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Aim: To make clear the repertoire of Killer immunoglobulin-like receptor at the level of DNA and RNA in NK-92MI and K562 cells and compare KIRs gene genotype and expression patterns in both cells., Methods: KIRs gene genotype, include KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, was analyzed by PCR. KIRs gene phenotype, include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR2DS1, KIR2DS3, KIR2DS2/4 gene, was determined by RT-PCR and sequencing., Results: KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DS2, 2DS4*003-006, 2DS5, 3DL1, 3DL2 and 3DL3 gene were present in K562 cells, but all corresponding receptors were not expressed by K562 cells. KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*001/002, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in NK-92MI cells, but only transcripts of KIR2DL1, KIR2DL3 and KIR2DS2/4 were detectable., Conclusion: KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in both K562 and NK-92MI cells. All corresponding KIR receptors were not expressed by K562 cells and only partial KIRs by NK-92MI cells. The repertoire of KIRs were specific for K562 and NK-92MI cells.

Objective: To study the effects of high mobile group box-1 protein (HMGB1) and lipopolysaccharide (LPS) singly or in combination on release of cytokines from human liver carcinoma cell line (HepG2)., Methods: HepG2 cells were cultured, and purified HMGB1 protein was prepared by chromatography on Ni(2+)-NTA Sepharose column under natural conditions with recombinant expression plasmid pET14b-HMGB1. Different concentrations of HMGB1 (0, 0.01, 0.1, 1, 10 mg/L) and LPS (0, 0.1, 1, 10, 100 mg/L) were added into the cultured cells for 24 hours, respectively. Then the supernatant were collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 by using LiquiChip system. Lastly, HepG2 cells were co-stimulated with 10 mg/L LPS and 1 mg/L HMGB1 for 24 hours. The supernatant were collected to determine the levels of above ten of cytokines ., Results: The expression and release of IL-6 and IL-8 increased from HepG2 cells after being stimulated by LPS in a dose-dependent manner, but there were no changes in other eight kinds of cytokines (P<0.05 or P<0.01). Low concentration of HMGB1 could up-regulate the expression of IL-6 and IL-8 in HepG2 cells (both P<0.01). But the extent of induction decreased with higher concentration of HMGB1. Similar to LPS, there was no effect of HMGB1 on the expression of other eight kinds of cytokines from cultured HepG2 cells. Furthermore, high concentration of HMGB1 could obviously inhibit the upregulation of IL-6 and IL-8 by high concentration of LPS when the HepG2 cells were co-stimulated with LPS and HMGB1 (both P<0.01)., Conclusion: HepG2 cells could only express and release a few kinds of cytokines when the cells were stimulated with pro-inflammatory agents, such as LPS or HMGB1. Two kinds of cytokines, IL-6 and IL-8 could be up-regulated by LPS and low concentration of HMGB1, and HMGB1 acted as an inhibitor of LPS to down-regulate the expression and release of IL-6 and IL-8 from HepG2 cells.

Objective: To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release., Methods: HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation., Results: The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng

Objective: To explore ZO-1 gene expression and methylation in leukemia cells and the involvement of ZO-1 gene in leukemogenesis., Methods: Restriction landmark genomic scanning (RLGS) was used to identify new leukemia related gene, and methylation specific PCR (MSP) for ZO-1 methylation status. ZO-1 specific siRNA was designed and prepared by in vitro transcription and transfected into K562 cells, the transfected cells were cultured for 48 hours before harvesting. The effect of ZO-1 siRNA was monitored by Northern blot. Cellular proliferation capacity was assayed by CCK-8, cell apoptosis by Annexin V-fluorescence in isothiocyanate (FITC) assay, and cell cycle by phosphatidylinositol (PI)., Results: The intensified spots in RLGS gel were subjected to bioinformatics analysis and one of the candidate spots was proved to be ZO-1 gene. In fresh leukemia cells, Molt4 cells and HL-60 cells, ZO-1 was hypermethylated, causing it reduced or silenced. ZO-1 gene was highly expressed with no methylation in normal peripheral blood MNC and K562 cells. There was a good correlation between promoter methylation and the gene silence. The silenced gene can be re-activated by demethylation treatment with 5-AZA-dC in most leukemia cell lines. RNA interference for ZO-1 gene in K562 cells did not interfere with cell proliferation, cell cycle and apoptosis., Conclusion: ZO-1 gene methylation might be involved in the tumorigenesis of acute leukemia.

