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Transcriptional Changes (transcriptional + change)
Selected AbstractsThe effect of a promoter polymorphism on the transcription of nitric oxide synthase 1 and its relevance to Parkinson's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2009Terrie Rife Abstract Transcriptional changes of the enzyme nitric oxide synthase I (NOS1) are believed to play a role in the development of many diseases. The gene for NOS1 has 12 alternative first exons (1A,1L). The 1F exon is one of the most highly utilized first exons in the brain and has a polymorphism ((TG)mTA(TG)n) located in its promoter region. The polymorphism's length has been suggested to affect NOS1 transcription and play a role in Parkinson's disease (PD); however, the actual influence of the polymorphism on NOS1 transcription has not been studied. To better characterize the links of the polymorphism with PD, a genotyping study was done comparing polymorphism length among 170 PD patients and 150 age-matched controls. The pattern of changes between the two group's allele frequencies shows statistical significance (P = 0.0359). The smallest polymorphism sizes are more predominant among PD patients than controls. To study the effects of this polymorphism on NOS1 gene transcription, reporter gene constructs were made by cloning the NOS1 1F promoter with polymorphism lengths of either 42, 54, or 62 bp in front of the luciferase gene and transfecting them into HeLa or Sk-N-MC cells. NOS1-directed reporter gene constructs with the 62-bp polymorphism increased transcription of luciferase 2.2-fold in HeLa and 1.8-fold in Sk-N-MC cells compared with reporter gene constructs with the 42-bp polymorphism. These data suggest that if smaller polymorphism size contributes to the higher NOS1 levels in PD patients, an as yet unknown transcriptional mechanism is required. © 2009 Wiley-Liss, Inc. [source] Transcriptional changes in insulin- and lipid metabolism-related genes in the hippocampus of olfactory bulbectomized miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2008Peter Gass Abstract Affymetrix chips were used to perform a hypothesis-free large-scale screening of transcripts in the hippocampus of olfactory bulbectomized mice, an established animal model of depression. Because only 11 transcripts were significantly changed, the statistically subsequent 25 transcripts below the significance level were additionally included in a first round of qRT-PCR evaluations. Furthermore, all 36 genes were then tested for mutual interactions or interactions with other molecules in a physiological context using PathwayArchitect software. Thirty of them were displayed in a network interacting with at least one partner molecule from the list or with other partner molecules known from the literature. All partner molecules from the most prominent 10 molecules of this network were then identified and put together into a new list. On those grounds, the hypothesis was made that metabolic network components of the insulin signaling pathway are perturbed in the disease. This pathway was subsequently tested by a second round of qRT-PCR, adding also a few additional candidate molecules belonging to this pathway. It turned out that the key target,FABP7,fell into the group of transcripts not significantly regulated within the chip data, and another key target,IRS1,did not show up in the chip experiments at all. In conclusion, our data reveal a problem with adhering to statistical significances in microarray experiments, insofar as molecules important for the disease may fall into the range of statistical noise. This approach may also be useful to find new targets for pharmacotherapy in affective disorders. © 2008 Wiley-Liss, Inc. [source] Selection of normalizer genes in conducting relative gene expression analysis of embryosBIRTH DEFECTS RESEARCH, Issue 8 2003Qin J. Zhang Abstract BACKGROUND In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T -gene, a gene known to be functionally relevant in posterior axial development. Reduction in T -gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos. Birth Defects Research (Part A), 2003. © 2003 Wiley-Liss, Inc. [source] Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regenerationDEVELOPMENTAL DYNAMICS, Issue 10 2006Matthew Grow Abstract Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues. Developmental Dynamics 235:2667,2685, 2006. © 2006 Wiley-Liss, Inc. [source] Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetesDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2009Sebastian Kreuz Abstract Background Acetyl-CoA carboxylases (ACC) 1 and 2 are central enzymes in lipid metabolism. To further investigate their relevance for the development of obesity and type 2 diabetes, expression of both ACC isoforms was analyzed in obese fa/fa Zucker fatty and Zucker diabetic fatty