence of IL-1β signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis. PMID:21576324

  14. UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis.

    PubMed

    Li, Nan; Teranishi, Mika; Yamaguchi, Hiroko; Matsushita, Tomonao; Watahiki, Masaaki K; Tsuge, Tomohiko; Li, Shao-Shan; Hidema, Jun

    2015-10-01

    Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 µmol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation. PMID:26272552

  15. Inferring haplotypes from genotypes on a pedigree with mutations, genotyping errors and missing alleles.

    PubMed

    Wang, Wei-Bung; Jiang, Tao

    2011-04-01

    Inferring the haplotypes of the members of a pedigree from their genotypes has been extensively studied. However, most studies do not consider genotyping errors and de novo mutations. In this paper, we study how to infer haplotypes from genotype data that may contain genotyping errors, de novo mutations, and missing alleles. We assume that there are no recombinants in the genotype data, which is usually true for tightly linked markers. We introduce a combinatorial optimization problem, called haplotype configuration with mutations and errors (HCME), which calls for haplotype configurations consistent with the given genotypes that incur no recombinants and require the minimum number of mutations and errors. HCME is NP-hard. To solve the problem, we propose a heuristic algorithm, the core of which is an integer linear program (ILP) using the system of linear equations over Galois field GF(2). Our algorithm can detect and locate genotyping errors that cannot be detected by simply checking the Mendelian law of inheritance. The algorithm also offers error correction in genotypes/haplotypes rather than just detecting inconsistencies and deleting the involved loci. Our experimental results show that the algorithm can infer haplotypes with a very high accuracy and recover 65%-94% of genotyping errors depending on the pedigree topology. PMID:21523936

  16. MyD88-Dependent Silencing of Transgene Expression During the Innate and Adaptive Immune Response to Helper-Dependent Adenovirus

    PubMed Central

    Suzuki, Masataka; Cerullo, Vincenzo; Bertin, Terry K.; Cela, Racel; Clarke, Christian; Guenther, Margaretha; Brunetti-Pierri, Nicola

    2010-01-01

    Abstract Activation of the host innate immune response after systemic administration of adenoviral vectors constitutes a principal impediment to successful clinical gene replacement therapies. Although helper-dependent adenoviruses (HDAds) lack all viral functional genes, systemic administration of a high dose of HDAd still elicits a potent innate immune response in host animals. Toll-like receptors (TLRs) are innate receptors that sense microbial products and trigger the maturation of antigen-presenting cells and cytokine production via MyD88-dependent signaling (except TLR3). Here we show that mice lacking MyD88 exhibit a dramatic reduction in proinflammatory cytokines after intravenous injection of a high dose of HDAd, and show significantly reduced induction of the adaptive immune response when compared with wild-type and TLR2-deficient mice. Importantly, MyD88–/– mice also show significantly higher and longer sustained transgene expression than do wild-type mice. Chromatin immunoprecipitation studies using wild-type and MyD88-deficient primary mouse embryonic fibroblasts showed significant MyD88-dependent transcriptional silencing of the HDAd-encoded transgenes. Our results demonstrate that MyD88 signaling, activated by systemic delivery of HDAd, initiates an innate immune response that suppresses transgene expression at the transcriptional level before initiation of the adaptive immune response. PMID:19824822

  17. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages.

    PubMed

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. PMID:23978445

  18. Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction.

    PubMed

    Purcell, Patricia; Oliver, Guillermo; Mardon, Graeme; Donner, Amy L; Maas, Richard L

    2005-12-01

    The Drosophila eyeless gene plays a central role in fly eye development and controls a subordinate regulatory network consisting of the so, eya and dac genes. All three genes have highly conserved mammalian homologs, suggesting possible conservation of this eye forming regulatory network. sine oculis (so) belongs to the so/Six gene family, and Six3 is prominently expressed in the developing mammalian eye. Eya1 and Dach1 are mammalian homologs of eya and dac, respectively, and although neither Eya1 nor Dach1 knockout mice express prenatal eye defects, possibilities exist for postnatal ocular phenotypes or for functional redundancy between related family members. To examine whether expression relationships analogous to those between ey, so, eya and dac exist in early mammalian oculogenesis, we investigated Pax6, Six3, Eya1 and Dach1 protein expression in murine lens and nasal placode development. Six3 expression in the pre-placode lens ectoderm is initially Pax6-independent, but subsequently both its expression and nuclear localization become Pax6-dependent. Six3, Dach1 and Eya1 nasal expression in pre-placode ectoderm are also initially Pax6-independent, but thereafter become Pax6-dependent. Pax6, Six3, Dach1 and Eya1 are all co-expressed in the developing ciliary marginal zone, a source of retinal stem cells in some vertebrates. An in vitro protein-protein interaction is detected between Six3 and Eya1. Collectively, these findings suggest that the Pax-Eya-Six-Dach network is at best only partly conserved during lens and nasal placode development. However, the findings do not rule out the possibility that such a regulatory network acts at later stages of oculogenesis. PMID:16024294

  19. Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells.