This study was aimed to obtain higher efficiency in gene transfection into K562 cells and to study the role of green fluorescence protein (GFP) as a reporter system. Transfection efficiencies with different methods including electroporation and lipofectamine 2000 transfection, were observed and calculated under fluorescent microscopy by using GFP as a reporter system. The results showed that the transfection efficiency with electroporation (10%) was higher than that with lipofectamine 2000 (1%). In conclusion, the electroporation is a more ideal method for introduction of foreign gene into K562 cells. GFP can be used as a reporter system for optimizing transfection of K562 cells.

Objective: To establish and evaluate the standardized protocol of multiple loci variable numbers tandem repeats (VNTR) analysis (MLVA) for genotyping Mycobacterium tuberculosis (M. tuberculosis)., Methods: 15 VNTR loci were chosen for genotyping 54 Chinese M. tuberculosis strains by PCR-electrophoresis-based VNTR analysis and the results were analyzed by software BioNumerics (Version 5.0)., Results: MLVA method was successfully established and standardized, including the standard protocol for bacterial culture, DNA isolation,PCR and agarose gel electrophoresis and the software analysis. 15 VNTR loci were confirmed, including ETRA, ETRB, ETRC, ETRD, ETRE, MIRU10, MIRU16, MIRU23, MIRU26, MIRU27, MIRU39, MIRU40, Mtub21, Mtub30 and Mtub39, to be suitable for MLVA analysis of M. tuberculosis., Conclusion: The standardized MLVA method has been established successfully. This method is simple and has powerful capacity for genotyping and strain differentiation, can be used for the network surveillance on pathogens of M. tuberculosis, and the data are comparable between laboratories. It is valuable for tracing the source and studying the trend of prevalence during investigation of M. tuberculosis infections.

Objective: To develop a standardized IS6110-restriction fragment length polymorphism (RFLP) method, used for evaluating the capacity of genotyping., Methods: IS6110-RFLP of 78 Mycobacterium (M.) tuberculosis strains were studied by bio-molecular techniques including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis, together with data analysis by software Gel-Pro analyzer 3.1 and BioNumerics (Version 5.0)., Results: IS6110-RFLP method was established and standardized successfully, including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis and usage of the analysis software with standard parameters. By this method, 78 M. tuberculosis isolates were classified into 75 genotypes which belonged to 11 different clusters. Of all the isolates, 66.7% (52/78) belonged to a main cluster., Conclusion: Standard IS6110-RFLP method was established successfully. This method had powerful capacity for genotyping and strain level identification and could be used for the surveillance on pathogens of M. tuberculosis in China.

Aim: To prepare monoclonal antibodies against HBsAg and estimate its binding capacity to both wild-type and varies of immune escape mutant-type HBsAg., Methods: Using self-made the anti-HBs G6 mAb and HRP-labeled goat anti-HBs antibody, A sandwich ELISA for detection both wild-type and varies of immune escape mutant-type HBsAg has been developed. Applying this assay, to detect 17 species of whole gene wild-type and recombination expressed HBsAg which varied in "a" determinant. to evaluate its clinical application characteristic of this assay, we used the other commercial reagents to Comparing with it., Results: One strain hybridoma cell lin secreted anti-HBs mAb (defined G6), was obtained. It grew stably and the titers of the culture supernatant and hydroperitoneum were 2 048 and 4 096x10(3). The sensitivity to the wild-type HBsAg of this assay was less than 0.125 microg/L. This anti-HBs mAb can bind with 12 in 15 species mutant HBsAg (P/N> or =2.5), and 2 of them were low absorbent value at 450 nm(A(450)) group which was 7.55 percent of the wild-type, 1 of them was middle A(450) value group which was 59.40 percent of the wild-type, 9 of them were high A(450) value group which was 92.1-109.4 percent of the wild-type. The low A(450) value group and the negative group were diversified in 120-124 basyl in I loop of the HBV "a" determinant. We also used other commercial reagents, which was widely used in clinic, to detect partial species mutant HBsAg, the average A(450) value was less than foregoing assay., Conclusion: The G6 anti-HBs mAb was able to bind most of the immune escape mutant HBsAg in the test. And there would be some special regulations between the mutant site and the recombination expressed HBsAg binding capability.

