ABSTRACT
<p><b>OBJECTIVE</b>To establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides.</p><p><b>METHODS</b>GILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System.</p><p><b>RESULTS</b>One lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load.</p><p><b>CONCLUSION</b>This infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.</p>
Subject(s)
Humans , Binding Sites , DNA , Chemistry , DNA-Binding Proteins , Chemistry , Metabolism , Electrophoretic Mobility Shift Assay , Methods , Fluorescent Dyes , Chemistry , Gene Expression Regulation , Infrared Rays , Protein Binding , Protein Interaction Domains and Motifs , Physiology , Proteins , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway.</p><p><b>METHODS</b>Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points.</p><p><b>RESULTS</b>The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01).</p><p><b>CONCLUSION</b>CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s</p>
Subject(s)
Humans , Apoptosis , Cycloheximide , Pharmacology , HL-60 Cells , Membrane Potentials , Mitochondria , Physiology , p38 Mitogen-Activated Protein Kinases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.</p><p><b>METHODS</b>The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.</p><p><b>RESULTS</b>The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.</p><p><b>CONCLUSIONS</b>Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.</p>
Subject(s)
Animals , Mice , Gene Rearrangement, T-Lymphocyte , Lymphocyte Count , Mice, Inbred BALB C , Mice, Inbred C57BL , Polymerase Chain Reaction , Receptors, Antigen, T-Cell , Genetics , Metabolism , T-Lymphocytes , Cell Biology , Thymus Gland , Cell Biology , Allergy and ImmunologyABSTRACT
Aim To study on the mitochondrial mass change in mouse thymocyte apoptosis.Method Control and Dexamethasone(DEX) groups were set;at 6h,we studied mitochondrial mass changes by NAO and Mitotracker Green(MG) staining flowcytometry and detected mitochondrial membrane potential change with DiOC_6(3) staining flowcytometry.We also used Annexin V-PE/MG double staining flocytometry to examine the mitochondrial changes in apoptosis progress.Results NAO staining results showed that 1 ?mol?L~(-1) DEX stimulation reduced the cardiolipin content of thymocyte mitochondria(P