    PubMed

    Paria, Biman C; Malik, Asrar B; Kwiatek, Angela M; Rahman, Arshad; May, Michael J; Ghosh, Sankar; Tiruppathi, Chinnaswamy

    2003-09-26

    We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha. PMID:12855710

  20. Multi-locus Genotypes Underlying Temperature Sensitivity in a Mutationally Induced Trait

    PubMed Central

    Lee, Jonathan T.; Taylor, Matthew B.; Shen, Amy; Ehrenreich, Ian M.

    2016-01-01

    Determining how genetic variation alters the expression of heritable phenotypes across conditions is important for agriculture, evolution, and medicine. Central to this problem is the concept of genotype-by-environment interaction (or ‘GxE’), which occurs when segregating genetic variation causes individuals to show different phenotypic responses to the environment. While many studies have sought to identify individual loci that contribute to GxE, obtaining a deeper understanding of this phenomenon may require defining how sets of loci collectively alter the relationship between genotype, environment, and phenotype. Here, we identify combinations of alleles at seven loci that control how a mutationally induced colony phenotype is expressed across a range of temperatures (21, 30, and 37°C) in a panel of yeast recombinants. We show that five predominant multi-locus genotypes involving the detected loci result in trait expression with varying degrees of temperature sensitivity. By comparing these genotypes and their patterns of trait expression across temperatures, we demonstrate that the involved alleles contribute to temperature sensitivity in different ways. While alleles of the transcription factor MSS11 specify the potential temperatures at which the trait can occur, alleles at the other loci modify temperature sensitivity within the range established by MSS11 in a genetic background- and/or temperature-dependent manner. Our results not only represent one of the first characterizations of GxE at the resolution of multi-locus genotypes, but also provide an example of the different roles that genetic variants can play in altering trait expression across conditions. PMID:26990313

  1. First nosocomial outbreak of vancomycin-resistant Enterococcus faecium expressing a VanD-like phenotype associated with a vanA genotype.

    PubMed

    Naas, Thierry; Fortineau, Nicolas; Snanoudj, Renaud; Spicq, Colette; Durrbach, Antoine; Nordmann, Patrice

    2005-08-01

    Although enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant Enterococcus faecium (VRE) isolates in the nephrology department at Bicêtre Hospital (K.-Bicêtre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 microg/ml, whereas teicoplanin MICs ranged from 4 to 48 microg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a vanA gene. Plasmid extraction and mating-out experiments indicated that the vanA gene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The vanA gene was part of a vanA-type operon for expression of resistance located on a Tn1546-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS16 in the vanY gene that encodes a d,d-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any. PMID:16081891

  2. Expression of the chemokine CXCL14 and cetuximab-dependent tumour suppression in head and neck squamous cell carcinoma.

    PubMed

    Kondo, T; Ozawa, S; Ikoma, T; Yang, X-Y; Kanamori, K; Suzuki, K; Iwabuchi, H; Maehata, Y; Miyamoto, C; Taguchi, T; Kiyono, T; Kubota, E; Hata, R-I

    2016-01-01

    Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression

  3. Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent.

    PubMed

    Hartlage-Rübsamen, Maike; Zeitschel, Ulrike; Apelt, Jenny; Gärtner, Ulrich; Franke, Heike; Stahl, Tobias; Günther, Albrecht; Schliebs, Reinhard; Penkowa, Milena; Bigl, Volker; Rossner, Steffen

    2003-01-15

    The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to beta-amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N-M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by beta-amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients. PMID:12509807

  4. Dose-dependent effects of metals on gene expression in the sydney rock oyster, Saccostrea glomerata.

    PubMed

    Taylor, Daisy A; Nair, Sham V; Thompson, Emma L; Raftos, David A

    2015-09-01

    In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring. PMID:24615909

  5. microRNA-dependent Temporal Gene Expression in the Ureteric Bud Epithelium during Mammalian Kidney Development

    PubMed Central

    Nagalakshmi, Vidya K.; Lindner, Volkhard; Wessels, Andy; Yu, Jing

    2014-01-01

    Background Our previous study on mouse mutants with the ureteric bud (UB) epithelium-specific Dicer deletion (Dicer UB mutants) demonstrated the significance of UB epithelium-derived miRNAs in UB development. Results Our whole-genome transcriptional profiling showed that the Dicer mutant UB epithelium abnormally retained transcriptional features of the early UB epithelium and failed to express many genes associated with collecting duct differentiation. Further, we identified a temporal expression pattern of early UB genes during UB epithelium development in which gene expression was detected at early developmental stages and became undetectable by E14.5. In contrast, expression of early UB genes persisted at later stages in the Dicer mutant UB epithelium and increased at early stages. Our bioinformatics analysis of the abnormally persistently expressed early genes in the Dicer mutant UB epithelium showed significant enrichment of the let-7 family miRNA targets. We further identified a temporal expression pattern of let-7 miRNAs in the UB epithelium that is anti-parallel to that of some early UB genes during kidney development. Conclusions We propose a model in which the let-7 family miRNAs silence the expression of a subset of early genes in the UB epithelium at later developmental stages in order to promote collecting duct differentiation. PMID:25369991

  6. Age-dependent expression of osteochondrosis-related genes in equine leukocytes

    PubMed Central

    Mendoza, L.; Piquemal, D.; Lejeune, J. P.; Vander Heyden, L.; Noguier, F.; Bruno, R.; Sandersen, C.; Serteyn, D.