Objective: To observe the expression and localization of endogenous C-reactive protein (CRP) in cells from different tissues under different conditions., Methods: Macrophages differentiated from THP-1 monocytes with phorbol ester (PMA) induction and human LO2 hepatocytes were stimulated with lipopolysaccharide (LPS). The culture supernatant of the LPS-stimulated THP-1 cells was collected and added into LO2 cell culture, and after incubation, the cells were lysed to extract the proteins for SDS-PAGE and Western blotting. The stimulated cells were also examined immunocytochemically for CRP expression., Results: Western blotting detected CRP in both of the unstimulated cell lysates, but in neither of the two cell supernatants. After LPS stimulation, CRP expression was significantly increased in the cell lysate of THP-1 cells with also a small amount present in the supernatant, but CRP expression and release in the LO2 cells showed no significant variation. Treatment of the LO2 cells with the culture supernatant of LPS-stimulated THP-1 cells resulted in positivity of CRP in the cell lysate and the culture supernatant. Immunocytochemistry identified CRP expression throughout the THP-1 cell body (most obvious in the nuclei), which increased after LPS stimulation. In LO2 hepatocytes, CRP expression was found only outside the nuclei and increased after stimulation with the culture supernatant of LPS-treated THP-1 cells, especially obvious around the membrane., Conclusion: CRP can not be up-regulated directly by LPS treatment in LO2 cells, but can be induced by certain cytokines (IL-6) secreted from LPS-stimulated THP-1 cells. The localization of CRP represents the characteristics of secreted protein in LO2 cells, but in THP-1 cells, CRP is found mainly in the cell nuclei.

The total proteins of Leymus chinensis were extracted from eight-week-old seedlings grown in the greenhouse by TCA-acetone precipitation method. After the lysis buffer was replaced by the start buffer, proteins were fractionated along the first dimension using chromatofocusing (CF). Subsequently the fractions with pI values between 8.5 and 4.0 collected after the first dimension separation were further fractionated by nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC, HPRP). With ProteoVue software the pI/UV map was generated to show the protein expression profile of Leymus chinensis. Some experiments were tested to optimize the fraction procedure. Three different elution gradients were employed to get the optimal chromatogram. Comparison of the protein expression pattern between 2D-PAGE and 2D-LC indicated that 2D-LC was a powerful tool in protein fraction. Reproducibility and veracity of the protein patterns were confirmed in different injections of the same sample. A method to fractionate the total protein of Leymus chinensis shoot with two-dimensional liquid chromatography (2D-LC) was founded.

Objective: To elucidate the characters of rpoB mutation in rifampin-resistant clinical isolates of Mycobacterium tuberculosis., Methods: 286 bp DNA fragment of rpoB gene including 81 bp code region (rifampin resistance deteremination region, RRDR) was analyzed by PCR-single-strand conformation polymorphism(SSCP). The 286 bp DNA fragment of each strain which had been proved to have mutation by PCR-SSCP was then sequenced. 110 strains of M. tuberculosis, including 73 rifampin-resistant strains, 11 rifampin-susceptible drug-resistant strains and 26 drug-susceptible strains were studied., Results: 47 rifampin-resistant strains were detected to have mutations by PCR-SSCP method. 76.6% rifampin-resistant strains showed that rpoB gene was carrying single point mutation analyzed by direct sequencing technique, which mainly located at 531-Ser (61.1%) and 526-His (25.0%). The combined mutation rate was 23.4%. In addition, 2 rifampin-susceptible drug-resistant strains and 1 drug-susceptible strain were mutated, detected by PCR-SSCP method. Sequencing results showed that the mutations located at 511-Leu, 526-His and 535-Pro., Conclusion: Mutations in the 81 bp RRDR of rpoB were the main reasons of M. tuberculosis resistant to rifampin. 531-Ser and 526-His were the most common positions of mutations.