rats at different ages in comparison to Zucker lean controls. Methods ACC1 and ACC2 transcript levels were measured by quantitative real-time polymerase chain reaction in metabolically relevant tissues of Zucker fatty, Zucker diabetic fatty and Zucker lean control animals. Quantitative real-time polymerase chain reaction was also applied to measure ACC tissue distribution in human tissues. For confirmation on a protein level, quantitative mass spectrometry was used. Results Disease-related transcriptional changes of both ACC isoforms were observed in various tissues of Zucker fatty and Zucker diabetic fatty rats including liver, pancreas and muscle. Changes were most prominent in oxidative tissues of diabetic rats, where ACC2 was significantly increased and ACC1 was reduced compared with Zucker lean control animals. A comparison of the overall tissue distribution of both ACC isoforms in humans and rats surprisingly revealed strong differences. While in rats ACC1 was mainly expressed in lipogenic and ACC2 in oxidative tissues, ACC2 was predominant in oxidative and lipogenic tissues in humans. Conclusion Our data support a potential role for both ACC isoforms in the development of obesity and diabetes in rats. However, the finding of fundamental species differences in ACC1 and ACC2 tissue expression might be indicative for different functions of both isoforms in humans and rats and raises the question to which degree these models are predictive for the physiology and pathophysiology of lipid metabolism in humans. Copyright © 2009 John Wiley & Sons, Ltd. [source] Integrative genomic analyses of neurofibromatosis tumours identify SOX9 as a biomarker and survival geneEMBO MOLECULAR MEDICINE, Issue 4 2009Shyra J. Miller Abstract Understanding the biological pathways critical for common neurofibromatosis type 1 (NF1) peripheral nerve tumours is essential, as there is a lack of tumour biomarkers, prognostic factors and therapeutics. We used gene expression profiling to define transcriptional changes between primary normal Schwann cells (n,=,10), NF1-derived primary benign neurofibroma Schwann cells (NFSCs) (n,=,22), malignant peripheral nerve sheath tumour (MPNST) cell lines (n,=,13), benign neurofibromas (NF) (n,=,26) and MPNST (n,=,6). Dermal and plexiform NFs were indistinguishable. A prominent theme in the analysis was aberrant differentiation. NFs repressed gene programs normally active in Schwann cell precursors and immature Schwann cells. MPNST signatures strongly differed; genes up-regulated in sarcomas were significantly enriched for genes activated in neural crest cells. We validated the differential expression of 82 genes including the neural crest transcription factor SOX9 and SOX9 predicted targets. SOX9 immunoreactivity was robust in NF and MPSNT tissue sections and targeting SOX9 , strongly expressed in NF1-related tumours , caused MPNST cell death. SOX9 is a biomarker of NF and MPNST, and possibly a therapeutic target in NF1. [source] Altered gene expression in the brain and ovaries of zebrafish (Danio Rerio) exposed to the aromatase inhibitor fadrozole: Microarray analysis and hypothesis generation,,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2009Daniel L. Villeneuve Abstract As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 ,g/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 ,g/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors. [source] Specific transcriptional responses induced by 8-methoxypsoralen and UVA in yeastFEMS YEAST RESEARCH, Issue 6 2007Michèle Dardalhon Abstract Treatment of eukaryotic cells with 8-methoxypsoralen plus UVA irradiation (8-MOP/UVA) induces pyrimidine monoadducts and interstrand crosslinks and initiates a cascade of events leading to cytotoxic, mutagenic and carcinogenic responses. Transcriptional activation plays an important part in these responses. Our previous study in Saccharomyces cerevisiae showed that the repair of these lesions involves the transient formation of DNA double-strand breaks and the enhanced expression of landmark DNA damage response genes such as RAD51, RNR2 and DUN1, as well as the Mec1/Rad53 kinase signaling cascade. We have now used DNA microarrays to examine genome-wide transcriptional changes produced after induction of 8-MOP/UVA photolesions. We found that 128 genes were strongly induced and 29 genes strongly repressed. Modifications in gene expression concern numerous biological processes. Compared to other genotoxic treatments, c. 42% of the response genes were specific to 8-MOP/UVA treatment. In addition to common DNA damage response genes and genes induced by environmental stresses, a large fraction of 8-MOP/UVA response genes correspond to membrane-related functions. [source] Correlated expression patterns of microRNA genes with age-dependent behavioural changes in honeybeeINSECT MOLECULAR BIOLOGY, Issue 