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Objective: To evaluate the clinical value of retrospective electrocardiogram (ECG) -editing technique in dual-source computed tomography (CT) coronary angiography in patients with arrhythmia., Methods: Totally 73 patients with arrhythmia during dual-source CT coronary angiography were included into this study. A retrospective gating technique and ECG-editing technique (Insert Sync; Disable Sync; Delete Sync; Shift R-peak) were used in patients who needed ECG-editing. Two experienced radiologists evaluated in consensus all pre-editing and post-editing reconstructed images and recorded scores according to the American Heart Association guidelines on coronary segmentation on a per segment basis. Image quality of all coronary segments was assessed using a four-point grading scale from excellent (4 scores) to non-assessable (1 score) ., Results: The overall mean image quality of 34 patients who did not need ECG-editing was 3.42 ± 0.20. In 39 patients who needed ECG-editing, the overall mean image quality before and after ECG-editing was 2.39?0.37 and 3.22?0.24. The mean image quality in every segment between pre-editing and post-editing was also significantly different (P<0.01) ., Conclusion: ECG-editing technique can remarkably improve image quality of coronary artery segments by reducing or even eliminating the artifact produced by arrhythmia during dual-source CT coronary angiography.

Objective: To explore the effect of a non-lethal heat shock, in comparison with the treatment of interferon-gamma (IFN gamma), on the expression of major histocompatibility complex transactivator (CTA) and its downstream target gene of the human leukocyte antigens (HLA)-DR in Jurkat cells., Methods: The changes of CTA mRNA in Jurkat cells before and after the treatment of heat shock or IFN gamma were detected using real time RT-PCR. The changes of CTA protein were detected with Western blot. The expression of HLA-DR was detected with flow cytometry. : CTA mRNA and protein were induced in Jurkat cells under heat shock, but not with IFN-gamma. The expression of HLA-DR gene significantly increased after recovery (P<0.01)., Conclusion: The expressions of CTA and HLA-DR in Jurkat cells remarkably increase after heat shock, indicating that heat shock may help reconstruct relevant genes in cells with immunologic gene deficiencies.

Objective: To explore the effects of PIH1D1 on its binding protein SNF5, a core subunit of the SWI/SNF chromatin remodeling complex., Method: The degradation pathway of SNF5 was identified with protein synthesis inhibitor cycloheximide (CHX) and a potent proteasome inhibitor MG132, and then the PIH1D1 eukaryotic expression plasmid was transfected to explore its effect on the stability of SNF5., Results: HEK293T cells were effectively treated with CHX (optimal concentration: 400 microg/ml) and MG132 (optimal concentration: 20 mmol/L). The degradation of SNF5 was mediated by the proteasome pathway. PIH1D1 regulated the protein level of SNF5 by attenuating its proteasome degradation., Conclusion: PIH1D1 may stabilize SNF5 by attenuating its proteasome degradation pathway.

Objective: To study the regulatory role of SirT7, one of class histone deacetylases, on the proliferation of mouse embryonal carcinoma cell line P19., Methods: We used an expression plasmid of SirT7 (151-402 amino acid residues) and its vector respectively to establish a stably expressed SirT7 and its control P19 cell lines. Recombinant DNA techniques, Western blot, cell growth curve, and flow cytometry were used in this paper., Results: Compared with the control cells, the P19 cells had significantly lower growth rate in stably expressed SirT7. G1 to S cell cycle arrests were only seen in the SirT7 over-expressed cell line., Conclusion: SirT7 play a dominant role in the grow inhibition of the P19 cells.

Objective: To study the role of Ezh2 in the all-trans retinoic acid RA induced P19 neural differentiation., Methods: The expression of Ngn1 in the RA induced P19 cells was detected at the mRNA and protein levels using real time RT-PCR and Western blot assays. The binding of Ezh2 and H3K27me3 on the Ngn 1 promoter was analyzed using chromatin immunoprecipitation assay., Result: In the RA induced P19 cells, the recruitment of Ezh2 and its methylated substrate H3K27me3 on the promoter of Ngn 1 gene elevated in the first 2 days, and then declined rapidly, followed by the initiation of neuronal differentiation., Conclusions: Ezh2 produces a repressive histone mark H3K27me3 in the early stage of RA induced P12 cells. By avoiding the premature expression of Ngn1 gene, Ezh2 can ensure the normal differentiation of P19 cells.

