A recombinant replication-deficient adenovirus has been blotting. As demonstrated by FACS analysis, up to 98% of generated that expresses a mutant of the Aquorea victoria HUVEC and approximately 70% of human smooth muscle green fluorescent protein (GFP) under the control of the cells could be transduced to express GFP. Since GFP can strong CMV promoter by insertion into the E1 region (AdVbe detected in cells without the need for prior fixing and GFP). High expression of GFP was found in different cell staining, the virus should be useful for optimizing in living types after infection with the recombinant virus that could cells the transduction efficiency of different cell types, of be easily detected by fluorescence microscopy. In human cells from different experimental animals, as well as studyumbilical vein endothelial cells (HUVEC), expression levels ing the kinetics and persistence of adenovirus-mediated had already reached a maximum after 2 days and were gene transfer in diverse experimental settings. stable for at least 7 days, as determined by Western Keywords: green fluorescent protein; recombinant adenovirus; marker gene; endothelium Gene transfer techniques using replication-deficient of them can be directly visualized in living cells without the need for prior incubation with substrates or staining recombinant adenoviral vectors (AdV) have considerable potential both at experimental and therapeutic levels. [1][2][3] procedures, a feature fulfilled by green fluorescent protein. Advantages include the ability to produce high-titer stocks, the high efficiency of gene transfer into a variety Originally isolated from the jellyfish Aquorea victoria as a 238 amino acid protein, 15 wild-type GFP emits a bright of cell types, and the ability to transduce cells that have a low mitotic index, eg the endothelium, kidney and cells green fluorescence upon excitation at both 395 and 475 nm. Crystallization has revealed a cylinder-like structure of the central nervous system. 4,5 Limitations of AdV include the relatively short time of expression of the of -sheets wrapped around a single central helix. 16 The fluorophore results from the autocatalytic cyclization (therapeutic) gene in vivo (a few weeks to several months, depending on the organ) due to the episomal maintebetween amino acids of the polypeptide backbone and oxidation of the ␣- bond of the central tyrosine. Due to nance of the adenoviral DNA in the cell, and the elicitation of an immune response by the host organism. [6][7][8] this rigid encapsulation, the fluorophore is remarkably insensitive to changes in environmental conditions, eg These features favor, in the first instance, applications where only a transient expression of the ectopic gene is pH, O 2 quenching of the excited state, and to proteolysis. The generation of mutants that show improved levels of desirable, eg inflammatory reactions.Despite its broad cell-type specificity, considerable diffluorescence as well as different excitation and emission maxima has further triggered the widesprea...
The cytokine and potent angiogenic factor vascular endothelial growth factor (VEGF) plays an important role in airway remodelling in various airway diseases such as idiopathic pulmonary fibrosis, pulmonary hypertension, lung cancer, asthma and chronic obstructive pulmonary disease (COPD). The effect of cigarette-smoking on VEGF expression, the modulatory role of extracellular signal-regulated kinase (ERK)-1,-2, p38mitogen-activated protein kinase (MAPK), histone acetylation and the anti-inflammatory effect of dexamethasone on TNFalpha-induced VEGF expression were examined in human airway smooth muscle cells (HASMC) of five non-smokers, 17 smokers without airflow limitation and 15 smokers with COPD. TNFalpha increased VEGF expression 5.4-fold and 4.0-fold in HASMC from non-smokers and smokers without airflow limitation, respectively, but only 2.5-fold in HASMC from smokers with COPD compared with non-stimulated HASMC. VEGF production was dependent on phosphorylation of ERK-1,-2 and p38MAPK, as was shown by examining the effects of PD 098059 (10 microM), an inhibitor of the upstream activator of MAPKkinase (MKK)-1, and SB 203580 (10 microM), an inhibitor of p38MAPK; there were no differences between non-smokers, smokers without airflow limitation and smokers with COPD in this respect. Dexamethasone (DEX; 10(-12)-10(-4) M) reduced TNFalpha-induced phosphorylation of ERK-1/-2 and prevented TNFalpha-induced VEGF generation without differences between non-smokers, smokers with and without COPD. There was an additional inhibitory effect of DEX (10(-12) M) on VEGF-release when PD 098059 was added. The basal and TNFalpha-induced acetylation status of the VEGF-promoter (chromatin immunoprecipitation [ChIP] assay) was increased in HASMC from smokers with COPD compared with smokers without airflow limitation and non-smokers. In comparison to non-stimulated HASMC, TNFalpha decreased the acetylation status of the VEGF-promoter by approximately 46% and approximately 43% in HASMC from non-smokers and smokers without COPD compared with approximately 68% in HASMC from smokers with COPD. The data suggest that HASMC express VEGF in response to TNFalpha and that this may be reduced in HASMC of smokers with COPD in a smoking-independent manner. VEGF expression is directly modulated by phosphorylation of ERK-1,-2 and p38MAPK and by histone acetylation and the acetylation status of the VEGF gene is increased in HASMC of smokers with COPD in a smoking-independent manner. TNFalpha reduced the acetylation status of the VEGF promoter in HASMC.
The transcription factor T-box-expressed-in-T-cells (T-bet) is required for T(H)1 lymphocyte differentiation, regulates the IL-12-induced expression of the T(H)1-specific cytokine IFNgamma and may be dysregulated in asthmatics. The modulatory role of extracellular signal-regulated kinase (ERK)-1/-2, p38mitogen-activated protein kinase (MAPK) and dexamethasone on IL-12 induced T-bet and IFNgamma expression was assessed in peripheral blood lymphocytes of 10 atopic asthmatics and 10 nonatopic normals. IFNgamma production was dependent on phosphorylation of ERK-1/-2 and p38MAPK, as examined by PD098059, an inhibitor of the upstream activator of MAPKkinase (MKK-1), and SB203580, an inhibitor of p38MAPK. The inhibitory effect of PD098059 on IFNgamma release was decreased in asthmatic T-cells compared with normals. The IL-12-induced T-bet expression and the inhibitory effect of SB203580 were increased in asthmatic T-cells compared with normals. Dexamethasone blocked the IL-12-induced T-bet expression in asthmatic T-cells completely and decreased IL-12-induced IFNgamma release by approximately 50%, which occurred to the same extent in asthmatic and normal T-cells. In conclusion, (1) p38MAPK-pathway rather than ERK-pathway may play a more basic role in the regulation of the increased T-bet expression in asthma, and (2) ERK- and p38MAPK-activation modulate IFNgamma expression independently of T-bet and this regulatory role of ERK-1/-2 on IFNgamma release is impaired in asthma. The therapeutic benefit of dexamethasone on T-bet and IFNgamma production seems to be critical.
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