4 2010S. K. Behura Abstract The hive-living honeybees (Apis mellifera) show age-dependent behavioural changes; young bees usually nurse the broods in the colony and the older bees engage in foraging activities. These developmentally regulated behavioural changes were previously shown to be correlated with genome-wide transcriptional changes in the honeybee brain. The indigenous small regulatory RNA molecules, known as microRNAs (miRNAs), are potent regulators of gene expression and also are developmentally regulated. Thus, we wanted to study if there might be correlation of differential expression of miRNA genes in the brain with age-dependent behavioural changes of the bees. We determined expression patterns of a set (n= 20) of predicted miRNA genes, by quantitative real-time PCR assays, in the brains of young and old bees that were engaged in nursing or foraging activities in the colony, respectively. Our data show correlated up-regulation of miRNA-124, miRNA-14, miRNA-276, miRNA-13b, let-7 and miRNA-13a in the young nurse bees. miRNA-12, miRNA-9, miRNA-219, miRNA-210, miRNA-263, miRNA-92 and miRNA-283 showed correlated expression patterns in the old forager bees. The modular changes of miRNA genes in the young nurse and old forager bees suggest possible roles of miRNAs in age-dependent behavioural changes in bees. The correlated expression of intronic miRNA genes and their host genes as well as of miRNA genes physically clustered in the genome are also observed. [source] The effects of mating and instrumental insemination on queen honey bee flight behaviour and gene expressionINSECT MOLECULAR BIOLOGY, Issue 2 2010S. D. Kocher Abstract Mating is fundamental to most organisms, although the physiological and transcriptional changes associated with this process have been largely characterized only in Drosophila melanogaster. In this study, we use honey bees as a model system because their queens undergo massive and permanent physiological and behavioural changes following mating. Previous studies have identified changes associated with the transition from a virgin queen to a fully mated, egg-laying queen. Here, we further uncouple the mating process to examine the effects of natural mating vs. instrumental insemination and saline vs. semen insemination. We observed effects on flight behaviour, vitellogenin expression and significant overlap in transcriptional profiles between our study and analogous studies in D. melanogaster, suggesting that some post-mating mechanisms are conserved across insect orders. [source] Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1AGING CELL, Issue 3 2009Sang-Kyu Park Summary Oxidative stress has been hypothesized to play a role in normal aging. The response to oxidative stress is regulated by the SKN-1 transcription factor, which also is necessary for intestinal development in Caenorhabditis elegans. Almost a thousand genes including the antioxidant and heat-shock responses, as well as genes responsible for xenobiotic detoxification were induced by the oxidative stress which was found using transcriptome analysis. There were also 392 down-regulated genes including many involved in metabolic homeostasis, organismal development, and reproduction. Many of these oxidative stress-induced transcriptional changes are dependent on SKN-1 action; the induction of the heat-shock response is not. When RNAi to inhibit genes was used, most had no effect on either resistance to oxidative stress or longevity; however two SKN-1-dependent genes, nlp-7 and cup-4, that were up-regulated by oxidative stress were found to be required for resistance to oxidative stress and for normal lifespan. nlp-7 encodes a neuropeptide-like protein, expressed in neurons, while cup-4 encodes a coelomocyte-specific, ligand-gated ion channel. RNAi of nlp-7 or cup-4 increased sensitivity to oxidative stress and reduced lifespan. Among down-regulated genes, only inhibition of ent-1, a nucleoside transporter, led to increased resistance to oxidative stress; inhibition had no effect on lifespan. In contrast, RNAi of nhx-2, a Na+/H+ exchanger, extended lifespan significantly without affecting sensitivity to oxidative stress. These findings showed that a transcriptional shift from growth and maintenance towards the activation of cellular defense mechanisms was caused by the oxidative stress; many of these transcriptional alterations are SKN-1 dependent. [source] Acute Activation of Hippocampal Glucocorticoid Receptors Results in Different Waves of Gene Expression Throughout TimeJOURNAL OF NEUROENDOCRINOLOGY, Issue 4 2006M. C. Morsink Abstract Several aspects of hippocampal cell function are influenced by adrenal-secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)-induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR-mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR-activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down-regulated