Objective: To explore the regulatory effect of the GAGA element-related protein (GRP) on the Drosophila GAGA-dependent promoter activity in Jurkat cells., Methods: Drosophila GAGA (dGAGA) factor (dGAF), GRP, and either the chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the GAGA element-containing promoter of ftz gene or its mutant GAGA control were selectively co-transfected into Jurkat cells. Promoter activity analyses were performed by analyzing the RNA expression of the CAT in a real-time-RT PCR system, with pRC-CMV-betaGal co-transfected with the CAT reporter as a transfection efficiency control. Electrophoretic mobility shift assays (EMSA) were carried out to examine the binding profile of Jurkat nuclear extracts with a biotin labeled probe-containing dGAGA., Results: In Jurkat cells, GRP, either singly or combined with dGAF, elevated the activity of wild type dGAGA-containing ftz promoter dose-dependently in certain range. However, when the level of GRP was excessively high, it reduced or even fully inhibited the promoter activity of the ftz gene. On the contrary, either GRP or dGAF could not activate the ftz promoter with mutant GAGA. EMSA profile showed a specific band composed of the GAGA element and its binding proteins from Jurkat cells., Conclusions: Human GAGA element-binding proteins exist in Jurkat cells. Its may either directly regulate the gene via GAGA elements or mediate the biphasic regulation of relevant gene in a GRP dose-dependent way.

Objective: To construct the eukaryotic expression plasmid of mouse histone lysine methyltransferase Setd7 and detect its effect on neuron development., Methods: The clone of mouse Setd7 was obtained and inserted into the eukaryotic expression vector pCMV-3tag-6-Flag. The plasmid was transfected into HEK 293T and identified by Western blot. Real-time PCR was used to detect the effect of Setd7 on the neuron differentiation marker gene Ngn 1 mRNA expression. Dual luciferase reporter system was used to detect the effect of Setd7 on Ngn 1 mRNA expression. Real-time PCR was used to detect the effect of Setd 7 siRNA plasmid on Ngn 1 mRNA expression., Results: An eukaryotic expression plasmid of Setd 7 was successfully constructed. Setd7 induced Ngn 1 mRNA expression and increased Ngn 1 promoter activity. Also, the knockdown of Setd 7 inhibited Ngn 1 mRNA expression., Conclusion: Histone lysine methyltransferase Setd7 can enhance neuron differentiation marker gene Ngn 1 transcription.

Objective: A CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock., Methods: A competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro., Results: The in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene., Conclusion: The heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.

Objective: To identify novel binding proteins of hSNF5, a subunit of chromatin remodeling complex in human fetal brain., Methods: The yeast two-hybrid system was used for this study. Positive cDNA clones were sequenced. Sequence homology and putative functional domains were analyzed and compared with databank., Results: Nine positive clones obtained were analyzed, among which the sequence of one clone was 97% homologous to the 3' mRNA of a hypothetical protein FLJ20643, while other four clones were related to protein coding sequences existed in the GenBank. The rest four clones were not in frame with any known protein coding sequence., Conclusions: Clones encoding for hSNF5 binding protein exists in cDNA library of human brain. These proteins may recruit chromatin remodeling complex via hSNF5 to modulate the transcription of their target gene and the related cell functions.

Objective: To study the role of a BTB/POZ domain protein in the expression of hsp90alpha gene., Methods: The eukaryotic expression plasmids of sense- and antisense-GAGA related protein (GRP) or empty vector were transfected into Jurkat cells with pREP4 episomal vector plasmids carrying the hsp90alpha promoter sequence from -1756 to +37 and control plasmids pMCAT. Total RNA was extracted. The relative promoter activity of hsp90alpha-CAT reporter gene was determined by competitive RT-PCR assay., Results: GRP markly increased the relative promoter activity of hsp90alpha-CAT reporter gene during heat shock., Conclusion: GRP may promote the expression of hsp90alpha gene by participating in chromatin remolding.

Objective: To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo., Methods: Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein., Results: The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples., Conclusions: p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.

Objective: To explore GAGA-like element binding protein in human cells., Methods: Yeast one-hybrid system was used to screen the GAGA-like element binding proteins in HTLV-1 transformed Jurkat cell cDNA fusion library. Total RNA extracted from Jurkat cells was first labeled by reverse transcription, and was taken as cDNA probe to hybridize with the candidate positive clones., Results: 9 positive clones were obtained, and 6 out of the 9 clones were positively hybridized with the cDNA probe., Conclusions: 6 candidate clones encoding for GAGA-like element binding proteins were obtained from Jurkat cells for further investigation.

Objective: To investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation., Methods: The binding site was first inserted into pBS-SK. After the plasmid annealing and elongation with mutagenic and selective primers, nuclease digestion and bacteria transformation was performed twice to select the positive mutated plasmid. Electrophoretic mobility shift assays (EMSA) was employed to detect the binding of hsp90 beta gene fragment containing mutated p53 binding site and Jurkat cell nuclear extract transfected by p53 expression vector., Results: The sequence analysis profile confirmed a successful mutation of two bases on the core sequence of the second half binding site. EMSA results showed the specific DNA-protein complex band disappeared after the mutation., Conclusions: The core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.