Objective: To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction., Methods: The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting., Results: Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent., Conclusion: The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.

Objective: To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology., Methods: The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method., Results: The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2., Conclusion: This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.

Objective: Variable Number of Tandem Repeats (VNTRs) analysis was a recently developed method which could serve as a 'real-time' genotyping tool for Mycobacterium tuberculosis. One hundred and thirteen M. tuberculosis isolates from the patients with tuberculosis in Beijing were analysed using the reference method to study the characters of genetic diversity and genotype., Methods: Thirteen tandem repeat loci (ETR-A, ETR-C, ETR-D, MIRU10, MIRU16, MIRU27, MIRU31, MIRU40, Mtub21, Mtub30, Mtub38, Qublla, Qubllb) in the total genome of MTB were analyzed by PCR and agarose gel electrophoresis method. The characters of the polymorphism of DNA fingerprinting of one hundred and thirteen MTB strains were analyzed with Gel-Pro analyzer 3.1 software and BioNumerics 3.0 software. Results One hundred and thirteen MTB strains were characterized and classified in to four genotype families(type I , type II , type NV, type V ) based on thirteen tandem repeat loci. One hundred and four isolates(92.0%) belonged to type I , the other three genotypes scattered, five strains(4.4%) remaining with type II , while type IV and type V having the same quantity 1.8% (2/113). M. tuberculosis H37Rv belonged to a unattached genotype(type ll ). Conclusion There was obvious length polymorphism in the M. tuberculosis isolates which implied that type I was the epidemic strain clusters in M. tuberculosis in Beijing. VNTRs analysis seemed to be a simple, rapid, sensitive and valuable tool for epidemiological studies of M. tuberculosis complex organisms.

Objective: To establish human integrin-alpha v adenovirus expressed in human liver cancer SMMC-7721 cells and analyze the characteristics of integrin-alpha adhesion to liver cancer cells., Methods: Human integrin-alpha v cDNA was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack-integrin-alpha v was cotransformed with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK-293A cells to package and amplify recombinant adenovirus particles. Western blot showed that integrin-alpha v gene was exactly transcript and expressed in SMMC-7721 cells. Twenty four hours after transfection, the effect of integrin-alpha on adhesion of SMMC-7721 cells was detected by adhesion experiment., Results: Recombinant adenovirus vector of integrin-alpha v gene was successfully constructed and high titers of recombinant adenovirus was obtained. Adhesion cell count was significantly higher in integrin-alpha v transfected cells than in untransfected and Ad-null-transfected cells (P<0.01)., Conclusion: Over expression of integrin-alpha v promotes adhesion of SMMC-7721 cells, is an important molecular mechanism of tumor metastasis.

