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Specific DNA Sequences (specific + dna_sequence)
Selected AbstractsTranscription factor binding study by capillary zone electrophoretic mobility shift assayELECTROPHORESIS, Issue 1-2 2003Zsolt Ronai Abstract Regulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein-DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1-specific fluorescein-labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti-Sp1 antibody. The applied electric field strength did not affect the stability of DNA-protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS-box transcription factor 4 (OsMADS4) and its consensus sequence is also reported. [source] Cryopreservation of fish sperm: applications and perspectivesJOURNAL OF APPLIED ICHTHYOLOGY, Issue 5 2010E. Cabrita Summary Cryopreservation is of interest not only for fish farming but also for the conservation and genetic improvement of resources. This technique has been well established in some freshwater fish species mainly, salmonid, sturgeons and carps, however, only in the last decade research was focused in marine fish species. The benefits of sperm cryopreservation include: (i) synchronization of gamete availability of both sexes, (ii) sperm economy; (iii) simplification of broodstock management, (iv) transport of gametes from different fish farms, and (v) germplasm storage for genetic selection programs or conservation of species. These issues would certainly benefit the aquaculture industry. The tremendous impact that biotechnology is having in aquaculture has been particularly obvious in recent years. Several species are being used as research models not only for aquaculture development applications but also for medical research. Sperm cryopreservation can give an important contribution in the germ storage of all transgenic lines. However, in all applications in fish sperm, cryopreservation needs to overcome a lack in standardization of methodologies and procedures, a correct assay of seminal quality and the development of tools to characterize cryoinjury. Many efforts have recently been made in the study of DNA using different approaches such as the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labelling), SCSA (sperm chromatin structure assay) and the analysis of specific DNA sequences using RT-PCR, since DNA damage may impair fertility or embryo development. Cryopreservation of gametes would certainly benefit from a higher concern on male improvement, basically through nutrition or selection of resistant stocks (e.g. stress resistant individuals or highly adapted to captivity) producing gametes of higher quality. There is a huge window of opportunities for improve the resistance of cells to cryopreservation through diet supplementation of certain compounds such as amino acids (taurine and hypotaurine), vitamins (Vit. E and C) and lipids or through a direct supplementation of the extender media. An equilibrium of those compounds will improve spermatozoa and seminal plasma composition protecting cells against oxidative stress (lipid peroxidation, protein oxidation, DNA fragmentation, enzyme protection) that is gaining each day more importance in cryodamage research. [source] Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization techniqueMOLECULAR ORAL MICROBIOLOGY, Issue 1 2008M. Tachibana-Ono Background/aims:,Porphyromonas gingivalis, a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P. gingivalis. Methods:, The subtractive hybridization technique was employed to identify the genes specific to P. gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P. gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains. Results:, We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50. Conclusions:, The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P. gingivalis strains. [source] Inhibition of NF-,B signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2010Hiroshi Okamoto Objective Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and is characterized mainly by symmetric polyarticular joint disorders. The pathologic processes are mediated by a number of cytokines, chemokines, cell adhesion molecules, and matrix metalloproteinases. The expression of most of these molecules is controlled at the transcriptional level. In addition, activation of NF-,B is involved in RA pathogenesis. This study was performed to explore the role of a novel serine/threonine kinase inhibitor, fasudil, in the control of the NF-,B activation pathway and to investigate the therapeutic effects of fasudil on arthritis development in a rat model of RA. Methods Fibroblast-like synoviocytes (FLS) from RA patients and human endothelial cells (ECs) were established and maintained. To study the role of fasudil on cytokine expression, various cytokines expressed in the RA FLS and human ECs were measured by enzyme-linked immunosorbent assay following stimulation of the cells with interleukin-1, (IL-1,) in the presence of various concentrations of fasudil. The role of fasudil on NF-,B activation was studied using a reporter gene assay, Western blotting of I,B,, immunofluorescence analysis of the p65 subunit of NF-,B, and electrophoretic mobility shift assay. The in vivo effects of fasudil on arthritis were studied in a rat adjuvant-induced arthritis (AIA) model. Results Fasudil inhibited cytokine expression in RA FLS and human ECs and also inhibited the activation of ECs, in a dose-dependent manner. Fasudil inhibited IL-1,,induced activation of NF-,B independent of the inhibition of I,B, degradation and nuclear translocation of NF-,B, and inhibited IL-1,,induced DNA binding of NF-,B. Finally, in vivo, fasudil ameliorated arthritis in rats with AIA, without any adverse effects. Conclusion Serine/threonine kinase inhibitor fasudil inhibits the development of arthritis in a rat model of RA, and also inhibits the NF-,B signaling required for binding of NF-,B to specific DNA sequences through, for example, the phosphorylation of p65, suggesting that a specific target of fasudil might be a novel NF-,B kinase. Thus, fasudil serves as a novel strategy for the treatment of RA. [source] | ||||||||||