genes 1 h after GR-activation to both up and down regulated genes 3 h after GR-activation. After 5 h, the response was almost back to baseline. Additionally, real-time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR-responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid-mediated effects on hippocampal cell function. [source] LIGHT REGULATION OF PHYCOBILISOME BIOSYNTHESIS AND CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTORJOURNAL OF PHYCOLOGY, Issue 2000K. Terauchi Ambient light quality changes dramatically affect the composition of light harvesting structures, the phycobilisomes, in many cyanobacterial species. In the cyanobacterium Fremyella diplosiphon, shifts in the ratio of red to green light lead to transcriptional changes and altered synthesis of several phycobilisome components. This process is called complementary chromatic adaptation (CCA). These two colors have opposite effects: red light activates an operon encoding the biliprotein phycocyanin (PC) and inactivates the operon encoding phycoerythrin (PE), whereas green light activates PE synthesis and shuts down PC synthesis. The effects of red and green light on CCA are photoreversible. Thus, CCA is similar to transcriptional processes that are controlled by phytochromes, a family of eukaryotic red/far red photoreversible photoreceptors. We are using molecular genetics to determine the mechanisms by which F. diplosiphon senses changes in the color of light of its environment. Initial mutant generation and complementation lead to the discovery of three CCA regulatory components that are part of a complex two component system. The most interesting of these is RcaE (regulator of chromatic adaptation), a histidine kinase-class protein containing a region in its amino-terminal half with similarity to the chromophore binding domains of phytochromes. Within this region, RcaE contains a cysteine residue in a similar location as that used for covalent attachment of the open-chain tetrapyrrole chromophore in phytochromes. We will present recent data characterizing RcaE, including in vivo analysis of the chromophore that is attached to RcaE, as well as results from our recent isolation of a new CCA regulatory component. [source] Identification of distinct and common gene expression changes after oxidative stress and gamma and ultraviolet radiation,MOLECULAR CARCINOGENESIS, Issue 2 2003Alexandra N. Heinloth Abstract The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (,) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G1 checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E,associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either , radiation (5 Gy), oxidative stress (75 ,M of tert-butyl hydroperoxide (t -butyl-OOH)), or UV radiation (UVC) (7.5 J/m2) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to , radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E,associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after , radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, , radiation had its major influence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage. Published 2003 Wiley-Liss, Inc. [source] General and specific host responses to bacterial infection in Peyer's patches: a role for stromelysin-1 (matrix metalloproteinase-3) during Salmonella enterica infectionMOLECULAR MICROBIOLOGY, Issue 1 2007Scott A. Handley Summary Salmonella enterica serovar Typhimurium (S. typhimurium) and Yersinia enterocolitica are enteric pathogens capable of colonizing and inducing inflammatory responses in Peyer's patches (PPs) and mesenteric lymph nodes (MLNs). Although the tissue colonization pattern is similar between these two pathogens, their pathogenic lifestyles are quite different. For example, while S. typhimurium is primarily an intracellular pathogen, Y. enterocolitica survives primarily extracellularly. We determined and compared the transcriptional changes occurring in response to S. typhimurium and Y. enterocolitica colonization of PP using Affymetrix GeneChip technology. Both pathogens elicited a general inflammatory response indicated by the upregulation of cytokines and chemokines. However, specific differences were also observed, most notably in the transcriptional regulation of gamma interferon (IFN-,) and IFN-,-regulated genes in response to S. typhimurium but not Y. enterocolitica. Of particular note, a group of genes encoding matrix metalloproteinases (MMPs) had increased transcript numbers in the PPs following infection with both pathogens. The experiments described here compare oral S. typhimurium or Y. enterocolitica infection in stromelysin-1 (MMP-3)-deficient mice (mmp-3,/,) with mice possessing functional MMP-3 (mmp-3+/+). There was little difference in the survival of MMP-3-deficient mice infected with Y. enterocolitica when compared