Objective: To observe the effects of high mobility group box 1 (HMGB1) protein on the release of cytokines of human umbilic vein endothelial cells (HUVECs) and the expression of interleukin-6 (IL-6) induced by lipopolysaccharide (LPS)., Methods: HUVECs were cultured and then divided into the following groups. (1) Recombinant HMGB1 protein of the terminal concentration of 15 ng/ml was added into the culture fluid of HUVECs, 24 hours later the supernatant was collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and interleukin-10, IL-12, IL-1beta, IL-2, IL-4, IL-6, and IL-8, by using LiquiChip system. (2) HMGB1 protein of the terminal concentrations of 3, 15, or 75 ng/ml was added into the culture fluid of the HUVECs, 24 hours later the supernatants were collected to detect the level of IL-6. (3) HMGB1 protein of the terminal concentration of 15 ng/ml was added into the culture fluid of HUVECs, 1, 3, 6, 12, and 24 hours after the stimulation the supernatant was collected to detect the level of IL-6. (4) The culture fluid of HUVECs was added with HMGB1 protein of the terminal concentration of 15 ng/ml and/or LPS of the concentration of 10 ng/ml, 24 hours later the supernatant was collected to detect the level of IL-6. Culture fluid not added with HMGB1 protein was used as control in any kind of test., Results: (1) The levels of GM-CSF, IFN-gamma, IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) secreted by the HMGB1 stimulated HUVECs were 5, 7, 4.2, 27.8, 12.8, and 5.4 times that of those control group (all P < 0.01) (2) The levels of IL-6 secreted by the HUVECs stimulated with HMGB1 protein of the concentrations of 3, 15 and 75 ng/ml respectively were 155 pg/ml +/- 33 pg/ml, 901 pg/ml +/- 184 pg/ml, and 1508 pg/ml +/- 379 pg/ml respectively, all significantly higher than that of the control group (32 pg/ml +/- 21 pg/ml, all P < 0.01). (3) Since 3 h after the stimulation of HMGB1 protein the level of IL-6 began to increase gradually. 6, 12, and 24 hours after the stimulation the levels of IL-6 secreted by the HUVEC were 75 pg/ml +/- 22 pg/ml, 453 pg/ml +/- 78 pg/ml, and 901 pg/ml +/- 184 pg/ml respectively, all significantly higher than that of the control group (32 pg/ml +/- 21 pg/ml, all P < 0.01). (4) When the HUVECs were stimulated individually by LPS (10 ng/ml) or HMGB1 (15 ng/ml), the levels of IL-6 increased to 289 pg/ml +/- 42 pg/ml and 901 pg/ml +/- 183.6 pg/ml respectively (both P < 0.01); when the HUVECs were costimulated by LPS and HMGB1, the level of IL-6 increased to 2360 pg/ml +/- 299 pg/ml, which showing that there was a synergistic effect between HMGB and LPS (F = 69.405, P < 0.001)., Conclusion: HMGB1 activates HUVECs to produce multiple inflammatory cytokines and induces HUVEC to secret IL-6 in a dose- and time-dependent manner. HMGB1 can also act synergistically with LPS in inducing IL-6 release, which may play an important role in the development of sepsis.

Context: Estrogen-related receptor beta (ERRbeta) was one of the first two orphan nuclear receptors reported and is believed to play important roles in estrogen-regulated pathways. Embryo lethality of ERRbeta-null mice indicated that ERRbeta is essential for embryo development., Objective: Two novel splicing isoforms of human (h) ERRbeta, hERRbeta2-Delta10 and short-form hERRbeta, were identified during the cloning of previously reported hERRbeta-hERRbeta2. We aim to investigate the functional differences of these three human ERRbeta-splicing isoforms., Results and Conclusions: A genomic sequence comparison within and flanking the ERRbeta genes of eight species demonstrated that short-form hERRbeta lacks an F domain and is the matched homolog of mouse and rat ERRbeta proteins in humans. However, hERRbeta2-Delta10 and the previously reported hERRbeta2 isoforms are primate specific. RT-PCR analysis showed that short-form hERRbeta has a wide distribution in the 24 of 27 human tissues and cell lines tested, whereas hERRbeta2 and hERRbeta2-Delta10 were only expressed in testis and kidney. The three human ERRbeta-splicing isoforms have different transcriptional activities when measured on an estrogen response element-driven luciferase reporter in transfection assays. The localization of a nuclear localization signal of short-form hERRbeta was also determined. Interestingly, the F domain of hERRbeta2 alters the function of the nuclear localization signal. Therefore, the ERRbeta isoforms are likely to have diverse biological functions in vivo, and characterizing the three isoforms of ERRbeta will lead to an understanding of the multiple levels of gene regulation involved in steroid receptor-signaling pathways in humans and may provide novel therapeutic targets for human diseases.

Objective: To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection., Method: The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection., Results: LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously., Conclusion: Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.

