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Oligonucleotide Microarray (oligonucleotide + microarray)
Terms modified by Oligonucleotide Microarray Selected AbstractsOligonucleotide microarrays for the detection and identification of viable beer spoilage bacteriaJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.G. Weber Abstract Aims:, The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Methods and Results:, Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. Conclusions:, This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. Significance and Impact of the Study:, The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population. [source] Identification of candidate genes involved in the radiosensitivity of esophageal cancer cells by microarray analysisDISEASES OF THE ESOPHAGUS, Issue 4 2008R. Ogawa SUMMARY., Radiotherapy plays a key role in the control of tumor growth in esophageal cancer patients. To identify the patients who will benefit most from radiation therapy, it is important to know the genes that are involved in the radiosensitivity of esophageal cancer cells. Hence, we examined the global gene expression in radiosensitive and radioresistant esophageal squamous cell carcinoma cell lines. Radiosensitivities of 13 esophageal cancer cell lines were measured. RNA was extracted from each esophageal cancer cell line and a normal esophageal epithelial cell line, and the global gene expression profiles were analyzed using a 34 594-spot oligonucleotide microarray. In the clonogenic assay, one cell line (TE-11) was identified to be highly sensitive to radiation, while the other cell lines were found to be relatively radioresistant. We identified 71 candidate genes that were differentially expressed in TE-11 by microarray analysis. The up-regulated genes included CABPR, FABP5, DSC2, GPX2, NME, CBR3, DOCK8, and ABCC5, while the down-regulated genes included RPA1, LDOC1, NDN, and SKP1A. Our investigation provided comprehensive information on genes related to radiosensitivity of esophageal cancer cells; this information can serve as a basis for further functional studies. [source] Development and experimental validation of a nifH oligonucleotide microarray to study diazotrophic communities in a glacier forefieldENVIRONMENTAL MICROBIOLOGY, Issue 8 2009Laurence Duc Summary Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom® One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection. [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] Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2007Patrick Larkin Abstract Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17,-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17,-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species. [source] The antiepileptic drug levetiracetam selectively modifies kindling-induced alterations in gene expression in the temporal lobe of ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004Jessie Gu Abstract Gene expression profiling by microarrays is a powerful tool for identification of genes that may encode key proteins involved in molecular mechanisms underlying epileptogenesis. Using the Affymetrix oligonucleotide microarray, we have surveyed the expression levels of more than 26,000 genes and expressed sequence tags (ESTs) in the amygdala-kindling model of temporal lobe epilepsy. Furthermore, the effect of the antiepileptic drug levetiracetam (LEV) on kindling-induced alterations of gene expression was studied. Treatment of rats with LEV during kindling acquisition significantly suppressed kindling development. For gene expression profiling, six groups of rats were included in the present study: (i) and (ii) sham-operated rats treated with saline or LEV; (iii) and (iv) electrode-implanted but non-kindled rats treated with saline or LEV; (v) and (vi) kindled rats treated with saline or LEV. Treatment was terminated after 11 or 12 daily amygdala stimulations, when all vehicle-treated rats had reached kindling criterion, i.e. a stage 5 seizure. Twenty-four hours later, the ipsilateral temporal lobe was dissected for mRNA preparation. Six temporal lobe preparations from each group were analysed for differential gene expression. In control (non-kindled) rats, LEV treatment was devoid of any significant effect on gene expression. In saline-treated kindled rats, a large number of genes were observed to display mRNA expression alterations compared with non-kindled rats. LEV treatment induced marked effects on gene expression from kindled rats. Previously described epilepsy-related genes, such as neuropeptide Y (NPY), thyrotropin-releasing hormone (TRH) and glial fibrillary acidic protein (GFAP) were confirmed to be up-regulated by kindling