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mRNA Isolated (mrna + isolated)
Selected AbstractsAlterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells,,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2005Gregory S. Akerman Abstract Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis. Environ. Mol. Mutagen., 2005. Published 2005 Wiley-Liss, Inc. [source] Charcot-Marie-Tooth neuropathy type 1A combined with Duchenne muscular dystrophyEUROPEAN JOURNAL OF NEUROLOGY, Issue 10 2007P. Vondracek We report a 24-year-old male with an unusual combination of two inherited neuromuscular disorders , Charcot-Marie-Tooth (CMT) disease type 1A and Duchenne muscular dystrophy (DMD). A phenotypic presentation of this patient included features of both these disorders. Nerve conduction studies revealed demyelinating peripheral neuropathy. Electromyography showed a profound myogenic pattern. The serum creatine kinase level was highly elevated. Muscle biopsy revealed a dystrophic picture with deficient dystrophin immunostaining. CMT1A duplication on chromosome 17p11.2 was found. The frame-shift mutation c.3609,3612delTAAAinsCTT (p.K1204LfsX11) was detected in the dystrophin gene by analysing mRNA isolated from the muscle tissue. The patient inherited both these mutations from his mother. The combination of CMT1A and DMD has not been reported as yet. [source] Monoterpene biosynthesis in lemon (Citrus limon)FEBS JOURNAL, Issue 13 2002cDNA isolation, functional analysis of four monoterpene synthases Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (,)-,-pinene and ,-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2+ as bivalent metal ion cofactor resulted in higher activity than Mg2+, with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 µm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present. [source] Analysis of vitamin D-regulated gene expression in LNCaP human prostate cancer cells using cDNA microarraysTHE PROSTATE, Issue 3 2004Aruna V. Krishnan Abstract BACKGROUND 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] exerts growth inhibitory, pro-differentiating, and pro-apoptotic effects on prostate cells. To better understand the molecular mechanisms underlying these actions, we employed cDNA microarrays to study 1,25(OH)2D3 -regulated gene expression in the LNCaP human prostate cancer cells. METHODS mRNA isolated from LNCaP cells treated with vehicle or 50 nM 1,25(OH)2D3 for various lengths of time were hybridized to microarrays carrying approximately 23,000 genes. Some of the putative target genes revealed by the microarray analysis were verified by real-time PCR assays. RESULTS 1,25(OH)2D3 most substantially increased the expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene. Our analysis also revealed several novel 1,25(OH)2D3 -responsive genes. Interestingly, some of the key genes regulated by 1,25(OH)2D3 are also androgen-responsive genes. 1,25(OH)2D3 also down-regulated genes that mediate androgen catabolism. CONCLUSIONS The putative 1,25(OH)2D3 target genes appear to be involved in a variety of cellular functions including growth regulation, differentiation, membrane transport, cell,cell and cell,matrix interactions, DNA repair, and inhibition of metastasis. The up-regulation of IGFBP-3 gene has been shown to be crucial in 1,25(OH)2D3 -mediated inhibition of LNCaP cell growth. 1,25(OH)2D3 regulation of androgen-responsive genes as well as genes involved in androgen catabolism suggests that there are interactions between 1,25(OH)2D3 and androgen signaling pathways in LNCaP cells. Further studies on the role of these genes and others in mediating the anti-cancer effects of 1,25(OH)2D3 may lead to better approaches to the prevention and treatment of prostate cancer. © 2004 Wiley-Liss, 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] 3132: The presence and suggested role of mesothelial proteins in the human corneal endotheliumACTA OPHTHALMOLOGICA, Issue 2010K JIRSOVA Purpose To determine whether proteins that are characteristic of the human mesothelial cell phenotype are expressed in human corneal endothelium. Methods Cadaverous human corneo-scleral discs and pathological corneal specimens (melted and rejected corneas) were used. The detection of mesothelin, proteinase inhibitor-9 (PI-9), calretinin and HBME-1 protein (a membrane protein of mesothelial cells) was performed on cryosections and corneal endothelial imprints using indirect immunofluorescent or enzymatic immuncytochemistry. The staining intensity was assessed using fluorescent or light microscopy, and the percentage of positive cells was calculated. Semi-quantitative RT PCR of PI-9, mesothelin and calretinin was performed using mRNA isolated from endothelial imprints. Results A strong signal for mesothelin was present in the corneal epithelium, while less intensive staining was visible in the endothelium. These results were confirmed using qRT-PCR. Immunostaining for PI-9 was observed in almost all corneal epithelial cells and in approximately 50% of the endothelial cells. An increased number of PI-9-positive cells was detected in stromal infiltrates of most melted corneal explants, while almost no positivity was observed in rejected explants. Calretinin was detected in the corneal epithelium and less intensively in the corneal endothelium, where both cytoplasmic and nuclear localisation were demonstrated. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Conclusion The proteins expressed constitutively in mesothelial cells (PI-9, mesothelin, calretinin and HBME-1 protein) are present in adult human corneal endothelium, confirming that this layer expresses markers typical of mesothelial cells. [source] | ||||||||||