A secretive expression vector of Pichia pastoris system which can be used for the direct cloning of PCR products was constructed,and was verified through the expression of recombinant cellobiohydrolase II in Pichia pastoris.A randomly selected fragment was amplified with properly designed primers by PCR.The XhoI and Eam1105Ⅰ restriction sites were included in the 5'end of the fragment,and the Eam1105Ⅰ and XbaI restriction sites were included in its 3'end.The PCR amplified product was inserted into the P.pastoris expression plasmid pPICZ?A through XhoI and XbaI restriction sites and the resultant plasmid was digested with Eam1105Ⅰ,and lastly the big fragment was recovered,generating the P.pastoris expressive Tvector pPICZ?T.Then the cellobiohydrolase II of T.reesei was successfully expressed in P.pastoris with this expressive Tvector.Such results indicated that the constructed expression Tvector was convenient for PCR product cloning,and was effective for heterologous protein expression in P.pastoris.On the other hand,the application of the expression Tvector avoided the introduction of additional amino acids at the Nterminus of the expressed protein,which generally occurred when normal expression vectors were used in secretive expression system.

Fibroblast growth factor-21 (FGF-21) is an important metabolism regulator, however, whether FGF-21 has effects on cardiovascular remains unclear. In this study, H2O2-induced injury in H9c2 cells was used as a cell model, the anti-apoptosis potential and mechanism of FGF-21 against oxidative injury were evaluated by MTT assay, flow cytometry assay and real-time PCR. The results showed that FGF-21 could increase the cell survival of H2O2-induced injury in H9c2 cells and prevent H9c2 cells from oxidative stress-induced apoptosis. Furthermore, FGF-21 can elevate SOD activity and regulate Bcl-2/Bax expression in H9c2 cells. The results suggest that FGF-21 have protective effect against the H2O2-induced apoptosis in H9c2 cells.
Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblast Growth Factors ; pharmacology ; Hydrogen Peroxide ; toxicity ; Malondialdehyde ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

Objective To explore the anatomic character of brachial plexus nerve by ultrasonography,and provide a basis for ultrasound-guided brachial plexus block.Methods Twenty-four healthy volunteers were chosen for high-frequency sonography examination,the features of brachial plexus distributions were studied in the axilla location and the interscalene location.Results Brachial plexus of all the 24 cases were detected.At the axilla level,main braches of brachial plexus nerves were very close to the axillary artery.At the interscalene level,the brachial plexus was consistently found between the scalenus anterior and medius muscles in the interscalene groove.Conclusions Using high-frequency sonography,the main braches of brachial plexus can be observed clearly.

Bel1, a transactivator of prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have shown that Bel1 bears a nuclear localization signal (NLS), but its amino acid sequence remains unclear and the corresponding importins have not been identified. In this report, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment could direct nuclear localization, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. Point mutation experiments revealed that K218, R219, and R221 are essential for the nuclear localization of Bel1. The results of the GST-pulldown showed that the Bel1 fragment with residues 215-223, which bears the NLS, interacts with KPNA1, KPNA6, and KPNA7. This result suggests that KPNA1, KPNA6, and KPNA7 maybe involved in Bel1 nuclear translocation.
Cell Line ; Cell Nucleus ; genetics ; metabolism ; virology ; Humans ; Nuclear Localization Signals ; genetics ; metabolism ; Protein Binding ; Protein Transport ; Retroviridae Infections ; genetics ; metabolism ; virology ; Retroviridae Proteins ; chemistry ; genetics ; metabolism ; Spumavirus ; chemistry ; genetics ; physiology ; Trans-Activators ; chemistry ; genetics ; metabolism ; alpha Karyopherins ; genetics ; metabolism

AIMTo isolate and purify gonyautoxins from Alexandrium mimutum Halim Amtk2 strain.

METHODSThe ethanol extracts of culture Alexandriun minutum Halim Amtk2 were isolated by means of gel filtration chromatography, the toxin fraction obtained was then purified by ion exchange chromatography.

RESULTSFrom 100 liter of cultivation liquid of Alexandrium mimutum Halim Amtk2 (6.74 +/- 0.31) x 10(9) cells were obtained. The ethanol extracts of Alexandriun minutum Halim purified by gel filtration chromatography obtained gonyautoxins mixture 29.59 mg. 4.06 mg of the mixture was further purified by two steps of ion exchange chromatography, and obtained pure GTX-4 (0.40 +/- 0.002) mg, GTX-1 (5.95 +/- 0.03) x 10(-2) mg, GTX-3 (6.92 +/- 0.05) x 10(-4) mg and GTX-2 (0.11 +/- 0.005) mg.