and partially normalized by LEV treatment. Real-time quantitative polymerase chain reaction confirmed NPY, TRH and GFAP expression data from chip experiments. Furthermore, a number of novel genes were identified from the gene chip experiments. A subgroup of these genes demonstrated correlation between expression changes and kindled phenotype measurements. In summary, this study identified many genes with potentially important roles in epileptogenesis and highlighted several important issues in using the gene chip technology for the study of animal models of CNS disorders. [source] Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent mannerFEBS JOURNAL, Issue 14 2003Toshiko Yamada-Okabe Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4-1BB, which is known to initiate a signal cascade activating NF-,B, was significantly up-regulated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF-,B reporter luciferase, even in the presence of the 4-1BB-Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4-1BB. Furthermore, up-regulation of 4-1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4-1BB serves as the mediator of signals from T3 to activate caspases. [source] Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCRINSECT SCIENCE, Issue 5 2008Gen-Hong Wang Abstract Gene expression quantification at mRNA level is very important for post-genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction (qRT-PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT-PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, swl4876, and swl3956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism. [source] Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteriaJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.G. Weber Abstract Aims:, The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Methods and Results:, Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. Conclusions:, This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. Significance and Impact of the Study:, The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population. [source] Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozoleJOURNAL OF FISH BIOLOGY, Issue 9 2008D. L. Villeneuve The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and seven genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology. [source] Decreased Triadin and Increased Calstabin2 Expression in Great Danes with Dilated CardiomyopathyJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2009M.A. Oyama Background: Dilated cardiomyopathy (DCM) is a common cardiac disease of Great Dane dogs, yet very little is known about the underlying molecular abnormalities that contribute to disease. Objective: Discover a set of genes that are differentially expressed in Great Dane dogs with DCM as a way to identify candidate genes for further study as well as to better understand the molecular abnormalities that underlie the disease. Animals: Three Great Dane dogs with end-stage DCM and 3 large breed control dogs. Methods: Prospective study. Transcriptional activity of 42,869 canine DNA sequences was determined with a canine-specific oligonucleotide microarray. Genome expression patterns of left ventricular tissue samples from affected Great Dane dogs were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with expression from large breed dogs with noncardiac disease. Results: Three hundred and twenty-three transcripts were differentially expressed (,2-fold change). The transcript with the greatest degree of upregulation (+61.3-fold) was calstabin2 (FKBP12.6), whereas the transcript with the greatest degree of downregulation (,9.07-fold) was triadin. Calstabin2 and triadin are both regulatory components of the cardiac ryanodine receptor (RyR2) and are critical to normal intracellular Ca2+ release and excitation-contraction coupling. Conclusion and clinical importance: Great Dane dogs with DCM demonstrate abnormal calstabin2 and triadin expression. These changes likely affect Ca2+ flux within cardiac cells and may contribute to the pathophysiology of disease. Microarray-based analysis identifies calstabin2, triadin, and RyR2 function as targets of future study. [source] Deoxyribonucleic acid-based diagnostic techniques to detect Helicobacter pyloriALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11 2004A. Ruzsovics Summary Helicobacter pylori is an important cause of many gastrointestinal disorders, ranging from chronic gastritis to gastric lymphoma and adenocarcinoma. The deoxyribonucleic acid-based assays have the potential to be a powerful diagnostic tool given its ability to specifically identify H. pylori deoxyribonucleic acid. Markers used to include general H. pylori structures and pathogenetic