    2015-01-01

    Introduction:  Osteochondrosis (OC) is a developmental disease in horses which has a significant impact on the horse's welfare and performance. The early disturbance in the process of endochondral ossification progresses to inflammatory and repair processes in older horses. Previously, differentially expressed genes in leukocytes of OC-affected horses have been identified. The aim of the present study is to detect age-related changes in these differentially expressed genes. Materials and Methods:  The expression of OC-related genes was analysed by real-time PCR and subsequent statistical analysis (ΔΔCT) in the leukocytes of 135 Belgian Warmblood horses divided into three different age groups: <12 months (n=47), 18–24 months (n=50) >30 months (n=38). Results:  Relative expression of genes of horses less than 12 months of age showed significant induction of the genes MGAT4A, PRKCG, MHCI, ApoB, ApoB3G, B4GALT6 and a significantly lower expression of the genes OAS3. Horses of 18–24 months of age, showed a significantly higher expression of the genes TBC1D9, MGAT4A, IFIH1, MHCIIa and MMP1. Horses of more than 30 months of age showed a significantly higher expression of the genes MGAT4A, HP, SECTM1 compared with their age-matched control groups. Conclusions:  The study demonstrates that OC-related genes are differentially expressed in horses of different ages compared with their age-matched controls. Some of the genes may be implicated in cell signalling and differentiation as well as carbohydrate and lipid metabolism and inflammation. However, the causal relationship between the differentially expressed genes and the development and progression of the OC lesions needs to be determined. PMID:26392886

  7. Glial Expression of the Caenorhabditis elegans Gene swip-10 Supports Glutamate Dependent Control of Extrasynaptic Dopamine Signaling.

    PubMed

    Hardaway, J Andrew; Sturgeon, Sarah M; Snarrenberg, Chelsea L; Li, Zhaoyu; Xu, X Z Shawn; Bermingham, Daniel P; Odiase, Peace; Spencer, W Clay; Miller, David M; Carvelli, Lucia; Hardie, Shannon L; Blakely, Randy D

    2015-06-24

    Glial cells play a critical role in shaping neuronal development, structure, and function. In a screen for Caenorhabditis elegans mutants that display dopamine (DA)-dependent, Swimming-Induced Paralysis (Swip), we identified a novel gene, swip-10, the expression of which in glia is required to support normal swimming behavior. swip-10 mutants display reduced locomotion rates on plates, consistent with our findings of elevated rates of presynaptic DA vesicle fusion using fluorescence recovery after photobleaching. In addition, swip-10 mutants exhibit elevated DA neuron excitability upon contact with food, as detected by in vivo Ca(2+) monitoring, that can be rescued by glial expression of swip-10. Mammalian glia exert powerful control of neuronal excitability via transporter-dependent buffering of extracellular glutamate (Glu). Consistent with this idea, swip-10 paralysis was blunted in mutants deficient in either vesicular Glu release or Glu receptor expression and could be phenocopied by mutations that disrupt the function of plasma membrane Glu transporters, most noticeably glt-1, the ortholog of mammalian astrocytic GLT1 (EAAT2). swip-10 encodes a protein containing a highly conserved metallo-β-lactamase domain, within which our swip-10 mutations are located and where engineered mutations disrupt Swip rescue. Sequence alignments identify the CNS-expressed gene MBLAC1 as a putative mammalian ortholog. Together, our studies provide evidence of a novel pathway in glial cells regulated by swip-10 that limits DA neuron excitability, DA secretion, and DA-dependent behaviors through modulation of Glu signaling. PMID:26109664

  8. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

    SciTech Connect

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to ethanol

  9. Transmission and persistence of Ceratonova shasta genotypes in Chinook salmon.

    PubMed

    Hurst, Charlene N; Wong, Peter; Hallett, Sascha L; Ray, R Adam; Bartholomew, Jerri L

