0.05), which were lower than that with manual compression. The failure rates were 8.3% and 9.2% respectively, but the vascular complications were less than 1%. Hematoma and femoral artery infection were seen in the PCI group, but happened less compared with manual compression. The removing of the arterial sheaths will not be limited by the anticoagulation and antiplatelet therapies, and it could be performed immediately after CAG and PCI, making hemostasis more easy to archeive. It could also reduce the burden of the medical staff, and be accepted by the patients. Conclusion Femoral arterial closure following PCI using regular and intensive anticoagulation and antiplatelet therapies could be safe and effective with vascular complication rates similar to or lower than with manual pressure.

Balb/c mice were immunized by intraperitoneal iniection of group A erythrocytes. Immunized spleen cells were fused with Sp2/0 murine myeloma cells from the solid tumour. After 10～15 days of incubation at 37℃ CO_2, the supernatants were screened for the agglutinabal antibodies. Three hybridoma cell lines secreting high titer and specific monoclonal anti-A antibodies were established.These hybridoma cells have the ability of constant seceting antibodies which belong to the IgM class. The specificity monocloal anti-A sera was the same as the human anti-A sera.

Objective: To investigate whether FasL expression by exogenous FasL gene transfer had regulatory effect on drug sensitivity of tumor cells. Methods: Exogenous Fasl, gene transfer was mediated by lipofectAMINE. The FasL expression was detected bv Immunohistochemistry and RT-PCR. The growth inhibition of cytotoxic drugs on tumor cells was measured by MTT assay. Fas mRNA expression was analyzed bv Semi-quantitative RT-PCR. Results: The eukaryotic expression vector, PcDNA3-FasL and PcDNA3 were successfully transteeted into MCF-7 cells. The transfectant cells was named as MCF-7/FL or MCF-7/Vt respectively. FasL was expressed on MCF-7/FL but not on MCF-7/Vt or MCF-7. The cytolysis ot MCF-7/FL induced by adriamycin at concentration of 0.625 ~ 2.5 ?g/ml, cisplatin of 1 .25 ~ 10 ?g/ml, or methotrexate of 3 ~ 6 ?g/ml respectively were all significantly enhanced compared with the cytolysis of MCF-7. Blocking FasL by using F(ab' )2-anti APO-1 antibody markedly reduced this up-regulated cytolysis induced by these drugs The significant up-regulation of Fas mRNA on MCF-7/FL was observed by Semi-quantitative RT-PCR after these drugs treatment. Conclusion: These findings indicated that FasL expression by gene transfer had up-regulation effect on drug sensitivity of tumor cells, this effect was associated with enhanced Fas expression induced by cytotoxic drugs.

The expression of microRNA (miRNA) is closely related to radio-chemosensitivity in glioma stem cells (GSCs).Moreover,the growth of glioma stem cells could be inhibited comprehensively by increasing radio-chemosensitivity and apoptosis,simultaneously with the regulation of a single miRNA,which has been confirmed by some researches.Thereby microRNA is prospective for the adoption as a specific agent in targeted therapy of glioma,so as to increase the radio-chemosensitivity in glioma stem cells.

Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.

This article introduces a new educational model-Ability Standard Teaching Mode,which emphasizes that students should participate in teaching and promotes the students to change their thinking style from memory and imitation style to thought and creativity style.

Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

In order to study the role of cofactor engineering in enhancing the production of S-adenosylmethionine (SAM), we altered the form and concentration of cofactor in Saccharomyces cerevisiae through gene recombination. Effects of cofactor on product synthesis, carbon and energy metabolism were analyzed aiming to provide a theoretical basis for a successful metabolic engineering of SAM producing strains. Because NADPH metabolism in mitochondrion and cytoplasm of S. cerevisiae is relatively independent, the effect of intracellular NADPH availability on the production of SAM was studied in different compartments of S. cerevisiae BY4741. The expression of NADH kinase in mitochondria (POS5 encoded) and cytoplasm (POS5Δ17 encoded) was separately confirmed using a laser scanning confocal microscope. NADPH regulation strategy enhanced SAM production. Compared with the control strain, the intracellular SAM concentration of strain NBYSM-1 was increased by 3.28 times, and the intracellular SAM concentration of strain NBYSM-2 was increased by 1.79 times at 24 h fermentation. In addition, SAM titer and NADPH/NADP⁺ ratio in strain NBYSM-1 were significantly higher than that of strain NBYSM-2. Therefore, NADPH regulation strategy will be a valuable tool for SAM production and could further improve the synthesis of a large range of cofactor-driven chemicals.

BACKGROUND:Little data have been available concerning the mechanism of drag resistance and anti-apoptosis in leukemic cells of leukemia children.The majority of studies focus on normal bone marrow mesenchymal stem cells (MSCs) and established stroma cells,but not interaction of MSCs and leukemic cells in leukemia children.OBJECTIVE:To explore the effect of MSCs in leukemia children on the proliferation and apoptosis of leukemic cell strain K562/AO_2.DESIGN,TIME AND SETTING:In vitro cytology experiment was performed at the laboratory of Department of Pediatrics,First Affiliated Hospital of Guangzhou Medical College from December 2007 to August 2008.MATERIALS:MSCs were provided by 30 leukemia children admitted to First Affiliated Hospital of Guangzhou Medical College,including 22 acute lymphoblastic leukemia and 8 acute myeloblastic leukemia.Written informed content was obtained from all families.K562/AO_2 was provided by Tianjin Institute of Hematopathy.METHODS:MSCs were isolated and cultured by Ficoll density gradient method.They were cultured in two conditions:the co-culture of MSCs and K562/AO_2 and K562/AO_2 suspension alone.In co-culture group,1×10~8 /L K562/AO_2 cells at log phase were added to confluent MSCs,and free floating K562/AO_2 cells were discarded after 24 hours.MAIN OUTCOME MEASURES:Effect of MSCs on the growth of K562/AO_2 cells was observed;effect of addamycln on K562/AO_2 cell apoptosis was detected by AnnexinV-FITC method.Cell cycle was determined by flow cytomtry,mdrl gene of K562/AO_2 cell was detected by Taqman-MGB probe real-time PCR.RESULTS:Compared with K562/AO_2 alone,the K562/AO_2 cell co-cultured with MSCs grew slower and the log phase of growth was not significant;the rate of apoptosis in earlier period was significantly decreased (P ＜ 0.05);co-cultured K562/AO_2 G_0-G_1 phase increased,but S phase decreased.No changes in mdr1 gene in cells were found between two culture conditions (P ＞ 0.05).CONCLUSION:In vitro cytology has demonstrated that leukemia children MSCs induce drug resistance of K562/AO_2 cells by changing K562/AO2 cell cycle through adhesion to avoid pro-apoptotic effect of drugs but not related with mdr1 gene.

TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.