factors like ureaseA, cagA, vacA, iceA. Deoxyribonucleic acid or bacterial ribonucleic acid for polymerase chain reaction assays can be collected from gastric biopsy, gastric juice, stool, buccal specimens. Polymerase chain reaction can yield quantitative and genotyping results with sensitivity and specificity that approaches 100%. A clear trend in the direction of the determination of quantitative H. pylori infection by real-time polymerase chain reaction can be observed. Fluorescent in situ hybridization is suggested for routine antibiotic resistance determination. To identify the organism, deoxyribonucleic acid structure and its virulence factors may be feasible by using oligonucleotide microarray specifically recognizing and discriminating bacterial deoxyribonucleic acid and various virulence factors. Deoxyribonucleic acid-based H. pylori diagnosis yields higher sensitivity, however, specificity requires sophisticated labour environment and associated with higher costs. [source] Myocardial Gene Expression of Angiogenic Factors in Human Chronic Ischemic Myocardium: Influence of Acute Ischemia/Cardioplegia and ReperfusionMICROCIRCULATION, Issue 3 2006YONGZHONG WANG ABSTRACT Objective: Angiogenic therapies in animals have demonstrated the development of new blood vessels within ischemic myocardium. However, results from clinical protein and gene angiogenic trials have been less impressive. The present study aimed to investigate the expression of angiogenic genes in human chronic ischemic myocardium and the influence of acute ischemia/cardioplegia and reperfusion on their expression. Methods: Myocardial biopsies were taken from chronic ischemic and nonischemic myocardium in 15 patients with stable angina pectoris during coronary bypass surgery. Tissue samples were evaluated by oligonucleotide microarray and quantitative real-time PCR for the expression of angiogenic factors. Results: There was identical baseline expression of VEGF-A and VEGF-C mRNA in chronic ischemic myocardium compared with nonischemic myocardium. Reperfusion increased the gene expression of VEGF-A and VEGF-C mRNA both in nonischemic and ischemic myocardium. VEGF-A protein was detected mainly in the extracellular matrix around the cardiomyocytes in ischemic myocardium. Conclusion: These data suggest that the nonconclusive VEGF gene therapy trials chronic coronary artery disease was not due to a preexisting upregulation of VEGF in chronic ischemic myocardium. There might be room for further therapeutic angiogenesis in chronic ischemic myocardium. [source] Identification of tryptase- and chymase-related gene clusters in human mast cells using microarraysALLERGY, Issue 3 2006C. Dahl Tryptase and chymase are the two major granular proteases present in human mast cell (MC)s. We used oligonucleotide microarray to measure the levels of approximately 22 000 transcripts in cord blood-derived MCs at 4 weeks, 8 weeks, 12 weeks and 18 weeks in culture. Tryptase (TPSB2) was expressed at the highest level among all transcripts and its expression level reached a plateau at 8 weeks. On the other hand, the expression level of chymase (CMAI) doubled every 4,6 weeks. A similar tendency was found at the protein levels with FACS analysis. After filtering the transcripts with MC-specificity, hierarchical clustering analysis identified 494 and 81 transcripts in the same clusters with tryptase and chymase, respectively. MC-specific genes, KIT and HDC were found in the tryptase cluster. In the chymase cluster, a critical suppressor for cell senescence, BMI1 and the several related genes were found, suggesting that chymase expression may be closely related to cell senescence/quiescence events. [source] A microarray-based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal diseasePRENATAL DIAGNOSIS, Issue 11 2009Tianjiao Chu Abstract Objectives We describe a novel microarray-based approach for the high-throughput discovery of epigenetic biomarkers for use in the noninvasive detection of fetal genetic disease. Methods We combined a 215 060-probe custom oligonucleotide microarray with a comprehensive library preparation method and novel statistical tools to compare DNA methylation patterns in chorionic villus samples (CVS) with gestational age-matched maternal blood cell (MBC) samples. Our custom microarray was designed to provide high-resolution coverage across human chromosomes 13, 18 and 21. Results We identified 6311 MspI/HpaII sites across all three chromosomes that displayed tissue-specific differential CpG methylation patterns. To maximize the probability of identifying biomarkers that have clinical utility we filtered our data to identify MspI/HpaII sites that are within 150 bp of a highly polymorphic