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Differential Screening (differential + screening)

Distribution by Scientific Domains

Medical Sciences	50%
Life Sciences	30%
Chemistry	20%

Selected Abstracts

Inflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA display

INFLAMMATORY BOWEL DISEASES, Issue 5 2003
Kouhei Fukushima M.D., Ph.D.
Abstract Intestinal epithelial cells are actively involved in the pathogenesis of inflammatory bowel disease resulting in an altered functional phenotype. The modulation of epithelial gene expression may occur as a consequence of proliferative, metabolic, immune, inflammatory, or genetic abnormalities. Differential screening of epithelial-cell-derived cDNA libraries (from control, ulcerative colitis, and Crohn's disease epithelial cells) and differential display of mRNA were used for investigation of disease-associated gene expression and modulation. Intestinal epithelial gene expression was successfully analyzed by both approaches. Using differential screening with clones encoding mitochondrial genes, quantitative overexpression was observed in both ulcerative colitis and Crohn's disease, while a unique expression of small RNA was noticed in Crohn's disease cells using Alu-homologous clones. Differential display demonstrated that several genes were differentially displayed among control, ulcerative colitis, and Crohn's disease epithelial cells. This was confirmed by immunohistochemical staining of pleckstrin, desmoglein 2 and voltage-dependent anion channel in control and inflammatory bowel disease mucosal samples. In summary, several inflammatory bowel disease-related associations were found. Since both differential screening and display have advantages and limitations, the combination of both techniques can generate complementary information, facilitate search for novel genes, and potentially identify genes uniquely associated with inflammatory bowel disease. [source]

Gene Expression Analysis in Cucumber Leaves Primed by Root Colonization with Pseudomonas chlororaphis O6 upon Challenge-Inoculation with Corynespora cassiicola

PLANT BIOLOGY, Issue 2 2004
M. S. Kim
Abstract: Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C. cassiicola, using plants colonized by O6. To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization. Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor. Expression of these genes was not induced by O6 colonization alone. Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection. Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation. [source]

Molecular biology of grape berry ripening

AUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 2 2000
SIMON P. ROBINSON
Abstract Grapevines produce non-climacteric fruit that exhibit a double sigmoidal pattern of growth. Ripening occurs during the second growth phase when grapes change colour, start to soften, accumulate reducing sugars, metabolise organic acids and synthesise flavour compounds. Unlike many other fruit, grapes ripen while the berries are still expanding, and as with most non-climacteric fruit, ripening does not appear to be controlled by ethylene. Sugars and amino acids that accumulate in grapes during ripening are imported via the phloem, while many secondary metabolites are synthesised within the berry itself. Grapes import sucrose but accumulate hexoses. Conversion of sucrose to hexoses is most likely catalysed by invertase. cDNAs encoding vacuolar invertases have been isolated from grape berries. Expression of these genes and an increase in invertase activity occur before veraison, so it seems unlikely that synthesis of this enzyme is a controlling factor for sugar accumulation during ripening. Proteins that transport sugars into the berry vacuole may regulate sugar accumulation, and cDNAs encoding both sucrose and hexose transporters have been isolated from ripening grape berries. Determination of the role of these transporters may reveal the pathway of sugar accumulation in grapes. Anthocyanins are only synthesised in the skin of red grapes after veraison. Analysis of the patterns of expression of genes in the flavonoid pathway has shown that there is a dramatic increase in expression of many of these genes in skin cells at veraison. Expression of the gene encoding a glycosyl transferase involved in the lasts steps of anthocyanin synthesis was absolutely correlated with anthocyanin synthesis and may explain the lack of anthocyanin synthesis in white grapes and in the flesh of most red grapes. We infer that the synthesis of anthocyanins is regulated at the transcription level and is likely to be controlled by regulatory genes. Softening of fruit generally results from changes in the properties of cell walls. Analysis of the cell walls of grapes during ripening suggests that there are no dramatic changes in polysaccharide composition but modification of specific components may contribute to softening. A number of proteins are newly synthesised in grapes during ripening and several of these proteins have now been identified. The most abundant are pathogenesis-related (PR) proteins, including chitinases and thaumatin-like proteins. Expression of genes encoding a number of PR proteins increased dramatically in grapes during ripening. It is not clear what role the PR proteins play during ripening but they may provide resistance to pathogens. Differential screening of a post-veraison grape berry cDNA library has also identified ripening-related genes, some of which encode proline-rich cell wall proteins. Other grape ripening-related genes have homologues that are induced by stress in other plants. These studies indicate that a dramatic change in gene expression occurs in grape berries at veraison and suggest that ripening involves a coordinated increase in transcription of a number of different genes. [source]

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene

FEBS JOURNAL, Issue 7 2001
Muriel Erent
The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase C,. NDP kinase C, had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase C,, based on the crystal structure of NDP kinase B, indicated that NDP kinase C, had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase C, readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo. [source]

Stage-specific gene expression in early differentiating oligodendrocytes

GLIA, Issue 2 2002
Francesca Blasi
Abstract The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A+ mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A+ mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288. GLIA 39:114,123, 2002. © 2002 Wiley-Liss, Inc. [source]

Inflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA display

Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent

THE PLANT JOURNAL, Issue 5 2000
Ailan Guo
Summary Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA. [source]