Objective: To establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells., Methods: RT-PCR system was established with gene cloning, gene recombination, in vitro transcription and RT-PCR techniques; The detection of expression level of 4 hsp genes in SW13 cell was carried out with this system., Results: In SW13 cell, hsp70 and hsp90 alpha were typical heat shock induced genes, while hsp60 and hsp90 beta were efficiently expressed and further induced by heat-shock to various extent., Conclusions: In our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.

Objective: To study the effect of Rac-MEKK-JNK (Rac-mitogen activated protein kinase kinase kinase-C-jun N-terminal protein kinase) signal pathway on heat shock-induced hsp90 beta gene expression and the impact of Hsp90 on the regulation of the pathway., Methods: DN-Rac, DN-MEKK or DN-JNK were cotransfected with hsp90 beta CAT reporter plasmid beta 3.1 into Jurkat or LETPa-2 cells individually, the CAT mRNA expression was then determined quantitatively by competitive RT-PCR based system. Western blot was carried out to detect the expression level and phosphorylation of c-Jun in Jurkat and LETPa-2 cells that were transfected with DN-Rac, DN-MEKK or DN-JNK. By in vitro kinase activity assay and Western blot, the effect of geldnamycin (GA) on heat induced JNK activity were evaluated., Results: In Jurkat cell transfected with DN-Rac, DN-MEKK or DN-JNK, heat shock induced relative CAT mRNA expression level was decreased to (72.8 +/- 5)%, (60 +/- 13.2)% and (47.7 +/- 12.1)% of the control respectively; while in LETPa-2 cell hsp90 beta 3.1 reporter gene expression was accordingly suppressed to (16.17 +/- 5.1)%, (50.2 +/- 8.7)% and (47.5 +/- 10)% of control. C-Jun expression and phosphorylation were inhibited by the transfection of either one of DN-Rac, DN-MEKK or DN-JNK. With GA treatment, heat shock induced JNK activity was repressed, while the expression level of JNK or c-Jun was not obviously changed., Conclusions: Rac-MEKK-JNK pathway promotes heat shock induced hsp90 beta gene expression and hsp90 may participate in the regulation of heat shock activated Rac-MEKK-JNK signal pathway in both Jurkat and LETPa-2 cells.

Objective: To investigate the role of STAT1 on the regulation of human hsp90 alpha gene expression., Methods: We first transfected Jurkat cells with the STAT1 expression construct and analyzed the expression of hsp90 alpha gene expression via quantitative RT-PCR system. Then we co-transfected the STAT1 expression construct and the CAT reporter gene driven by different length of 5' flanking sequence of hsp90 alpha gene. Western blot was carried out to detect the level of tyrosine phosphorylation in Jurkat cells with and without heat shock treatment (42 degrees C 1 h). By electrophoretic mobility shift assays (EMSA), we evaluated the DNA binding activity of a STAT1 responsible element located in the regulatory region of hsp90 alpha gene in Jurkat cell nuclear extracts., Results: The mRNA level of hsp90 alpha gene in Jurkat cells was decreased when transfected by STAT1 expression construct, over-expression of STAT1 down-regulates the expression of CAT reporter gene with the present of a distal fragment from -1756 to -1463 within the 5' flanking regulatory sequences of hsp90 alpha gene. The tyrosine phosphorylation of STAT1 was detectable in Jurkat cells and increased when subjected to heat shock. Electrophoretic mobility shift assays (EMSA) results showed that STAT1 could bind to its responsible element in the regulatory region of hsp90 alpha gene., Conclusion: STAT1 could negatively regulate the human hsp90 alpha gene expression.

Objective: To study gene expression changes of cells in response to heat stress, we isolated total RNA from Jurkat cells before and after heat shock treatment., Methods: cDNA was labeled with alpha-32P-dATP during reverse transcription of RNA and then used as probe to cDNA expression array. Autoradiogram images were analyzed by ESTblot software., Results: After heat shock, the expression level of some forty genes increased while that of sixteen genes decreased. In addition to the elevated expression of heat shock genes, expression of c-Jun and CLK-1 increased most remarkably. The genes with notably decreased expression were integrin alpha-4 and transforming growth factor beta. The elevated expression of c-Jun and hsp90 alpha was further confirmed by Northern blot analysis., Conclusions: The expression of some genes in Jurkat cells changes after heat shock treatment. Heat shock induces elevated expression of hsp, c-Jun, CLK-1 gene while decreases expression of integrin alpha-4 and transforming growth factor beta.