single nucleotide polymorphism (SNP) so that its allelic ratio may be determined for the detection of fetal aneuploidy. Our microarray design and the computational tools used for data analysis are available for download as is the entire data set. Conclusions This high-resolution analysis of DNA methylation patterns in the human placenta during the first trimester of pregnancy identifies numerous potential biomarkers for the diagnosis of fetal aneuploidy on chromosomes 13, 18 and 21. Copyright © 2009 John Wiley & Sons, Ltd. [source] Assessment of the swine protein-annotated oligonucleotide microarrayANIMAL GENETICS, Issue 6 2009J. P. Steibel Summary The specificity and utility of the swine protein-annotated oligonucleotide microarray, or Pigoligoarray (http://www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed including HGNC identities and comparative mapping alignments with human orthologs. Hybridization results based on the Pigoligoarray's sets of control, perfect match (PM) and deliberate mismatch (MM) probes provide an important means of assessing non-specific hybridization. Simple descriptive diagnostic analyses of PM/MM probe sets are introduced in this paper as useful tools for detecting non-specific hybridization. Samples of RNA from liver, brain stem, longissimus dorsi muscle and uterine endothelium from four pigs were prepared and hybridized to the arrays. Of the total 20 400 oligonucleotides on the Pigoligoarray, 12 429 transcripts were putatively differentially expressed (DE). Analyses for tissue-specific expression [over-expressed in one tissue with respect to all the remaining three tissues (q < 0.01)] identified 958 DE transcripts in liver, 726 in muscle, 286 in uterine endothelium and 1027 in brain stem. These hybridization results were confirmed by quantitative PCR (QPCR) expression patterns for a subset of genes after affirming that cDNA and amplified antisense RNA (aRNA) exhibited similar QPCR results. Comparison to human ortholog expression confirmed the value of this array for experiments of both agricultural importance and for tests using pigs as a biomedical model for human disease. [source] Using gene chips to identify organ-specific, smooth muscle responses to experimental diabetes: potential applications to urological diseasesBJU INTERNATIONAL, Issue 2 2007Jason D. Hipp OBJECTIVE To identify early diabetes-related alterations in gene expression in bladder and erectile tissue that would provide novel diagnostic and therapeutic treatment targets to prevent, delay or ameliorate the ensuing bladder and erectile dysfunction. MATERIALS AND METHODS The RG-U34A rat GeneChip® (Affymetrix Inc., Sunnyvale, CA, USA) oligonucleotide microarray (containing ,8799 genes) was used to evaluate gene expression in corporal and male bladder tissue excised from rats 1 week after confirmation of a diabetic state, but before demonstrable changes in organ function in vivo. A conservative analytical approach was used to detect alterations in gene expression, and gene ontology (GO) classifications were used to identify biological themes/pathways involved in the aetiology of the organ dysfunction. RESULTS In all, 320 and 313 genes were differentially expressed in bladder and corporal tissue, respectively. GO analysis in bladder tissue showed prominent increases in biological pathways involved in cell proliferation, metabolism, actin cytoskeleton and myosin, as well as decreases in cell motility, and regulation of muscle contraction. GO analysis in corpora showed increases in pathways related to ion channel transport and ion channel activity, while there were decreases in collagen I and actin genes. CONCLUSIONS The changes in gene expression in these initial experiments are consistent with the pathophysiological characteristics of the bladder and erectile dysfunction seen later in the diabetic disease process. Thus, the observed changes in gene expression might be harbingers or biomarkers of impending organ dysfunction, and could provide useful diagnostic and therapeutic targets for a variety of progressive urological diseases/conditions (i.e. lower urinary tract symptoms related to benign prostatic hyperplasia, erectile dysfunction, etc.). [source] Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2,-deoxycytidine treatment and oligonucleotide microarrayCANCER SCIENCE, Issue 1 2006Satoshi Yamashita To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2,-deoxycytidine (5-aza-dC). After 5-aza-dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16-fold or more, using an oligonucleotide microarray with 39 000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty-two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421 ± 75 (95% confidence