    2014-12-01

    Ceratonova shasta is a myxozoan parasite of salmon and trout transmitted by waterborne actinospores. Based on DNA sequence data and host specificity, 4 distinct parasite genotypes are recognized. Genotypes I and II are common in the lower reaches of the Klamath River, Oregon-California, but only infection by genotype I causes mortality in Chinook salmon. We conducted sentinel fish exposures and determined genotype composition in river water during exposure, and in fish gills, intestine, and tank water post-exposure to determine whether: (1) transmission of parasites having different genotypes is host-specific and (2) all transmitted genotypes persist in the host through to release as waterborne stages. Initial parasite transmission to the fish host appears indiscriminant, since we detected both genotypes I and II in 83.6% of the fish gills sampled. However, only genotype I was detected in fish that succumbed to infection, while both genotypes persisted in fish that survived. Persistence was likely dependent on exposure dose, initial infection type (mixed or single) and infection outcome (mortality or survival). The transmission of both genotypes to a majority of Chinook salmon and the persistence of multiple genotypes raises questions about how infection with mixed genotypes could result in within-host interactions that affect disease severity. PMID:24945751

  10. Expression of CD24 in Human Bone Marrow-Derived Mesenchymal Stromal Cells Is Regulated by TGFβ3 and Induces a Myofibroblast-Like Genotype

    PubMed Central

    Schäck, Luisa Marilena; Buettner, Manuela; Wirth, Alexander; Krettek, Christian; Hoffmann, Andrea; Noack, Sandra

    2016-01-01

    Human bone marrow-derived stromal cells (hBMSCs) derived from the adult organism hold great promise for diverse settings in regenerative medicine. Therefore a more complete understanding of hBMSC biology to fully exploit the cells' potential for clinical settings is important. The protein CD24 has been reported to be involved in a diverse range of processes such as cancer, adaptive immunity, inflammation, and autoimmune diseases in other cell types. Its expression in hBMSCs, which has not yet been analyzed, may add an important aspect in the understanding of hBMSC biology. The present study therefore analyzes the expression, regulation, and functional implication of the surface protein CD24 in hBMSCs. Methods used are stimulation studies with TGF beta as well as shRNA-mediated knockdown and overexpression of CD24 followed by microarray, immunocytochemistry, and flow cytometric analyses. To our knowledge, we demonstrate for the first time that the expression of CD24 is an inherent property of hBMSCs. Importantly, the data links the upregulation of CD24 to the adoption of a myofibroblast-like gene expression pattern in hBMSCs. We demonstrate that CD24 is an important modulator in transforming growth factor beta 3 (TGFβ3) signaling with a reciprocal regulatory relationship between these two proteins. PMID:26788063

  11. Expression and Self-Assembly of Virus-Like Particles from Two Genotypes of Marine Vesiviruses and Development of an ELISA for the Detection of Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequences encoding the major capsid protein (VP1) from two marine vesivirus isolates (Steller sea lion viruses V810 and V1415) were engineered for expression of virus-like particles (VLPs) in the baculovirus system. The resulting VLPs were morphologically similar to native vesivirus virions. Purif...

  12. Dietary deficiency increases presenilin expression, gamma-secretase activity, and Abeta levels: potentiation by ApoE genotype and alleviation by S-adenosyl methionine.

    PubMed

    Chan, Amy; Tchantchou, Flaubert; Rogers, Eugene J; Shea, Thomas B

    2009-08-01

    Apolipoprotein E4 (ApoE4) is a risk factor for Alzheimer's disease (AD). Whether this risk arises from a deficient function of E4 or the lack of protection provided by E2 or E3 is unclear. Previous studies demonstrate that deprivation of folate and vitamin E, coupled with dietary iron as a pro-oxidant, for 1 month displayed increased presenilin 1 (PS-1) expression, gamma-secretase, and Abeta generation in mice lacking ApoE (ApoE-/- mice). While ApoE-/- mice are a model for ApoE deficiency, they may not reflect the entire range of consequences of E4 expression. We therefore compared herein the impact of the above deficient diet on mice expressing human E2, E3, or E4. As folate deficiency is accompanied by a decrease in the major methyl donor, S-adenosyl methionine (SAM), additional mice received the deficient diet plus SAM. E2 was more protective than murine ApoE or E3 and E4. Surprisingly, PS-1 and gamma-secretase were over-expressed in E3 to the same extent as in E4 even under a complete diet, and were not alleviated by SAM supplementation. Abeta increased only in E4 mice maintained under the complete diet, and was alleviated by SAM supplementation. These findings suggest dietary compromise can potentiate latent risk factors for AD. PMID:19457069

  13. Stability of expression of reference genes among different lentil (Lens culinaris) genotypes subjected to cold stress, white mold disease, and Aphanomyces root rot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lentils have served as an important plant source of dietary protein for over 8000 years. The development of improved lentil varieties is accelerated by a better understanding of the genetic basis of desirable traits, which can be gained by examining patterns of gene expression among phenotypically d...

