OBJECTIVETo observe the expression of phospholipid scramblase 1 (PLSCR1) in matrine (MAT) induced differentiation of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) cells, and to explore its correlation to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal pathway.

METHODSNB4 (an APL cell line sensitive to ATRA) and NB4-R1 (a resistant strain of ATRA) were observed as subjects in this study. Effects of combined treatment of 0.1 mmol/L MAT and 1 [mol/L ATRA on the differentiation of two cell lines were detected using nitroblue tetrazolium (NBT) reduction test and flow cytometry (CD11b). Expressions of PML/RARot and PLSCR1 protein/gene were detected using Western blot and Real-time fluorescence quantitative PCR assay. Meanwhile, H89, PKA antagonist, was used to observe cell differentiation antigen and changes of aforesaid proteins and genes.

RESULTSMAT combined ATRA could significantly elevate positive rates of NBT and CD11 b in NB4-R1 cells, and significantly down-regulate the expression of PML/RARapha-fusion protein/gene (P < 0.05, P < 0.01). ATRA used alone could obviously enhance the expression of PLSCRI in NB4 cells at protein and mRNA levels (P < 0.01). But the expression of PLSCR1 was up-regulated in NB4-R1 cells, but with statistical.difference only at the protein level (P <0. 01). In combination of MAT, PLSCR1 protein expression was further elevated in the two cell lines (P < 0.01). Besides, there was statistical difference in mRNA expressions in NB4-R1 cells (P < 0.05). All these actions could be reversed by treatment of 10 micromol/L H89 (P < 0.05, P < 0.01).

CONCLUSIONMAT combined ATRA could significantly induce the differentiation of NB4-R1 cells, and inhibit the expression of PML/RARalpha fusion gene/protein, which might be associated with up-regulating PLSCR1 expression.

Alkaloids ; Antineoplastic Agents ; Cell Differentiation ; Cell Line, Tumor ; Down-Regulation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Phospholipid Transfer Proteins ; metabolism ; Quinolizines ; RNA, Messenger ; Signal Transduction ; Tretinoin ; Tumor Cells, Cultured ; Up-Regulation

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines. Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells. Then the expression level of Mad2 gene was detected by Western blotting. Flow cytometry revealed that SKOV3 cells were not fully arrested in G(2)/M phase in contrast to A2780 cells in the presence of paclitaxel. However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G(2)/M phase and the expression of Bcl-2 was significantly changed. These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.

Fe3O4-grafted nitrogen-doped graphene (Fe3O4/N-G) nanomaterials were synthesized by chemical co-precipitation method, and its adsorption properties were discussed preliminarily.It was demonstrated that the adsorption of parachlormetaxylenol on Fe3O4/N-G was not limited to uniform monolayer adsorption and the adsorption kinetic followed the pseudo-second-order kinetic mode.Then, an ultrasound-assisted magnetic solid-phase extraction with Fe3O4/N-G as the magnetic adsorbent has been developed for the determination of four compounds including triclosan, chloroxylenol, hexachlorobenzene and 2,2′,4,4′,5,5′-hexachlorobiphenyl in environmental water samples, in combination with gas chromatography coupled to tandem mass spectrometry.Several factors related to extraction efficiencies, such as the amount of adsorbent, extraction time, sample pH and desorption conditions were investigated.The proposed preparation procedure was as follows: 6.0 mg of Fe3O4/N-G was dispersed into 100 mL of water sample under ultrasound.After 15 s, the Fe3O4/N-G carrying four compounds was separated from the water sample by an external magnetic field.Then, the targets were eluted from Fe3O4/N-G with 3 mL of ethanol and 2 mL of dichloromethane, sequentially.Finally, the eluent was dried under a mild stream of nitrogen and reconstituted with methanol and dichloromethane (1∶1, V/V) for the subsequent GC-MS/MS analysis.Under the optimized condition, an excellent linearity was observed in the range of 0.1-10 ng/L for the four compounds, with the correlation coefficients ranging from 0.9983 to 0.9999.The limits of detections (S/N=3) ranged from 0.05 to 0.6 ng/L and the limits of quantity (S/N=10) ranged from 0.2 to 2.4 ng/L.The mean recoveries at three spiked levels ranged from 68.2% to 99.6%.The relative standard deviations (RSDs) of intraday and interday were in the range of 3.3%-6.9% and 3.4%-9.4% (n=6), respectively.The proposed method was demonstrated to be simple and feasible for the trace analysis of antimicrobial agents and organochlorine contaminants in environmental water samples.