interval) methylation-silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor-related genes with promoter CGI that were upregulated four-fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor-related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one primary gastric cancer. A metastasis suppressor gene, MTSS1, was located in a genomic region with frequent loss of heterozygosity (8q22), and was expressed abundantly in the normal gastric mucosa, suggesting its role in gastric carcinogenesis. (Cancer Sci 2006; 97: 64 ,71) [source] Role of ocular pigment epithelial cells in regional ocular immunityACTA OPHTHALMOLOGICA, Issue 2008S SUGITA Purpose To whether soluble factors by retinal pigment epithelial cells (RPE) promote the generation of T regulatory cells in vitro. Methods Primary cultured RPE cells were established from normal C57BL/6 mice. T cells were co-cultured with RPE, x-irradiated, and used as regulators (RPE Treg cells). Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by [3H],thymidine incorporation. Expression of cytotoxic T lymphocyte antigen-2, (CTLA-2,) and cathepsin L on RPE and T cells was evaluated with oligonucleotide microarray, RT-PCR, immune staining, western blots and flow cytometry. Recombinant mouse CTLA-2, and anti-mouse CTLA-2, abs were used for the assay. For induction of experimental autoimmune uveitis (EAU), mice were immunized with interphotoreceptor retinoid-binding protein peptide emulsified in complete Freund's adjuvant. Results RPE converted CD4+ T cells into Treg cells by producing and secreting CTLA-2,, a cathepsin L inhibitor. CTLA-2, secreted by RPE cells selectively inhibited cathepsin L in the T cells and the cathepsin L-lacking T cells exhibited Treg phenotype, i.e. expression of Foxp3 and production of transforming growth factor beta (TGF,). CTLA-2, enhanced their production of active forms of TGF,. In addition, CD4+ T cells from EAU-induced cathepsin L knockout (KO) donors contained high population of Foxp3+ T cells and EAU in cathepsin L KO mice was significantly less than those in wild type mice. Furthermore, treatment with recombinant CTLA-2, significantly suppressed EAU. Conclusion These results indicate that immunosuppressive factors derived from RPE participate in the establishment of immune regulation in the posterior segment of the eye. [source] Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strainsFEMS YEAST RESEARCH, Issue 6 2007Philippe Marullo Abstract Natural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers the possibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was only effective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO2 production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms. [source] ERK5 is a target for gene amplification at 17p11 and promotes cell growth in hepatocellular carcinoma by regulating mitotic entryGENES, CHROMOSOMES AND CANCER, Issue 2 2009Keika Zen Using high-density oligonucleotide microarrays, we investigated DNA copy-number aberrations in cell lines derived from hepatocellular carcinomas (HCCs) and detected a novel amplification at 17p11. To identify the target of amplification at 17p11, we defined the extent of the amplicon and examined HCC cell lines for expression of all seven genes in the 750-kb commonly amplified region. Mitogen-activated protein kinase (MAPK) 7, which encodes extracellular-regulated protein kinase (ERK) 5, was overexpressed in cell lines in which the gene was amplified. An increase in MAPK7 copy number was detected in 35 of 66 primary HCC tumors. Downregulation of MAPK7 by small interfering RNA suppressed the growth of SNU449 cells, the HCC cell line with the greatest amplification and overexpression of MAPK7. ERK5, phosphorylated during the G2/M phases of the cell cycle, regulated entry into mitosis in SNU449 cells. In conclusion, our results suggest that MAPK7 is likely the target of 17p11 amplification and that the ERK5 protein product of MAPK7 promotes the growth of HCC cells by regulating mitotic entry. © 2008 Wiley-Liss, Inc. [source] Glia-induced neuronal differentiation by transcriptional regulationGLIA, Issue 11 2007Christian Göritz Abstract There is increasing evidence that different phases of brain development depend on neuron,glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia-derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia-conditioned medium (GCM) and cholesterol up- and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes. © 2007 Wiley-Liss, Inc. [source] Plasticity in the adult rat pancreas: Transdifferentiation of exocrine to hepatocyte-like cells in primary cultureHEPATOLOGY, Issue 6 2004Jessy