  14. Integrated analysis of miRNA and mRNA paired expression profiling of prenatal skeletal muscle development in three genotype pigs

    PubMed Central

    Tang, Zhonglin; Yang, Yalan; Wang, Zishuai; Zhao, Shuanping; Mu, Yulian; Li, Kui

    2015-01-01

    MicroRNAs (miRNAs) play a vital role in muscle development by binding to messenger RNAs (mRNAs). Based on prenatal skeletal muscle at 33, 65 and 90 days post-coitus (dpc) from Landrace, Tongcheng and Wuzhishan pigs, we carried out integrated analysis of miRNA and mRNA expression profiling. We identified 33, 18 and 67 differentially expressed miRNAs and 290, 91 and 502 mRNA targets in Landrace, Tongcheng and Wuzhishan pigs, respectively. Subsequently, 12 mRNAs and 3 miRNAs differentially expressed were validated using quantitative real-time PCR (qPCR), and 5 predicted miRNA targets were confirmed via dual luciferase reporter or western blot assays. We identified a set of miRNAs and mRNA genes differentially expressed in muscle development. Gene ontology (GO) enrichment analysis suggests that the miRNA targets are primarily involved in muscle contraction, muscle development and negative regulation of cell proliferation. Our data indicated that more mRNAs are regulated by miRNAs at earlier stages than at later stages of muscle development. Landrace and Tongcheng pigs also had longer phases of myoblast proliferation than Wuzhishan pigs. This study will be helpful to further explore miRNA-mRNA interactions in myogenesis and aid to uncover the molecular mechanisms of muscle development and phenotype variance in pigs. PMID:26496978

  15. Dopamine and Serotonin Transporter Genotypes Moderate Sensitivity to Maternal Expressed Emotion: The Case of Conduct and Emotional Problems in Attention Deficit/Hyperactivity Disorder

    ERIC Educational Resources Information Center

    Sonuga-Barke, Edmund J. S.; Oades, Robert D.; Psychogiou, Lamprini; Chen, Wai; Franke, Barbara; Buitelaar, Jan; Banaschewski, Tobias; Ebstein, Richard P.; Gil, Michael; Anney, Richard; Miranda, Ana; Roeyers, Herbert; Rothenberger, Aribert; Sergeant, Joseph; Steinhausen, Hans Christoph; Thompson, Margaret; Asherson, Philip; Faraone, Stephen V.

    2009-01-01

    Background: Mothers' positive emotions expressed about their children with attention deficit/hyperactivity disorder (ADHD) are associated with a reduced likelihood of comorbid conduct problems (CP). We examined whether this association with CP, and one with emotional problems (EMO), is moderated by variants within three genes, previously reported…

  16. Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice

    EPA Science Inventory

    Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and...

  17. Quantum dots induced interferon beta expression via TRIF-dependent signaling pathways by promoting endocytosis of TLR4.

    PubMed

    Ho, Chia-Chi; Luo, Yueh-Hsia; Chuang, Tsung-Hsien; Lin, Pinpin

    2016-02-17

    Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation and remodeling in the mouse lung. Expression of interferon beta (IFN-β), involved in tissue remodeling, was induced in the mouse lung. The objective of this study was to understand the mechanism of QD705 induced interferon beta (IFN-β) expression. QD705-COOH and QD705-PEG increased IFN-β and IP-10 mRNA levels during day1 to 90 post-exposure in mouse lungs. QD705-COOH increased IFN-β expression via Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) dependent Toll-like receptor (TLR) signaling pathways in macrophages RAW264.7. Silencing TRIF expression with siRNA or co-treatment with a TRIF inhibitor tremendously abolished QD705s-induced IFN-β expression. Co-treatment with a TLR4 inhibitor completely prevented IFN-β induction by QD705-COOH. QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented IFN-β induction. Thus, activation of the TRIF dependent TLRs pathway by promoting endocytosis of TLR4 is one of the mechanisms for immunomodulatory effects of nanoparticles. PMID:26925925

  18. Heat Stress- and Heat Shock Transcription Factor-Dependent Expression and Activity of Ascorbate Peroxidase in Arabidopsis1

    PubMed Central

    Panchuk, Irina I.; Volkov, Roman A.; Schöffl, Friedrich

    2002-01-01

    To find evidence for a connection between heat stress response, oxidative stress, and common stress tolerance, we studied the effects of elevated growth temperatures and heat stress on the activity and expression of ascorbate peroxidase (APX). We compared wild-type Arabidopsis with transgenic plants overexpressing heat shock transcription factor 3 (HSF3), which synthesize heat shock proteins and are improved in basal thermotolerance. Following heat stress, APX activity was positively affected in transgenic plants and correlated with a new thermostable isoform, APXS. This enzyme was present in addition to thermolabile cytosolic APX1, the prevalent isoform in unstressed cells. In HSF3-transgenic plants, APXS activity was detectable at normal temperature and persisted after severe heat stress at 44°C. In nontransgenic plants, APXS was undetectable at normal temperature, but could be induced by moderate heat stress. The mRNA expression profiles of known and three new Apx genes were determined using real-time PCR. Apx1 and Apx2 genes encoding cytosolic APX were heat stress and HSF dependently expressed, but only the representations of Apx2 mRNA met the criteria that suggest identity between APXS and APX2: not expressed at normal temperature in wild type, strong induction by heat stress, and HSF3-dependent expression in transgenic plants. Our data suggest that Apx2 is a novel heat shock gene and that the enzymatic activity of APX2/APXS is required to compensate heat stress-dependent decline of APX1 activity in the cytosol. The functional roles of modulations of APX expression and the interdependence of heat stress and oxidative stress response and signaling mechanisms are discussed. PMID:12068123

  19. Genotypic Variation under Fe Deficiency Results in Rapid Changes in Protein Expressions and Genes Involved in Fe Metabolism and Antioxidant Mechanisms in Tomato Seedlings (Solanum lycopersicum L.)