To evaluate the feasibility of gene therapy using bcl-2 as target in multiple drug resistance of leukemia, the small interfering RNA eukaryotic expression vector specific to human bcl-2 gene was constructed by gene recombination, then transfected into HL-60/VCR cells. Stable transfectants were obtained by G418 screening. The growth curve and drug sensitivity were detected by using MTT. The expression of Bax and ZNRD1 was analyzed by Western blot. The results showed that mU6pro-bcl-2 siRNA was successfully constructed and transfected into HL-60/VCR cells. The IC(50) of transfected cells to vincristine and adriamycin was significantly reduced as compared with that of the control. The expression of ZNRD1 in transfected cells was decreased as compared with that of the control, while Bax not. It is concluded that the bcl-2 siRNA restores the sensitivity of HL-60/VCR cells to conventional chemotherapeutic agents to a certain degree.

Objective: To construct the eukaryotic expression vector for HPC2 for expression in HEK293 cells., Methods: HPC2 from pcDNA3/HPC2 were inserted into the flag-tagged vector pcDNA3-flag by subcloning method. The recombinant plasmid pcDNA3-flag/HPC2 was then transfected into HEK293 cells using a routine lipofectamine method. The cell lysate was used for Western blotting to examine the expression of the target protein., Results and Conclusion: Double restriction enzyme digestion and DNA sequencing indicated successful construction of the eukaryotic expression vector for HPC2 and the fusion protein was highly expressed in HEK293 cells, which provides an important basis for functional study of HPC2.

Objective: To study the effect of Hongjingtian (Gadol) injection on cardiac hemodynamics and myocardial oxygen consumption for analyzing its underlying mechanism in the treatment of coronary heart disease., Method: A total of 20 dogs anesthetized with pentobarbital sodium (30 mg x kg(-1), i.v.) were evenly randomized into control group, low-dose Gadol (LDG) group, high-dose Gadol (HDG) group and Herbesser Injection group. The blood flow volume (BFV) of the left coronary artery and cardiac output (CO), left ventricular pressure (LVP), maximum ascending rate (dp/dtmax) of LVP, mean arterial pressure (MAP) of the femoral artery, oxygen contents of the coronary artery and coronary vein (venous sinus), oxygen consumption index (OCI), cardiac index (CI), coronary artery resistance (CAR) and total peripheral resistance (TPR) as well as oxygen utilization rate (OUR) were detected respectively., Result: After venous injection of Gadol, CAR, MAP, TPR, OCI, myocardial oxygen consumption and heart rate lowered significantly (P < 0.05-0.01), while BFV and blood oxygen content of the venous sinus increased considerably (P < 0.05-0.01) in comparison with pre-injection. No significant differences were found in LVP and myocardial contractivity between control group and LDG, and between control and HDG groups respectively., Conclusion: It showed dilation of the coronary artery and reduction of the cardiac afterload after injection of Gadol. Besides, CO and stroke volume increased considerably and the cardiac effective work was raised without any significant simultaneous increase of both myocardial contractility and LVP. A decrease in the myocardial oxygen consumption and reduction of OCI indicates an improvement of the oxygen supply of the myocardium, and a favorable regulation of the compliance of the cardiac vessels. As a result, the cardiovascular performance was ameliorated. The abovementioned improvement of these indexes may contribute to the therapeutic effect of Gadol in the treatment of coronary heart disease in clinic.