Lardon Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte-like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse-transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer-binding protein-ß protein, a liver-enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and ,-fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX-treated cells using oligonucleotide microarrays and found that multiple liver-specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX-treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes. (HEPATOLOGY 2004;39:1499,1507.) [source] Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 3 2007Anje A. te Velde PhD Abstract Background: Mouse models of inflammatory bowel diseases (IBD) are used to unravel the pathophysiology of IBD and to study new treatment modalities, but their relationship to Crohn's disease (CD) or ulcerative colitis (UC) is speculative. Methods: Using Agilent mouse TOX oligonucleotide microarrays, we analyzed colonic gene expression profiles in three widely used models of experimental colitis. In 2 of the models (TNBS and DSS-induced colitis), exogenous agents induce the colitis. In the third model the colitis is induced after transfer of a T-cell population (CD4+CD45RBhigh T cells) that lacks regulatory cells into an immunodeficient host. Results: Compared with control mice, in DSS, TNBS, and the CD45RB transfer colitis mice, 387, 21, and 582 genes were more than 2-fold upregulated in the intestinal mucosa. Analyses of exclusively shared gene expression profiles between the different models revealed that DSS/transfer colitis share 69 concordantly upregulated genes, DSS/TNBS 6, and TNBS/transfer colitis 1. Seven genes were upregulated in all three models. The CD45RB transfer model expression profile included the most genes that are known to be upregulated in IBD. Of 32 genes that are known to change transcriptional activity in IBD (TNF, IFN -,, Lt,, IL - 6, IL - 16, IL - 18R1, IL - 22, CCR2, 7, CCL2, 3, 4, 5, 7, 11, 17, 20, CXCR3, CXCL1, 5, 10, Mmp3, 7,9, 14, Timp1, Reg3,, and Pap, S - 100a8, S - 100a9, Abcb1, and Ptgs2), 2/32 are upregulated in TNBS, 15/32 are upregulated or downregulated in DSS and 30/32 are upregulated or downregulated in the CD45RB transfer colitis. Conclusion: The pattern of gene expression in the CD45RB transfer model most closely reflects altered gene expression in IBD. (Inflamm Bowel Dis 2007) [source] Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteriaJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.G. Weber Abstract Aims:, The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Methods and Results:, Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. Conclusions:, This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. Significance and Impact of the Study:, The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population. [source] Calcium control of gene regulation in rat hippocampal neuronal culturesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Giulietta Pinato Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20,µM gabazine applied for 30,min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs. J. Cell. Physiol. 220: 727,747, 2009. © 2009 Wiley-Liss, Inc. [source] Gene expression of AGS cells stimulated with released proteins by Helicobacter pyloriJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 4 2008Nayoung Kim Abstract Background and Aim:, Interactions between released proteins by Helicobacter pylori (H. pylori) and the cells of gastric epithelium to which it adheres may contribute to gastric inflammation and epithelial damage. The present study was performed to evaluate the gene expression of AGS gastric cancer cells stimulated with released proteins by H. pylori. Methods:, Gene expression of AGS cells to the stimulation by H. pylori -released proteins (G27 strain) were monitored using oligonucleotide microarrays. Results:, Eighty-eight genes (0.88%) and eight genes (0.08%) were up- or downregulated, respectively, by treating AGS cells with H. pylori -released proteins but not by H. pylori adhesion after 12 h of coculture. Out of the selected 40 up- and five downregulated genes, 29 upregulated genes classified as general RNA polymerase II transcription factor activity (GTF2B, PPARGC1A), SH3/SH2 adaptor activity (CRKL), transferase activity (ACLY, CRKL, PIGC, PLK4), and oxidoreductase activity (IDH1) were confirmed to be upregulated by released proteins and not by H. pylori adhesion by real-time reverse transcription,polymerase chain reaction. When the concentrated H. pylori -cultured supernatant prepared by our protocol was treated by boiling, the upregulations of 26 of these 29 genes (89.7%) except for CD160, ZNF268, and PSAT1 disappeared. This confirmed that most of these upregulations were caused by released