with littermate controls. Surprisingly though, mmp-3,/, mice were markedly more resistant to S. typhimurium infection than the control mice. S. typhimurium was able to colonize mmp-3,/, mice, albeit in a delayed fashion, to equivalent levels as mmp-3+/+ mice. Nevertheless, significantly lower levels of inflammatory cytokines were detected in tissues and serum in the mmp-3,/, mice in comparison with mmp-3+/+ mice. We hypothesize that MMP-3 is involved in initiating an early and lethal cytokine response to S. typhimurium colonization. [source] Host immunity modulates transcriptional changes in a multigene family (yir) of rodent malariaMOLECULAR MICROBIOLOGY, Issue 3 2005Deirdre A. Cunningham Summary Variant antigens, encoded by multigene families, and expressed at the surface of erythrocytes infected with the human malaria parasite Plasmodium falciparum and the simian parasite Plasmodium knowlesi, are important in evasion of host immunity. The vir multigene family, encoding a very large number of variant antigens, has been identified in the human parasite Plasmodium vivax and homologues (yir) of this family exist in the rodent parasite Plasmodium yoelii. These genes are part of a superfamily (pir) which are found in Plasmodium species infecting rodents, monkeys and humans (P. yoelii, P. berghei, P. chabaudi, P. knowlesi and P. vivax). Here, we show that YIR proteins are expressed on the surface of erythrocytes infected with late-stage asexual parasites, and that host immunity modulates transcription of yir genes. The surface location and expression pattern of YIR is consistent with a role in antigenic variation. This provides a unique opportunity to study the regulation and expression of the pir superfamily, and its role in both protective immunity and antigenic variation, in an easily accessible animal model system. [source] The plant pathogenic fungus Gaeumannomyces graminis var. tritici improves bacterial growth and triggers early gene regulations in the biocontrol strain Pseudomonas fluorescens Pf29ArpNEW PHYTOLOGIST, Issue 2 2009M. Barret Summary ,,In soil, some antagonistic rhizobacteria contribute to reduce root diseases caused by phytopathogenic fungi. Direct modes of action of these bacteria have been largely explored; however, commensal interaction also takes place between these microorganisms and little is known about the influence of filamentous fungi on bacteria. ,,An in vitro confrontation bioassay between the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) and the biocontrol bacterial strain Pseudomonas fluorescens Pf29Arp was set up to analyse bacterial transcriptional changes induced by the fungal mycelium at three time-points of the interaction before cell contact and up until contact. For this, a Pf29Arp shotgun DNA microarray was constructed. Specifity of Ggt effect was assessed in comparison with one of two other filamentous fungi, Laccaria bicolor and Magnaporthe grisea. ,,During a commensal interaction, Ggt increased the growth rate of Pf29Arp. Before contact, Ggt induced bacterial genes involved in mycelium colonization. At contact, genes encoding protein of stress response and a patatin-like protein were up-regulated. Among all the bacterial genes identified, xseB was specifically up-regulated at contact by Ggt but down-regulated by the other fungi. ,,Data showed that the bacterium sensed the presence of the fungus early, but the main gene alteration occurred during bacterial,fungal cell contact. [source] Differential gene expression in senescing leaves of two silver birch genotypes in response to elevated CO2 and tropospheric ozonePLANT CELL & ENVIRONMENT, Issue 6 2010SARI KONTUNEN-SOPPELA ABSTRACT Long-term effects of elevated CO2 and O3 concentrations on gene expression in silver birch (Betula pendula Roth) leaves were studied during the end of the growing season. Two birch genotypes, clones 4 and 80, with different ozone growth responses, were exposed to 2× ambient CO2 and/or O3 in open-top chambers (OTCs). Microarray analyses were performed after 2 years of exposure, and the transcriptional profiles were compared to key physiological characteristics during leaf senescence. There were genotypic differences in the responses to CO2 and O3. Clone 80 exhibited greater transcriptional response and capacity to alter metabolism, resulting in better stress tolerance. The gene expression patterns of birch leaves indicated contrasting responses of senescence-related genes to elevated CO2 and O3. Elevated CO2 delayed leaf senescence and reduced associated transcriptional changes, whereas elevated O3 advanced leaf senescence because of increased oxidative stress. The combined treatment demonstrated that elevated CO2 only temporarily alleviated the negative effects of O3. Gene expression data alone were insufficient to explain the O3 response in birch, and additional physiological