Objective: To construct a eukaryotic expression vector for His-tagged human soluble receptor for advanced glycation end products (hsRAGE), and study the function and biological activity of the fusion protein., Methods: The coding sequence of hsRAGE was amplified from the recombinant pCI-neo/RAGE by PCR method. The fragment was cloned into pET-14b vector and identified by digestion with restriction enzymes and sequence analysis. After transformation of the vector into E. coli BL21(DE3), the fusion protein was expressed successfully in response to IPTG induction in the form of inclusion body. After extraction from the bactrerial cells and washing, the fusion protein was dissolved in 8 mol/L urea and purified using immobilized metal ion affinity chromatography. Renaturing of the fusion protein occurred after gradually removal of the denaturants by dialysis. The protein was subsequently identified by Western blotting, enzyme-linked immunosorbent assay (ELISA) and blockade experiments in the endothelial cells., Results: The recombinant hsRAGE/pET-14b was verified by enzyme digestion and sequencing. A fusion protein with a molecular weight of about 45 kD was purified, which could interact with anti-RAGE antibody and AGE and block AGE-induced nuclear factor (NF)-kB activation in the endothelial cells., Conclusions: His-hsRAGE fusion protein has been cloned, expressed and purified successfully and identified to possess immunogenicity, binding activity to AGE and blocking effect on AGE-RAGE.

Objective: To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody., Methods: The total RNA was extracted from mouse liver cells, and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was then cloned into the prokaryotic expression vector pET14b. After enzyme digestion and DNA sequencing, the plasmid was transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of His-tagged mtTFA. The expressed fusion protein was purified with Ni(2+)-NTA His-bind resin and quantified with Bradford method., Results: The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing, and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG. The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained., Conclusions: The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained, which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.

Objective: To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli., Methods: ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography., Results: The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained., Conclusion: The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

Objective: To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor for advanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its interacting proteins., Methods: The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted into pGEX-KG vector, a general GST fusion protein expression vector. After PCR identification, endonuclease digestion and DNA sequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced by isopropyl-beta-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin., Results: The recombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing. After protein expression was achieved in E. coli, a molecular mass of 35 kD GST fusion protein was purified, whose molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)., Conclusion: The expression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusion protein is obtained, which can be of value for further studies.

Objective: To screen and identify proteins that interact with p38 mitogen-activated protein (MAP) kinases by means of T7 phage-display screening system., Method: His-tagged fusion protein of p38 MAP kinase was used to coat a 96-well ELISA plate and Ni-NTA resin, which served as the media for screening human lung and liver T7 phage cDNA libraries., Results: After 4 rounds of biopanning, 86 independent plaques were selected and processed by EDTA. The inserted gene fragments from these plaques were amplified by PCR, the products purified by a gel recovery method. The sequences of the insertions were identified and analyzed with BLAST program in GenBank. Forty-six clones were found to encode proteins., Conclusion: T7 phage-display screening system is convenient, rapid and effective for screening the P38 MAP kinase-binding proteins.

Objective: To construct the fluorescence protein vector pairs with toll-like receptor 4 (TLR4) signal peptide, namely pECFP-C1-SP and pEYFP-C1-SP, therefore to make the application of fluorescence resonance energy transfer (FRET) possible in the study of the membrane protein interaction during lipopolysaccharide (LPS) recognition., Methods: pECFP-C1-SP and pEYFP-C1-SP were constructed by means of site-directed mutagenesis, and the constructed plasmids were transiently transfected into NIH3T3 cells via lipofectamin to observe their intracellular expressions under a fluorescence microscope., Results: DNA sequence analysis attested the validity of the constructed fluorescence vectors with signal peptide for FRET, and the expression of the vectors was located principally on the cell membrane as observed under fluorescence microscope., Conclusion: The constructed vectors TLR4 signal peptide are valid and capable of expressing on the cell membrane, therefore they can be effectively used in the study of the interaction between the membrane proteins.

Objective: To construction of vector of his-tagged cytoplasmic fragment of human Toll like receptor 4 (hTLR4) and its expression in E.coli., Methods: hTLR4 cytoplasmic cDNA codon domain was amplified by polymerase chain reaction (PCR) and cloned into pET-DsbA2.0 plasmid expressing His-DsbA fusion protein. After being identified by the assay of restrictional enzyme and sequencing, His-Dsb A fusion proteins were induced with isopropy-beta-D-thiogalactoside (IPTG) and further purified., Results: A fusion protein with molecular weight of 42 kd was obtained., Conclusion: hTLR4 which was constructed and expressed successfully under nondenaturing conditions provides a tool for further studies.