    PubMed Central

    Muneer, Sowbiya; Jeong, Byoung Ryong

    2015-01-01

    To investigate Fe deficiency tolerance in tomato cultivars, quantification of proteins and genes involved in Fe metabolism and antioxidant mechanisms were performed in “Roggusanmaru” and “Super Doterang”. Fe deficiency (Moderate, low and –Fe) significantly decreased the biomass, total, and apoplastic Fe concentration of “Roggusanmaru”, while a slight variation was observed in “Super Doterang” cultivar. The quantity of important photosynthetic pigments such as total chlorophyll and carotenoid contents significantly decreased in “Roggusanmaru” than “Super Doterang” cultivar. The total protein profile in leaves and roots determines that “Super Doterang” exhibited an optimal tolerance to Fe deficiency compared to “Roggusanmaru” cultivar. A reduction in expression of PSI (photosystem I), PSII (photosystem II) super-complexes and related thylakoid protein contents were detected in “Roggusanmaru” than “Super Doterang” cultivar. Moreover, the relative gene expression of SlPSI and SlPSII were well maintained in “Super Doterang” than “Roggusanmaru” cultivar. The relative expression of genes involved in Fe-transport (SlIRT1 and SlIRT2) and Fe(III) chelates reductase oxidase (SlFRO1) were relatively reduced in “Roggusanmaru”, while increased in “Super Doterang” cultivar under Fe deficient conditions. The H+-ATPase relative gene expression (SlAHA1) in roots were maintained in “Super Doterang” compared to “Roggusanmaru”. Furthermore, the gene expressions involved in antioxidant defense mechanisms (SlSOD, SlAPX and SlCAT) in leaves and roots showed that these genes were highly increased in “Super Doterang”, whereas decreased in “Roggusanmaru” cultivar under Fe deficiency. The present study suggested that “Super Doterang” is better tomato cultivar than “Roggusanmaru” for calcareous soils. PMID:26602920

  20. Genotypic Variation under Fe Deficiency Results in Rapid Changes in Protein Expressions and Genes Involved in Fe Metabolism and Antioxidant Mechanisms in Tomato Seedlings (Solanum lycopersicum L.).

    PubMed

    Muneer, Sowbiya; Jeong, Byoung Ryong

    2015-01-01

    To investigate Fe deficiency tolerance in tomato cultivars, quantification of proteins and genes involved in Fe metabolism and antioxidant mechanisms were performed in "Roggusanmaru" and "Super Doterang". Fe deficiency (Moderate, low and -Fe) significantly decreased the biomass, total, and apoplastic Fe concentration of "Roggusanmaru", while a slight variation was observed in "Super Doterang" cultivar. The quantity of important photosynthetic pigments such as total chlorophyll and carotenoid contents significantly decreased in "Roggusanmaru" than "Super Doterang" cultivar. The total protein profile in leaves and roots determines that "Super Doterang" exhibited an optimal tolerance to Fe deficiency compared to "Roggusanmaru" cultivar. A reduction in expression of PSI (photosystem I), PSII (photosystem II) super-complexes and related thylakoid protein contents were detected in "Roggusanmaru" than "Super Doterang" cultivar. Moreover, the relative gene expression of SlPSI and SlPSII were well maintained in "Super Doterang" than "Roggusanmaru" cultivar. The relative expression of genes involved in Fe-transport (SlIRT1 and SlIRT2) and Fe(III) chelates reductase oxidase (SlFRO1) were relatively reduced in "Roggusanmaru", while increased in "Super Doterang" cultivar under Fe deficient conditions. The H⁺-ATPase relative gene expression (SlAHA1) in roots were maintained in "Super Doterang" compared to "Roggusanmaru". Furthermore, the gene expressions involved in antioxidant defense mechanisms (SlSOD, SlAPX and SlCAT) in leaves and roots showed that these genes were highly increased in "Super Doterang", whereas decreased in "Roggusanmaru" cultivar under Fe deficiency. The present study suggested that "Super Doterang" is better tomato cultivar than "Roggusanmaru" for calcareous soils. PMID:26602920

  1. Anti-Müllerian hormone concentration in sheep and its dependence of age and independence of BMP15 genotype: an endocrine predictor to select the best donors for embryo biotechnologies.