and biochemical data were required to understand the true O3 sensitivity of these clones. [source] Novel markers of inflammation identified in tumor necrosis factor receptor,associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell lineARTHRITIS & RHEUMATISM, Issue 1 2009Susana L. Rebelo Objective To analyze the effects of tumor necrosis factor receptor,associated periodic syndrome (TRAPS),associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. Methods Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase,polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. Results Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte,macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD),dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. Conclusion TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients. [source] Real-time Polymerase Chain Reaction to Follow the Response of Muscle to TrainingARTIFICIAL ORGANS, Issue 8 2008Lauren M. Moore Abstract:, The adaptive response of muscle to changes in activity or loading can take many weeks. Changes in the levels of RNA within a muscle fiber can give an early indication of the nature of the response of that fiber to changes in activity or loading. We have designed a new primer set for quantitative polymerase chain reaction (PCR) that will allow us to follow these early transcriptional changes in rat muscle, and have shown that analysis can be performed by standard techniques on as little as 5 mg of muscle, an amount that can be obtained by needle biopsy. [source] Abiotic stress and plant responses from the whole vine to the genesAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 2010G.R. CRAMER Abstract Drought, salinity and extreme temperatures significantly limit the distribution of grapes around the world. In this review, the literature of grape responses to abiotic stress with particular reference to whole plant and molecular responses observed in recent studies is discussed. A number of short-term and long-term studies on grapevine shoots and berries have been conducted using a systems biology approach. Transcripts, proteins and metabolites were profiled. Water deficit, salinity and chilling altered the steady-state abundance of a large number of transcripts. Common responses to these stresses included changes in hormone metabolism, particularly abscisic acid (ABA), photosynthesis, growth, transcription, protein synthesis, signalling and cellular defences. Some of the transcriptional changes induced by stress were confirmed by proteomic and metabolomic analyses. More than 2000 genes were identified whose transcript abundance was altered by both water deficit and ABA. Different gene sets were used to map molecular pathways regulated by ABA, water deficit, salinity and chilling in grapevine. This work supports the hypothesis that ABA is a central regulator of abiotic stress tolerance mechanisms. ABA affects signalling pathways that trigger important molecular activities involving metabolism, transcription, protein synthesis, and cellular defence and also regulates important physiological responses such as stomatal conductance, photoprotection and growth. Systems biology approaches are providing more comprehensive understanding of the complex plant responses to abiotic stress. The molecular sets generated from mapping the ABA-inducible stress responses provide numerous targets for genetic and cultural manipulation for improved plant protection and grape quality. [source] Can molecular mechanisms of biological processes be extracted from expression profiles?BIOESSAYS, Issue 12 2001Case study: endothelial contribution to tumor-induced angiogenesis Whereas the genome contains all potential developmental programs, expression profiles permit the determination of genes that are actively transcribed under defined physiological conditions. In this article, the idea of extracting biological mechanisms from expression data is tested. Molecular processes of the endothelial contribution to angiogenesis are derived from recently published expression profiles. The analysis reveals the sensitivity limits of experimental detection of transcriptional changes and how sequence-analytic techniques can help to identify the function of genes in question. We conclude that the transcripts (http://mendel.imp.univie.ac.at/SEQUENCES/TEMS/) found to be up-regulated in angiogenesis are involved in extracellular matrix remodeling, cellular migration, adhesion, cell-cell communication rather than in angiogenesis initiation or integrative control. Comparison with tissue-specific patterns of EST occurrence shows that, indeed, the presumptive tumor-specific endothelial markers are more generally expressed by cell types involved in migration and matrix remodeling processes. This exemplary study demonstrates how bioinformatics approaches can be helpful in deriving mechanistic