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Selected AbstractsImpact of carbohydrate supplementation during endurance training on glycogen storage and performanceACTA PHYSIOLOGICA, Issue 2 2009L. Nybo Abstract Aim:, Glucose ingestion may improve exercise endurance, but it apparently also influences the transcription rate of several metabolic genes and it alters muscle metabolism during an acute exercise bout. Therefore, we investigated how chronic training responses are affected by glucose ingestion. Methods:, In previously untrained males performance and various muscular adaptations were evaluated before and after 8 weeks of supervised endurance training conducted either with (n = 8; CHO group) or without (n = 7; placebo) glucose supplementation. Results:, The two groups achieved similar improvements in maximal oxygen uptake and peak power output during incremental cycling (both parameters elevated by 17% on average) and both groups lost ,3 kg of fat mass during the 8 weeks of training. An equal reduction in respiratory exchange ratio (0.02 units) during submaximal exercise was observed in both groups. Beta-hydroxyacyl-CoA-dehydrogenase activity was increased in both groups, however, to a larger extent in the placebo group (45 ± 11%) than CHO (23 ± 9%, P < 0.05). GLUT-4 protein expression increased by 74 ± 14% in the placebo group and 45 ± 14% in CHO (both P < 0.05), while resting muscle glycogen increased (P < 0.05) to a larger extent in the placebo group (96 ± 4%) than CHO (33 ± 2%). Conclusion:, These results show that carbohydrate supplementation consumed during exercise training influences various muscular training adaptations, but improvements in cardiorespiratory fitness and reductions in fat mass are not affected. [source] Overexpression of c-Fos is sufficient to stimulate tyrosine hydroxylase (TH) gene transcription in rat pheochromocytoma PC18 cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Baoyong Sun Abstract The AP1 site within the tyrosine hydroxylase gene proximal promoter is essential for the response of the gene to numerous stimuli. Stimulation of this gene is often associated with induction of the AP1 transcription factor, c-Fos. However, many stimuli activate or induce multiple transcription factors that interact with this AP1 site or other sites within the gene's proximal promoter. Hence, it remains unclear whether c-Fos induction by itself is sufficient to stimulate the tyrosine hydroxylase gene. In this study we produce rat pheochromocytoma PC18 cells that overexpress c-Fos under control of the tet-inducible system. We demonstrate that induction of c-Fos leads to dramatic stimulation of tyrosine hydroxylase gene transcription rate measured using nuclear run-on assays. This stimulation is closely associated quantitatively with the induction of c-Fos and does not apparently require phosphorylation of c-Fos. The response is partially dependent on the AP1 site within the tyrosine hydroxylase proximal promoter. However, the response of the proximal promoter to c-Fos induction is relatively small compared with that of the endogenous gene. Consequently, our results suggest that c-Fos exerts its influence on the tyrosine hydroxylase gene via multiple mechanisms that are dependent and independent of the proximal promoter AP1 site. [source] The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] A novel sensor kinase,response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosaMOLECULAR MICROBIOLOGY, Issue 4 2004Michelle A. Laskowski Summary The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media. We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P. aeruginosa. RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains. Deletion of rtsM resulted in a defect in production and secretion of the type III effectors. Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the ,rtsM strain under TTSS-inducing conditions. Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact. The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in ,rtsM than in the wild-type parent, PA103. The defect in TTSS effector production and secretion of ,rtsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation. ,rtsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo. We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals. [source] Temperature effects on sex determination and ontogenetic gene expression of the aromatases cyp19a and cyp19b, and the estrogen receptors esr1 and esr2 in atlantic halibut (Hippoglossus hippoglossus)MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2006Solveig van Nes Abstract The aromatase (CYP19) and estrogen receptor (ESR) play important roles in the molecular mechanism of sex determination and differentiation of lower vertebrates. Several studies have proven these mechanisms to be temperature sensitive, which can influence the direction of phenotypic gender