    PubMed

    Lahoz, B; Alabart, J L; Cocero, M J; Monniaux, D; Echegoyen, E; Sánchez, P; Folch, J

    2014-01-15

    Embryo biotechnologies contribute significantly to the genetic enhancement of livestock, although their efficiency remains limited in sheep, mainly owing to variable ovarian responses to gonadotropins. At present, anti-Müllerian hormone (AMH), which is produced by the granulosa cells of the small antral follicles, is a reliable endocrine marker of the ovarian follicle reserve in many species. The expression of AMH in granulosa cells was shown to be stimulated by bone morphogenetic proteins (BMPs) in vitro, so a mutation affecting the BMP15 gene might modulate AMH production in vivo. The present study aimed to assess plasma AMH concentrations before puberty in two groups of Rasa Aragonesa ewes that were carrying (R+) or not carrying (++) the prolific FecX(R) allele and to relate them with their AMH concentrations at adulthood. Additionally, we sought to establish in both genotypes whether AMH measurements during a laparoscopic ovum pick-up (LOPU) program could be predictive of the number of ovarian follicles (≥3 mm) and recovered cumulus-oocyte complexes (COCs). No differences in AMH were found between the R+ and ++ ewes before puberty or during the adult age. Before puberty, the AMH concentration tended to increase from 3 to 4.5 months and to decline at 6 months to levels similar to those observed later in adults (333.8 ± 73.3, 483.2 ± 135.5, and 184.1 ± 38.2 pg/mL, respectively; P < 0.1), showing a large variability between individuals and between ages. A relationship between the AMH concentrations before puberty and during adulthood was not found, likely reflecting different follicular growth dynamics. In adults, the AMH concentration at the beginning of the FSH treatment was strongly correlated with the number of punctured follicles at LOPU in R+ and ++ ewes (r = 0.75 and 0.78, respectively; P < 0.001), and it was possible to accurate determine AMH cutoff values for both genotypes to identify high-responding ewes. On average, 5.1 extra follicles and 2

  2. Expression of BDNF and TrkB Phosphorylation in the Rat Frontal Cortex During Morphine Withdrawal are NO Dependent.

    PubMed

    Peregud, Danil I; Yakovlev, Alexander A; Stepanichev, Mikhail Yu; Onufriev, Mikhail V; Panchenko, Leonid F; Gulyaeva, Natalia V

    2016-08-01

    Nitric oxide (NO) mediates pharmacological effects of opiates including dependence and abstinence. Modulation of NO synthesis during the induction phase of morphine dependence affects manifestations of morphine withdrawal syndrome, though little is known about mechanisms underlying this phenomenon. Neurotrophic and growth factors are involved in neuronal adaptation during opiate dependence. NO-dependent modulation of morphine dependence may be mediated by changes in expression and activity of neurotrophic and/or growth factors in the brain. Here, we studied the effects of NO synthesis inhibition during the induction phase of morphine dependence on the expression of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF1) as well as their receptors in rat brain regions after spontaneous morphine withdrawal in dependent animals. Morphine dependence in rats was induced within 6 days by 12 injections of morphine in increasing doses (10-100 mg/kg), and NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) (10 mg/kg) was given 1 h before each morphine injection. The expression of the BDNF, GDNF, NGF, IGF1, and their receptors in the frontal cortex, striatum, hippocampus, and midbrain was assessed 40 h after morphine withdrawal. L-NAME treatment during morphine intoxication resulted in an aggravation of the spontaneous morphine withdrawal severity. Morphine withdrawal was accompanied by upregulation of BDNF, IGF1, and their receptors TrkB and IGF1R, respectively, on the mRNA level in the frontal cortex, and only BDNF in hippocampus and midbrain. L-NAME administration during morphine intoxication decreased abstinence-induced upregulation of these mRNAs in the frontal cortex, hippocampus and midbrain. L-NAME prevented from abstinence-induced elevation of mature but not pro-form of BDNF polypeptide in the frontal cortex. While morphine abstinence did not affect Trk

  3. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

  4. Serum amyloid A induces interleukin-33 expression through an IRF7-dependent pathway

    PubMed Central

    Sun, Lei; Zhu, Ziyan; Cheng, Ni; Yan, Qian; Ye, Richard D.

    2014-01-01

    Interleukin-33 (IL-33), an IL-1 family cytokine and nuclear alarmin, is constitutively expressed in epithelial barrier tissues and human blood vessels. However, little is known about the induced expression of IL-33 in monocytes and macrophages, which are major cytokine-producing cells of the innate immune system. Here we report the induction of IL-33 expression in both human monocytes and mouse macrophages from C57BL/6 mice by the acute-phase protein serum amyloid A (SAA). SAA induced transcriptional activation of the IL-33 gene, resulting in nuclear accumulation of the IL-33 protein. TLR2, one of the SAA receptors, was primarily responsible for the induction of IL-33. Progressive deletion of the human IL-33 promoter led to the identification of two potential binding sites for interferon regulatory factor 7 (IRF7), one of which (−277/−257) was found to be important for SAA-stimulated IL-33 promoter activity. IRF7 was recruited to the IL-33 promoter upon SAA stimulation, and silencing IRF7 expression in THP-1 cells abrogated SAA-induced IL-33 expression. SAA also promoted an interaction between TRAF6 and IRF7. Taken together, these results identify IRF7 as a critical transcription factor for SAA-induced IL-33 expression in monocytes and macrophages. PMID:24777946