proteins. Conclusion:, Host genes involving transcription, signaling and stress are significantly modulated by the proteins released by H. pylori. This might strengthen the gastroduodenal pathogenesis induced by H. pylori. [source] Gene expression in endoprosthesis loosening: Chitinase activity for early diagnosis?,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2008L. Morawietz Abstract The aim of the study was to identify markers for the early diagnosis of endoprosthesis loosening, for the differentiation between wear particle,induced and septic loosening and to gather new insights into the pathogenesis of endoprosthesis loosening. Gene expression profiles were generated from five periprosthetic membranes of wear particle,induced and five of infectious (septic) type using Affymetrix HG U133A oligonucleotide microarrays. The results of selected differentially expressed genes were validated by RT-PCR (n,=,30). The enzyme activity and the genotype of chitinase-1 were assessed in serum samples from 313 consecutive patients hospitalized for endoprosthesis loosening (n,=,54) or for other reasons, serving as control subjects (n,=,259). Eight hundred twenty-four genes were differentially expressed with a fold change greater than 2 (data sets on http://www.ncbi.nlm.nih.gov/geo/ GSE 7103). Among these were chitinase 1, CD52, calpain 3, apolipoprotein, CD18, lysyl oxidase, cathepsin D, E-cadherin, VE-cadherin, nidogen, angiopoietin 1, and thrombospondin 2. Their differential expression levels were validated by RT-PCR. The chitinase activity was significantly higher in the blood from patients with wear particle,induced prosthesis loosening (p,=,0.001). However, chitinase activity as a marker for early diagnosis has a specificity of 83% and a sensitivity of 52%, due to a high variability both in the disease and in the control group. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:394,403, 2008 [source] Gene Expression in Human Alcoholism: Microarray Analysis of Frontal CortexALCOHOLISM, Issue 12 2000Joanne M. Lewohl Background: Changes in brain gene expression are thought to be responsible for the tolerance, dependence, and neurotoxicity produced by chronic alcohol abuse, but there has been no large scale study of gene expression in human alcoholism. Methods: RNA was extracted from postmortem samples of superior frontal cortex of alcoholics and nonalcoholics. Relative levels of RNA were determined by array techniques. We used both cDNA and oligonucleotide microarrays to provide coverage of a large number of genes and to allow cross-validation for those genes represented on both types of arrays. Results: Expression levels were determined for over 4000 genes and 163 of these were found to differ by 40% or more between alcoholics and nonalcoholics. Analysis of these changes revealed a selective reprogramming of gene expression in this brain region, particularly for myelin-related genes which were down-regulated in the alcoholic samples. In addition, cell cycle genes and several neuronal genes were changed in expression. Conclusions: These gene expression changes suggest a mechanism for the loss of cerebral white matter in alcoholics as well as alterations that may lead to the neurotoxic actions of ethanol. [source] Proteome characterization of human T helper 1 and 2 cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2004Kirsi Rautajoki Abstract T helper (Th) cells can be polarized into two different main subtypes, Th1 and Th2 cells. Their activation is linked to the eradication of different pathogens and to dissimilar immunological dysfunctions, which implies differences also in their protein expression patterns. To identify these differences, CD4+ T cells were isolated from human cord blood, polarized in vitro to Th1 and Th2 and activated via CD3 and CD28. Cells were lysed, soluble proteins were separated with two-dimensional electrophoresis and differing protein spots were identified with peptide mass fingerprinting. The expression of 14 proteins differed in Th1 and Th2 cells after both 7 and 14 days of polarization. Twelve of the proteins could be identified, most of which are new in this context. Two proteins were differentially modified in the two cell types. Especially, N -terminal acetylation of cyclophilin A was stronger in Th1 than in Th2 cells. To compare the RNA and the protein levels of the identified genes, mRNA expression was measured with Affymetrix oligonucleotide microarrays (HG-U133A). The mRNA and protein expression level correlated only in six cases out of eleven, which highlights the complementary roles that proteomics and transcriptomics have in the elucidation of biological phenomena. [source] | ||||||||||||||||||||||||||||||||||