information from diverse sources of experimental data. BioEssays 23:1159,1175, 2001. © 2001 John Wiley & Sons, Inc. [source] Animal performance and stress: responses and tolerance limits at different levels of biological organisationBIOLOGICAL REVIEWS, Issue 2 2009Karin S. Kassahn ABSTRACT Recent advances in molecular biology and the use of DNA microarrays for gene expression profiling are providing new insights into the animal stress response, particularly the effects of stress on gene regulation. However, interpretation of the complex transcriptional changes that occur during stress still poses many challenges because the relationship between changes at the transcriptional level and other levels of biological organisation is not well understood. To confront these challenges, a conceptual model linking physiological and transcriptional responses to stress would be helpful. Here, we provide the basis for one such model by synthesising data from organismal, endocrine, cellular, molecular, and genomic studies. We show using available examples from ectothermic vertebrates that reduced oxygen levels and oxidative stress are common to many stress conditions and that the responses to different types of stress, such as environmental, handling and confinement stress, often converge at the challenge of dealing with oxygen imbalance and oxidative stress. As a result, a common set of stress responses exists that is largely independent of the type of stressor applied. These common responses include the repair of DNA and protein damage, cell cycle arrest or apoptosis, changes in cellular metabolism that reflect the transition from a state of cellular growth to one of cellular repair, the release of stress hormones, changes in mitochondrial densities and properties, changes in oxygen transport capacities and changes in cardio-respiratory function. Changes at the transcriptional level recapitulate these common responses, with many stress-responsive genes functioning in cell cycle control, regulation of transcription, protein turnover, metabolism, and cellular repair. These common transcriptional responses to stress appear coordinated by only a limited number of stress-inducible and redox-sensitive transcription factors and signal transduction pathways, such as the immediate early genes c-fos and c-jun, the transcription factors NF,B and HIF - 1,, and the JNK and p38 kinase signalling pathways. As an example of environmental stress responses, we present temperature response curves at organismal, cellular and molecular levels. Acclimation and physiological adjustments that can shift the threshold temperatures for the onset of these responses are discussed and include, for example, adjustments of the oxygen delivery system, the heat shock response, cellular repair system, and transcriptome. Ultimately, however, an organism's ability to cope with environmental change is largely determined by its ability to maintain aerobic scope and to prevent loss in performance. These systemic constraints can determine an organism's long-term survival well before cellular and molecular functions are disturbed. The conceptual model we propose here discusses some of the crosslinks between responses at different levels of biological organisation and the central role of oxygen balance and oxidative stress in eliciting these responses with the aim to help the interpretation of environmental genomic data in the context of organismal function and performance. [source] Microarray-based gene expression analysis as a process characterization tool to establish comparability of complex biological products: Scale-up of a whole-cell immunotherapy productBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Min Wang Abstract Whole-cell immunotherapies and other cellular therapies have shown promising results in clinical trials. Due to the complex nature of the whole cell product and of the sometimes limited correlation of clinical potency with the proposed mechanism of action, these cellular immunotherapy products are generally not considered well characterized. Therefore, one major challenge in the product development of whole cell therapies is the ability to demonstrate comparability of product after changes in the manufacturing process. Such changes are nearly inevitable with increase in manufacturing experience leading to improved and robust processes that may have higher commercial feasibility. In order to comprehensively assess the impact of the process changes on the final product, and thus establish comparability, a matrix of characterization assays (in addition to lot release assays) assessing the various aspects of the cellular product are required. In this study, we assessed the capability of DNA-microarray-based, gene-expression analysis as a characterization tool using GVAX cancer immunotherapy cells manufactured by Cell Genesys, Inc. The