Role of acetylated p53 in regulating the expression of map2 in retinoic acid-induced P19 cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2 (MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid (RA).</p><p><b>METHODS</b>Neuronal differentiation of P19 cells was initiated with 4-day RA treatment. Immunofluorescence, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells. Real-time PCR-based chromatin immunoprecipitation assay (ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter.</p><p><b>RESULTS</b>The expression of MAP2 was markedly increased in RA-induced P19 cells. The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment, compared with the cells without RA treatment (control). p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity. p300/CBP associated factor (PCAF) was found induced in RA-treated cells and enriched in the nucleus, which might contribute to the acetylation of p53 in the regulation of map2 gene.</p><p><b>CONCLUSIONS</b>Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells. PCAF is possibly involved in this process by mediating the acetylation of p53.</p>

Histone lysine methyltransferase Setd7 enhances Ngn 1 gene expression / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct the eukaryotic expression plasmid of mouse histone lysine methyltransferase Setd7 and detect its effect on neuron development.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Histone lysine methyltransferase Setd7 can enhance neuron differentiation marker gene Ngn 1 transcription.</p>

Role of Ezh2 in the all-trans retinoic acid induced P19 neural differentiation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of Ezh2 in the all-trans retinoic acid RA induced P19 neural differentiation.</p><p><b>METHODS</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Inhibitory role of SirT7 in the growth of P19 cell line / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the regulatory role of SirT7, one of class histone deacetylases, on the proliferation of mouse embryonal carcinoma cell line P19.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>SirT7 play a dominant role in the grow inhibition of the P19 cells.</p>

Heat shock induced the expression of major histocompatibility complex class transactivator and human leukocyte antigen-DR in Jurkat cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Effect of PIH1D1 on the degradation of its binding protein SNF5 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effects of PIH1D1 on its binding protein SNF5, a core subunit of the SWI/SNF chromatin remodeling complex.</p><p><b>METHOD</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>PIH1D1 may stabilize SNF5 by attenuating its proteasome degradation pathway.</p>

Regulatory effect of GAGA element-related protein on the Drosophila GAGA-dependent promoter activity in Jurkat Cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the regulatory effect of the GAGA element-related protein (GRP) on the Drosophila GAGA-dependent promoter activity in Jurkat cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

A signal generator using standard MIT-BIH arrhythmia database / 中国医疗器械杂志

This paper introduces the design of a device which combines the waves with notes from standard MIT-BIH Arrhythmia Database to generate a signal source. The device displays both waves and notes on LCD while the waves are being output, making things convenient for testing and observation. In addition, this generator includes some necessary test signals stated in our new National Industry Standard on Medical Instrumentation as well.

Expression of P-gp, GST-pi and Topo II alpha in gastric and colorectal cancers and their clinical significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the expression and clinical significance of P-gp, GST-pi and Topo II alpha in gastric and colorectal cancers.</p><p><b>METHODS</b>The expression of P-gp, GST-pi and Topo II alpha in 83 cases with gastric or colorectal cancer were examined by immunohistochemistry S-P.</p><p><b>RESULTS</b>The positive expression rates of P-gp, GST-pi, Topo II alpha in normal tissue and gastric and colorectal cancers were 69.9%, 65.1%, 50.6% and 83.1%, 85.5%, 45.8%, respectively. The positive rates of P-gp and GST-pi in gastric and colorectal cancer were significantly higher than those in normal gastric and colorectal tissue (P < 0.05). The expression of Topo II alpha in poorly differentiated cancers was significantly higher than that in well-and moderately differentiated cancers. There was no correlation between other items and clinicopathological parameters (P > 0.05).</p><p><b>CONCLUSION</b>P-gp, GST-pi and Topo II alpha play important role in multidrug resistance. Their mechanisms of drug resistance were different. The detection of expression of P-gp, GST-pi and Topo II alpha has an important guiding significance in chemotherapy for gastric and colorectal cancers.</p>

Enhanced action of a BTB/POZ domain protein on the expression of hsp90alpha gene in heat shock / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of a BTB/POZ domain protein in the expression of hsp90alpha gene.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>GRP markly increased the relative promoter activity of hsp90alpha-CAT reporter gene during heat shock.</p><p><b>CONCLUSION</b>GRP may promote the expression of hsp90alpha gene by participating in chromatin remolding.</p>

Screening, cloning, and analyzing for hSNF5 binding proteins in human fetal brain / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify novel binding proteins of hSNF5, a subunit of chromatin remodeling complex in human fetal brain.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Heat shock induced transcription of hsp90alpha gene on chromatin template / 中国医学科学院学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>A competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.</p>

Chromatin immunoprecipitation and its preliminary application for gene regulation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.</p><p><b>METHODS</b>Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.</p>

Heat shock activated Rac-MEKK-JNK pathway and hsp90 beta gene expression / 中国医学科学院学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Cloning cDNA encoding for GAGA-like element binding proteins in human Jurkat cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore GAGA-like element binding protein in human cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>9 positive clones were obtained, and 6 out of the 9 clones were positively hybridized with the cDNA probe.</p><p><b>CONCLUSIONS</b>6 candidate clones encoding for GAGA-like element binding proteins were obtained from Jurkat cells for further investigation.</p>

The role of p53 binding site on the trans binding of p53 to Hsp90 beta gene / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>The core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.</p>

Establishment and application of a RT-PCR system for quantifying mRNA of 4 hsp genes in human cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>In our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.</p>