Down-regulation of cathepsin K in synovium leads to progression of osteoarthritis in rabbits

ARTHRITIS & RHEUMATISM, Issue 8 2009
Daisuke Takahashi
Objective The hypothesis of this study was that synovial factors playing a pivotal role in the pathogenesis of osteoarthritis (OA) and thus gene expression in the synovium would be altered at the initial stage of OA. The aims of this study were to identify the candidate genes in synovium related to OA initiation, to evaluate cartilage degeneration after knockdown of the gene using small interfering RNA (siRNA) gene silencing in the knee joints at the initial stage of OA, and to determine the potential role of the knocked-down gene in OA initiation. Methods Genes overexpressed in synovium at the initial stage of disease in a rabbit model of anterior cruciate ligament transection (ACLT),induced OA were identified using the suppression subtractive hybridization technique and differential screening. Candidate gene expression in the synovium of the knees of rabbits with OA was manipulated with electroporation-assisted siRNA transduction 4 times before and after operation. Four weeks after surgery, histologic analysis was performed. Results Cathepsin K gene and protein expression was significantly up-regulated in synovium at the initial stage of OA in rabbits. Down-regulation of cathepsin K in synovium at the initial stage of OA significantly accelerated cartilage degeneration. Conclusion These results indicate that cathepsin K plays a protective role in cartilage degeneration at the initial stage of OA. We believe that the current results obtained from models of the early phase of OA will provide useful information for developing a novel strategy to prevent disease progression. [source]

Frequent aberrant methylation of the promoter region of sterile , motif domain 14 in pulmonary adenocarcinoma

CANCER SCIENCE, Issue 11 2008
Weihong Sun
Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-suppressor and tumor-related genes. In order to identify novel hypermethylated genes in early stage lung adenocarcinoma, we carried out methylated CpG island amplification, modified suppression subtractive hybridization, and methylation-specific polymerase chain reaction to identify aberrant methylation of CpG islands in the A/J mouse lung adenoma model, which histologically mimics the early stage of human pulmonary adenocarcinoma. Through methylated CpG island amplification, suppression subtractive hybridization, and differential screening, we detected five genes, three of which have human homologs. Two of them showed downregulation of their expression in human lung adenocarcinoma. Of these two genes, we selected sterile , motif domain 14 (SAMD14) and further analyzed its methylation status and expression level by methylation-specific polymerase chain reaction and quantitative real-time polymerase chain reaction. Most of the lung adenocarcinoma cell lines showed suppressed expression of SAMD14 together with hypermethylation at the promoter region, although an immortalized bronchial epithelium cell line (PL16B) did not show hypermethylation and did express SAMD14. The expression of SAMD14 in A549 was rescued by treatment with the demethylation agent 5-aza-2,-deoxycytidine. These data indicate that hypermethylation of the SAMD14 gene promoter region is associated with silencing of its expression. Hypermethylation at the CpG site of the SAMD14 promoter region was detected frequently in early invasive adenocarcinoma (8/24, 33.3%) but not in in situ adenocarcinoma (0/7, 0%) or normal lung tissue (0/31, 0%). Hypermethylation of the SAMD14 gene is a specific event in pulmonary adenocarcinogenesis and malignant progression. (Cancer Sci 2008; 99: 2177,2184) [source]

OCIA domain containing 2 is highly expressed in adenocarcinoma mixed subtype with bronchioloalveolar carcinoma component and is associated with better prognosis

CANCER SCIENCE, Issue 1 2007
Tadashi Ishiyama
Although lung adenocarcinoma is a major cause of cancer death worldwide, details of its molecular carcinogenesis and stepwise progression are still unclear. To characterize the sequential progression from bronchioloalveolar adenocarcinoma of the lung (BAC, in situ carcinoma) to adenocarcinoma mixed subtype with BAC component, polymerase chain reaction-based cDNA suppression subtractive hybridization (SSH) was carried out using two representative cases of BAC (non-invasive tumors) and adenocarcinoma mixed subtype with BAC (invasive tumors). Through differential screening, virtual reverse northern hybridization and quantitative real-time reverse-transcription,polymerase chain reaction (qRT-PCR) we selected five genes (TncRNA, OCIAD2, ANXA2, TMED4 and LGALS4) that were expressed at significantly higher levels in invasive adenocarcinoma mixed subtype with BAC than in BAC. After in situ hybridization and qRT-PCR analyses, we confirmed that only the OCIAD2 gene showed significantly higher expression in the tumor cells of invasive adenocarcinoma mixed subtype with BAC than in BAC (P = 0.026). We then carried out in situ hybridization of OCIAD2 in 56 adenocarcinoma mixed subtype with BAC component and assessed the correlation between OCIAD2 expression and clinicopathological features. In contrast to our expectation, the patients with OCIAD2 expression showed a better clinical outcome than those without OCIAD2 expression, and OCIAD2 expression showed an inverse correlation with lymphatic invasion, blood vessel invasion and lymph node metastasis. These results suggest that OCIAD2 begins to express at the progression from in situ to invasive carcinoma, and is associated with the favorable prognosis of adenocarcinoma mixed subtype with BAC component. (Cancer Sci 2007; 98: 50,57) [source]