GVAX immunotherapy product consists two prostate cancer cell lines (CG1940 and CG8711) engineered to secrete human GM-CSF. To demonstrate the capability of the assay, we assessed the transcriptional changes in the product when produced in the presence or absence of fetal bovine serum, and under normal and hypoxic conditions, both changes intended to stress the cell lines. We then assessed the impact of an approximately 10-fold process scale-up on the final product at the transcriptional level. These data were used to develop comparisons and statistical analyses suitable for characterizing culture reproducibility and cellular product similarity. Use of gene-expression data for process characterization proved to be a reproducible and sensitive method for detecting differences due to small or large changes in culture conditions as might be encountered in process scale-up or unanticipated bioprocess failures. Gene expression analysis demonstrated that cell products of representative lots under the same production process and at the same production scale were statistically identical. Large process changes that resulted from the artificial stress conditions used (absence of FBS and induction of hypoxia) displayed profoundly different gene expression patterns. We propose the use of simple t -test analysis in combination with the herein introduced expression ratio with mean intensity (ERMI) analysis as useful tools for process characterization by global gene expression analysis. Biotechnol. Bioeng. 2009; 104: 796,808 © 2009 Wiley Periodicals, Inc. [source] Comparative transcriptome analysis to unveil genes affecting recombinant protein productivity in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Joon Chong Yee Abstract Low temperature culture (33°C) has been shown to enhance the specific productivity of recombinant antibodies in Chinese hamster ovary (CHO) cells but did not affect antibody productivity in hybridoma (MAK) cells. We probed the transcriptional response of both cells undergoing temperature shift using cDNA microarrays. Among the orthologous gene probes, common trends in the expression changes between CHO and MAK are not prominent. Instead, many transcriptional changes were specific to only one cell line. Notably, oxidative phosphorylation and ribosomal genes were downregulated in MAK but not in CHO. Conversely, several protein trafficking genes and cytoskeleton elements were upregulated in CHO but remained unchanged in MAK. Interestingly, at 33°C, immunoglobulin heavy and light chain showed no significant changes in CHO, but the immunoglobulin light chain was downregulated in MAK. Overall, a clear distinction in the transcriptional response to low temperature was seen in the two cell lines. To further elucidate the set of genes responsible for increased antibody productivity, the expression data of low temperature cultures was compared to that of butyrate treatment which increased specific antibody productivity in both cell lines. Genes which are commonly differentially expressed under conditions that increased productivity are likely to reflect functional classes that are important in the productivity changes. This comparative transcriptome analysis suggests that vesicle trafficking, endocytosis and cytoskeletal elements are involved in increased specific antibody productivity. Biotechnol. Bioeng. 2009;102: 246,263. © 2008 Wiley Periodicals, Inc. [source] Gene expression profiling of the ageing rat vibrissa follicleBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2005C-L. Yang Summary Background, The application of gene expression profiling to the study of chronological ageing has the potential to illuminate the molecular mechanisms underlying a complex and active process. For example, ageing of the skin and its constituent organs has myriad phenotypic consequences, and a better understanding of the means by which these changes arise has important corollaries for intervention strategies. Objectives, We used a transcriptional profiling approach to investigate changes in gene expression associated with ageing of the large vibrissa follicle of the Wistar rat. Methods, Follicle mRNA isolated from male Wistar rats at 1 and 18 months of age was hybridized to Clontech Atlas 1.2 Rat cDNA macroarrays. Confirmation of array results was provided by the use of Northern blotting and immunohistochemistry. Results, Seven transcripts displayed at least a 1·6-fold increase in expression with age, of which APOD (2·5-fold), GSTM2 (2·0-fold) and NPY (1·8-fold) showed the greatest increases. Decreased expression was found in 19 transcripts, most notably in ALOX12 (13·3-fold) and GAP43 (12·6-fold) expression. Conclusions, Follicular ageing is characterized by transcriptional changes associated with diverse aspects of keratinocyte metabolism, proliferation and development. [source] | |||||||||||||