Objective To probe into clinical value of real-time three-dimensional echocardiography (RT-3DE) in evaluating global and regional left ventricular function in patients with acute inferior, posterior and lateral myocardial infarction(MI).Methods The study consisted of 41 normal subjects and 23 patients with acute MI.RT-3DE was performed and three-dimensional image data was analyzed offline with software.A series of global and regional left ventricular volume curves were plotted.Regional and global diastolic volumes, regional and global systolic volumes, regional and global ejection fractions, regional stroke volume to global diastolic volume were compared respectively between control group and MI group.Results By contrast with control group, global left ventricle function and regional left ventricle function in the zones with infarction and part of the zones close to infarction had significant difference( P＜0.05),however,no effective statistic change was found in the most of the distal zones of infarction( P＞0.05).Conclusions RT-3DE can accurately evaluate measurement the left ventricular global and regional function in patients with acute MI.

Objective To investigate the effect of lidocaine on the LPS-induced NF-κB activity in rat peritoneal macrophages. Methods The peritoneal macrophages obtained from male Wistar rats were placed in 12-well plates at 2 × 106 cell/ml after being cultured for 3 days. Each well contained 1 ml of cell suspension. The cells were randomized into control group (group C), LPS group and 3 LPS + lidocaine group S (group LL1.2.3)(n = 10 wells each). In group LPS and LL1,2,3, the cells were exposed to LPS 100 ng/ml. In group LL1,2,3 the cells were exposed to lidocaine 2, 20 200 kg/ml respectively in addition to LPS 100 ng/ml. After being incubated for 24 h, the HMGB1 concentration in the supernatant (by ElISA) and HMGB1 mRNA expression (by RT-PCR)and NF-κB activity in the cells were measured. Results LPS signiticantly increased HMGB1 concentration,HMGB1 mRNA expression and NF-κB activity in the supernatant. Lidocaine treatment significantly attenuated the LPS-induced increase in HMGB1 concentration HMGB1 mRNA expression and NF-κB activity in a dose-dependent manner. Conclusion Lidocaine can inhibit NF-κB activity in the rat peritoneal macrophages and in turn inhibit the synthesis and release of HMGB1.

Objective To observe the effect of cognition training on the motor and executive functioning of patients after a basal ganglia stroke.Methods Thirty patients with basal ganglia stroke were randomly divided into a treatment group and a control group.The control group received standard stroke rehabilitation training,while the treatment group received in addition 2 months of cognitive function training.The executive and motor functions of all of the subjects and their facility in the activities of daily living (ADL) were tested using the Tower of Hanoi,the Wisconsin card sorting test,a Stroop-3 test and the Fugl-Meyer assessment,the Berg balance scale and the modified Barthel index before and at the end of treatment.Results After two months of training,both within-group and between group comparisons showed that the treatment group had improved significantly more in executive function,cognition and motor function.Conclusion Cognition training can improve executive function,motor function and ADL performance after a basal ganglia stroke.

Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bub1 cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel (PTX). Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bub1 in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G(2)/M arrest, and inhibited Bub1 expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.

Supercritical fluid chromatography (SFC) meets with great favor due to its high efficiency, low organic solvent consumption, and the specialty for the identification of the isomeric species. This review de-scribes the advances of SFC in targeted and untargeted lipid profiling. The advancement of the SFC in-struments and the stationary phases are summarized. Typical applications of SFC to the targeted and untargeted lipid profiling are discussed in detail. Moreover, the perspectives of SFC in the lipid profiling are also proposed. As a useful and promising tool for investigating lipids in vitro and in vivo, SFC will predictably obtain further development.

Objective To detect anti-M_2 autoantibody using recombinant BCOADC-E_2.Methods We purified recombinant BCOADC-E_2 by Ni~(2+)affinity chromatography column and then detect anti-M_2 autoan- tibody in the sera of patients with primary biliary cirrhosis(PBC)by Western blot test(WBT)and enzyme linked immunosorbent assays(ELISA).Results Among 60 sera from PBC patients,33 were positive,all of controls were negative.Conclusion The recombinant BCOADC-E_2 can be used to detect anti-M_2 autoanti- body specifically and sensitively.It is helpful for the diagnosis of PBC.

OBJECTIVETo investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.

METHODSThe R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.

RESULTSFIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.

CONCLUSIONThe abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.

Factor IX ; genetics ; HEK293 Cells ; Hemophilia B ; genetics ; pathology ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Transfection