development. A temperature study was conducted to examine the effect of temperature on the sex differentiation in farmed Atlantic halibut. Sexually undifferentiated larvae were exposed to 7°C, 10°C, or 13°C during gonadal differentiation. Temperature effects on the transcription rate of the aromatase genes cyp19a (ovary type) and cyp19b (brain type) and the ESR genes esr1 and esr2 were examined by quantitative real-time PCR. With increasing temperatures, both cyp19a mRNA levels and the female incidence showed a decreasing trend, thus strongly indicating a relation between the expression of cyp19a and morphological ovary differentiation. In contrast to cyp19a, the levels of cyp19b, esr1, and esr2 mRNA strongly increased in all temperature groups throughout the study period, and did not show obvious temperature-related expression patterns. The present data provide evidence that posthatching temperature exposure significantly affects the expression of cyp19a mRNA during the developmental period and that high temperature possibly influences genetic sex determination in Atlantic halibut. Though, the female incidence never exceeded 50%, suggesting that only the homogametic (XX) female is thermolabile. So whereas temperature treatment is not likely suitable for direct feminization in halibut, the possibility for high-temperature production of XX neomales for broodstock to obtain all-female offspring by crossing with XX females is suggested. Mol. Reprod. Dev. 73: 1481,1490, 2006. © 2006 Wiley-Liss, Inc. [source] Transgene-induced silencing of Arabidopsis phytochrome A gene via exonic methylationTHE PLANT JOURNAL, Issue 6 2007Rekha Chawla Summary Transgene-induced promoter or enhancer methylation clearly retards gene activity. While exonic methylation of genes is frequently observed in the RNAi process, only sporadic evidence has demonstrated its definitive role in gene suppression. Here, we report the isolation of a transcriptionally suppressed epi-allele of the Arabidopsis thaliana phytochrome A gene (PHYA) termed phyA, that shows methylation only in symmetric CG sites resident in exonic regions. These exonic modifications confer a strong phyA mutant phenotype, characterized by elongated hypocotyls in seedlings grown under continuous far-red light. De-methylation of phyA, in the DNA methyl transferase I (met1) mutant background increased PHYA expression and restored the wild-type phenotype, confirming the pivotal role of exonic CG methylation in maintaining the altered epigenetic state. PHYA epimutation was apparently induced by a transgene locus; however, it is stably maintained following segregation. Chromatin immunoprecipitation assays revealed association with dimethyl histone H3 lysine 9 (H3K9me2), a heterochromatic marker, within the phyA, coding region. Therefore, transgene-induced exonic methylation can lead to chromatin alteration that affects gene expression, most likely through reduction in the transcription rate. [source] Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug aloneBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2001N. Di Gaetano We have shown previously that the anti-CD20 chimaeric monoclonal antibody rituximab exerts its effects on neoplastic B-lymphoma cell lines in part via complement-dependent cytotoxicity. In addition, membrane expression levels of complement inhibitory proteins CD55 and CD59 play a role in determining susceptibility to lysis. We have identified one t(14;18)-positive human B-cell non Hodgkin's lymphoma cell line (Karpas 422) that is resistant to rituximab and complement and used it for subsequent studies on the possible interaction between this novel therapeutic agent and established antineoplastic drugs. We have exposed Karpas to several chemotherapeutic agents (doxorubicin, idarubicin, cisplatin, taxol) for different time periods and subsequently exposed the cells to rituximab and human complement. The combination of these drugs with rituximab induced an additive cytotoxic effect. In contrast, exposure to fludarabine (1 µg/ml for 48,72 h) showed a synergistic effect, with cell lysis increasing from 10% to 20% using fludarabine or rituximab and complement alone to about 70% with both cytotoxic agents. Analysis of the mechanism for this synergistic effect showed that fludarabine downmodulates the membrane expression of CD55 (from 96% to 55% positive cells) without significantly altering CD20 levels. Northern analysis demonstrated that fludarabine induced a general downmodulation of steady state mRNA levels with no change in transcription rate detected in run-off assays. The study of the effect of fludarabine and rituximab in six freshly isolated B-cell chronic lymphocytic leukaemia (B-CLL) samples showed that, in most cases, fludarabine has an additive cytotoxic activity with rituximab and complement. This report gives a rational support for clinical studies with