Current Issue : January - March Volume : 2015 Issue Number : 1 Articles : 6 Articles
RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not\nreflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their\nresponses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results\nserve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to\nanalyze proteinââ?¬â??mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed\nin cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain\nantibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins\nis assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to\nliving cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or\npharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available\nGBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment.\nTherefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast\nand simple radioactivity-free method will find many useful applications in RNA biology....
Objective. Technically primates and dogs represent ideal models to investigate diseases characterized by abnormal intracortical\nremodeling.High expenses and ethical issues, however, restrict the use of those animals in research. Rodentmodels have been used\nas alternatives instead, but their value is limited, if none, because these animals lack intracortical bone remodeling. This study aimed\nat investigating the effect of ovariectomy onto the stimulation of intracortical remodeling in rat mandibles. Materials and Methods.\nSixteen 12-week-old Spraque-Dawly (SD) female rats were randomly assigned into two groups, receiving either ovariectomy or\nsham operation. All the rats were sacrificed 18 weeks postoperatively. The entire mandibles were harvested for microcomputed\ntomography (micro-CT) and histomorphometric assessments. Results.Micro-CT examination showed significantly decreased bone\nmineral density (0.95 �± 0.01 versus 1.01 �± 0.02 g/cm3, P < 0.001) and bone volume (65.78 �± 5.45 versus 87.41 �± 4.12%, P < 0.001)\nin ovariectomy group. Histomorphometric assessment detected a sixfold increased intracortical bone remodeling as well as an\nincreased bone modeling in mandibles of ovariectomized rats. Conclusion. For the first time, to the authorsâ�� knowledge, it was\ndetected that ovariectomy stimulates intracortical remodeling in rat mandibles. This animal model might be of use to study various\nbone diseases associated with an abnormal intracortical remodeling process....
Purpose Influenza CD8+ T-cell epitopes are conserved amongst\ninfluenza strains and can be recognized by influenza-specific cytotoxic\nT-cells (CTLs), which can rapidly clear infected cells. An\ninfluenza peptide vaccine that elicits these CTLs would therefore\nbe an alternative to current influenza vaccines, which are not\ncross-reactive. However, peptide antigens are poorly immunogenic\ndue to lack of delivery to antigen presenting cells, and\ntherefore need additional formulation with a suitable delivery\nsystem. In this study, the potential of virosomes as a delivery\nsystem for an influenza T-cell peptide was investigated.\nMethods The conserved human HLA-A2.1 influenza T-cell epitope\nM158ââ?¬â??66 was formulated with virosomes. The immunogenicity\nand protective effect of the peptide-loaded virosomes was\nassessed in HLA-A2 transgenic mice. Delivery properties of the\nvirosomes were studied in mice and in in vitro dendritic cell cultures.\nResults Immunization of HLA-A2.1 transgenic C57BL/6 mice\nwith peptide-loaded virosomes in the presence of the adjuvant\nCpG-ODN 1826 increased the number of peptide-specific\nCTLs. Vaccination with adjuvanted peptide-loaded virosomes\nreduced weight loss in mice after heterologous influenza infection.\nAssociation with fusion-active virosomes was found to be crucial\nfor antigen uptake by dendritic cells, and subsequent induction of\nCTLs in mice.\nConclusions These results show that influenza virosomes loaded\nwith conserved influenza epitopes could be the basis of a novel\ncross-protective influenza vaccine....
Background. Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology\nof CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder.\nTo reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia\npain (CPIP) model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we\nperformed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the\nproteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed\nbetween the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic\nplasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A,\ncereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model\nindicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the\ndevelopment and maintenance of CRPS....
Hepatocellular carcinoma is the fifth most common cancer worldwide and shows a complex clinical course, poor response to\npharmacological treatment, and a severe prognosis.Thus, the aim of this study was to investigate whether tacrolimus (FK506) has\nsynergistic antitumor effects with doxorubicin on two human hepatocellular carcinoma cell lines, Huh7 and HepG2. Cell viability\nwas analyzed by Sulforhodamine B assay and synergic effect was evaluated by the software CalcuSyn. Cell apoptosis was evaluated\nusingAnnexinVandDeadCell assay.Apoptosis-related protein PARP-1 cleaved and autophagy-related protein expressions (Beclin-\n1 and LC3B) were measured by western blotting analysis. Cytokines concentration in cellular supernatants after treatments was\nstudied by Bio-Plex assay. Interestingly the formulation with doxorubicin and tacrolimus induced higher cytotoxicity level on\ntumor cells than single treatment. Moreover, our results showed that the mechanisms involved were (i) a strong cell apoptosis\ninduction, (ii) contemporaneous decrease of autophagy activation, understood as prosurvival process, and (iii) downregulation of\nproinflammatory cytokines. In conclusion, future studies could relate to the doxorubicin/tacrolimus combination effects in mice\nmodels bearing HCC in order to see if this formulation could be useful in HCC treatment....
Sciatic nerve crush injury in rat animal model is one of the most common experimental models used in regenerative research.\nHowever, the availability of transgenic mouse for nerve regeneration studies is constantly increasing and, therefore, the shift from\nratmodel to mousemodel is, in some cases, necessary.Moreover, since most of the human nerve lesions occur in the upper limb, it\nis also advantageous to shift from sciatic nerve to median nerve. In this study we described an experimental model which involves\nlesions of the median nerve in the mouse. Data showed that the finger flexor muscle contraction strength, assessed to evaluate\nthe motor function recovery, and reached values not different from the control already 20 days after injury. The degree of nerve\nregeneration evaluated with stereological methods in light microscopy showed that, 25 days after injury, the number of regenerated\nmyelinated fibers was comparable to the control, but they were smaller with a thinnermyelin thickness. Stereological analysis made\nin electron microscopy confirmed these results, although the total number of fibers quantified was significantly higher compared\nto light microscopy analysis, due to the very small size of some fibers that can be detected only in electron microscopy...
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