  5. Impact of experience-dependent and -independent factors on gene expression in songbird brain.

    PubMed

    Drnevich, Jenny; Replogle, Kirstin L; Lovell, Peter; Hahn, Thomas P; Johnson, Frank; Mast, Thomas G; Nordeen, Ernest; Nordeen, Kathy; Strand, Christy; London, Sarah E; Mukai, Motoko; Wingfield, John C; Arnold, Arthur P; Ball, Gregory F; Brenowitz, Eliot A; Wade, Juli; Mello, Claudio V; Clayton, David F

    2012-10-16

    Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience. PMID:23045667

  6. Gene Expression Profile of Calcium/Calmodulin-Dependent Protein Kinase IIα in Rat's Hippocampus during Morphine Withdrawal

    PubMed Central

    Ahmadi, Shamseddin; Amiri, Shahin; Rafieenia, Fatemeh; Rostamzadeh, Jalal

    2013-01-01

    Introduction Calcium/calmodulin-dependent protein kinase II (CaMKII) which is highly expressed in the hippocampus is known to play a pivotal role in reward-related memories and morphine dependence. Methods In the present study, repeated morphine injections once daily for 7 days was done to induce morphine tolerance in male Wistar rats, after which gene expression profile of α-isoform of CaMKII (CaMKIIα) in the hippocampus was evaluated upon discontinuation of morphine injection over 21 days of morphine withdrawal. Control groups received saline for 7 consecutive days. For gene expression study, rats’ brains were removed and the hippocampus was dissected in separate groups on days 1, 3, 7, 14, and 21 since discontinuation of of morphine injection. A semi-quantitative RT-PCR method was used to evaluate the gene expression profile. Results Tolerance to morphine was verified by a significant decrease in morphine analgesia in a hotplate test on day 8 (one day after the final repeated morphine injections). Results showed that gene expression of CaMKIIα at mRNA level on day 1, 3, 7, 14 and 21 of morphine withdrawal was significantly altered as compared to the saline control group. Post hoc Tukey's test revealed a significantly enhanced CaMKIIα gene expression on day 14. Discussion It can be concluded that CaMKIIα gene expression during repeated injections of morphine is increased and this increase continues up to 14 days of withdrawal then settles at a new set point. Therefore, the strong morphine reward-related memory in morphine abstinent animals may, at least partly be attributed to, the up-regulation of CaMKIIα in the hippocampus over 14 days of morphine withdrawal. PMID:25337341

  7. Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    SciTech Connect

    Saraswat, L.D.; Filutowicz, M.

    1986-05-01

    The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N/sub 3/-(/sup 32/P)-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH/sub 2/-terminal sequence was confirmed by amino acid sequencing.

  8. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    PubMed Central

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

    2011-01-01

    Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

  9. Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

    PubMed Central

    Giehl, Klaudia; Keller, Christof; Muehlich, Susanne; Goppelt-Struebe, Margarete

    2015-01-01

    Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF

  10. Autoregressive Higher-Order Hidden Markov Models: Exploiting Local Chromosomal Dependencies in the Analysis of Tumor Expression Profiles

    PubMed Central

    Seifert, Michael; Abou-El-Ardat, Khalil; Friedrich, Betty; Klink, Barbara; Deutsch, Andreas

    2014-01-01

    Changes in gene expression programs play a central role in cancer. Chromosomal aberrations such as deletions, duplications and translocations of DNA segments can lead to highly significant positive correlations of gene expression levels of neighboring genes. This should be utilized to improve the analysis of tumor expression profiles. Here, we develop a novel model class of autoregressive higher-order Hidden Markov Models (HMMs) that carefully exploit local data-dependent chromosomal dependencies to improve the identification of differentially expressed genes in tumor. Autoregressive higher-order HMMs overcome generally existing limitations of standard first-order HMMs in the modeling of dependencies between genes in close chromosomal proximity by the simultaneous usage of higher-order state-transitions and autoregressive emissions as novel model features. We apply autoregressive higher-order HMMs to the analysis of breast cancer and glioma gene expression data and perform in-depth model evaluation studies. We find that autoregressive higher-order HMMs clearly improve the identification of overexpressed genes with underlying gene copy number duplications in breast cancer in comparison to mixture models, standard first- and higher-order HMMs, and other related methods. The performance benefit is attributed to the simultaneous usage of higher-order state-transitions in combination with autoregressive emissions. This benefit could not be reached by using each of these two features independently. We also find that autoregressive higher-order HMMs are better able to identify differentially expressed genes in tumors independent of the underlying gene copy number status in comparison to the majority of related methods. This is further supported by the identification of well-known and of previously unreported hotspots of differential expression in glioblastomas demonstrating the efficacy of autoregressive higher-order HMMs for the analysis of individual tumor expression