combinations of drugs, including monoclonal antibodies and fludarabine. [source] Prolonged expression of CD154 on CD4 T cells from pediatric lupus patients correlates with increased CD154 transcription, increased nuclear factor of activated T cell activity, and glomerulonephritisARTHRITIS & RHEUMATISM, Issue 8 2010Jay Mehta Objective To assess CD154 expression in patients with pediatric systemic lupus erythematosus (SLE) and to explore a transcriptional mechanism that may explain dysregulated expression of CD154. Methods Cell surface CD154 expression (pre- and postactivation) in peripheral blood CD4 T cells from 29 children with lupus and 29 controls matched for age, sex, and ethnicity was examined by flow cytometry. CD154 expression was correlated with clinical features, laboratory parameters, and treatments received. Increased CD154 expression on CD4 T cells from the SLE patients was correlated with CD154 message and transcription rates by real-time reverse transcription,polymerase chain reaction (RT-PCR) and nuclear run-on assays, respectively. Nuclear factor of activated T cell (NF-AT) transcription activity and mRNA levels in CD4 T cells from SLE patients were explored by reporter gene analysis and real-time RT-PCR, respectively. Results CD154 surface protein levels were increased 1.44-fold in CD4 T cells from SLE patients as compared with controls in cells evaluated 1 day postactivation ex vivo. This increase correlated clinically with the presence of nephritis and an elevated erythrocyte sedimentation rate. Increased CD154 protein levels also correlated with increased CD154 mRNA levels and with CD154 transcription rates, particularly at later time points following T cell activation. Reporter gene analyses revealed a trend for increased NF-AT, but decreased activator protein 1 and similar NF-,B, activity in CD4 T cells from SLE patients as compared with controls. Moreover, NF-AT1 and, in particular, NF-AT2 mRNA levels were notably increased in CD4 T cells from SLE patients as compared with controls. Conclusion Following activation, cell surface CD154 is increased on CD4 T cells from pediatric lupus patients as compared with controls, and this increase correlates with the presence of nephritis, increased CD154 transcription rates, and increased NF-AT activity. These results suggest that NF-AT/calcineurin inhibitors, such as tacrolimus and cyclosporine, may be beneficial in the treatment of lupus nephritis. [source] The nematode,arthropod clade revisited: phylogenomic analyses from ribosomal protein genes misled by shared evolutionary biasesCLADISTICS, Issue 2 2007Stuart J. Longhorn Phylogenetic analysis of major groups of Metazoa using genomic data tends to recover the sister relationships of arthropods and chordates, contradicting the proposed Ecdysozoa (the molting animals), which group the arthropods together with nematodes and relatives. Ribosomal protein genes have been a major data source in phylogenomic studies because they are readily detected as Expressed Sequence Tags (ESTs) due to their high transcription rates. Here we address the debate about the recovery of Ecdysozoa in genomic data by building a new matrix of carefully curated EST and genome sequences for 25 ribosomal protein genes of the small subunit, with focus on new insect sequences in addition to the Diptera sequences generally used to represent the arthropods. Individually, each ribosomal protein gene showed low phylogenetic signal, but in simultaneous analysis strong support emerged for many expected groups, with support increasing linearly with increased gene number. In agreement with most studies of metazoan relationships from genomic data, our analyses contradicted the Ecdysozoa (the putative sister relationship of arthropods and nematodes), and instead supported the affinity of arthropods with chordates. In addition, relationships among holometabolan insects resulted in an unlikely basal position for Diptera. To test for biases in the data that might produce an erroneous arthropod,chordate affinity we simulated sequence data on tree topologies with the alternative arthropod,nematode sister relationships, applying a model of amino acid sequence evolution estimated from the real data. Tree searches on these simulated data still revealed an arthropod,chordate grouping, i.e., the topologies used to simulate the data were not recovered correctly. This suggests that the arthropod,chordate relationships may be obtained erroneously also from the real data even if the alternative topology (Ecdysozoa) represents the true phylogeny. Whereas denser taxon sampling in the future may recover the Ecdysozoa, our analyses demonstrate that recent phylogenomic studies may be affected by as yet unspecified biases in amino acid sequence composition in the model organisms with available genomic data. © The Willi Hennig Society 2007. [source] | |||||||||||||