CONCLUSIONPure gonyautoxins can be obtained by means of gel filtration chromatography and ion exchange chromatography from ethanol extracts of cultured Alexandriun minutum Halim Amtk2 strain.

Animals ; Chromatography, Gel ; methods ; Chromatography, Ion Exchange ; Dinoflagellida ; chemistry ; Marine Toxins ; chemistry ; isolation & purification ; Molecular Structure ; Saxitoxin ; analogs & derivatives ; chemistry ; isolation & purification

Objective To compare the CLIP score, the JIS score, and the China staging system (CS) in the prediction of survival of patients with resectable hepatocellular carcinoma (HCC). Methods The Clinicopathologic and follow-up data of 224 patients who underwent hepatic resection for HCC from January 2000 to July 2005 were retrospectively studied. The patient distribution and the survival curve of each staging system were used to compare the ability to stratify and to discriminate prognosis. The likelihood ratio, chi-square test and the linear trend chi-square test were used to compare the homogeneity and the monotonicity of the relationship between stage and mortality rate of each staging system. The increase in the -2 log likelihood statistic on removal of any one staging system was in turn used as a means of ranking the individual staging systems according to their importance within the regression model. The statistical package used was SPSS version 16. 0 and Stata SE version 8.0. Results Based on the China staging system, the percentages of patients categorized as Ⅰa, Ⅰ b, Ⅱa,Ⅱb and Ⅲ a were 14. 3%, 17.4%, 21.9%, 31.7% and 14. 7% respectively, showing excellent stratification ability. However, nearly 81. 6% of the patients were classified as a CLIP score of 0-2, which showed poor stratification ability, and only 3. 1 % of the patients were classified as score 0 category of the JIS scoring system. In the follow-up period, the log-rank test and the corresponding Kaplan-Meier survival curves confirmed each staging system to be able to differentiate patient survival in the different stages. Individual pairwise comparisons revealed inconsistencies across the different staging systems. In particular, using the log-rank test, the JIS scoring system and the China staging system showed significant differences in patient survival on all pairwise comparisons. By contrast, the CLIP scoring system failed to differentiate significantly between score 2 and score 3 patients. The JIS scoring system could identify the best prognostic group who would benefit from curative and aggressive treatments, whereas the discriminatory value of the CLIP score was noted in the intermediate- and advanced-phase HCC patients. The China staging system was shown to have the best homogeneity, overall discriminatory capacity and monotonicity of gradient. The change in the -2 log likelihood statistic on removal of any staging system revealed that for this cohort of patients, the appropriate importance in the ranking of the independent contribution of each factor to the regression model was: CS> CLIP>JIS. Conclusion Among three clinical staging systems, the China staging system had the highest prognostic value, with better stratification and higher discriminatory capacity than the CLIP scoring system and the JIS scoring system for this cohort of patients who received partial hepatectomy for HCC. The CLIP scoring system performed better in identifying the worst prognostic patients.

AIMTo study the synthesis of 5-(3'-indolyl)-oxazoles and their antioxidative activity.

METHODSThe amides were prepared from tryptophan and different acid derivatives by the catalytic dehydration of dicyclohexyl carbodiimide (DCC). The characteristic heterocyclic ring system of 5-(3'-indolyl)-oxazoles was constructed by oxidative cyclization of amide, using dicholorodicyanoquinone (DDQ). Their antioxidative activity in vitro was tested using DPPH system.

RESULTSEleven 2-substituted phenyl-5-(3'-indolyl)-oxazoles were prepared, the compounds 21 and 22 have shown antioxidative activity 3-4 times stronger than that of Vit E, and the compound 29 showed antioxidative activity almost as same as Vit E.

CONCLUSIONThree 5-(3'-indoyl)-oxazole compounds synthesized showed potent antioxidative effect and they would be a good antioxidants.

Antioxidants ; chemical synthesis ; chemistry ; Indoles ; chemistry ; isolation & purification ; Molecular Structure ; Oxazoles ; chemistry ; isolation & purification

The aim of the manuscript was to optimize formulations and preparation technologies of cataplasm of white mustard seed varnish, and to evaluate its anti-asthma effect on rats. The single factor experiments included spreading thickness, types of crosslinking agents, dihydroxyaluminum aminoacetate amount, sodium polyacrylate amount, types of adhesive agents with human sense as the evaluation index. Blank cataplasm matrix was optimized by the orthogonal experiment with the amount of glycerine, citric acid, and sodium carboxymethylcellulose as the major influential factors. Initial adhesive force, peeling strength and human sense were as the evaluation index. The optimized formulation of blank cataplasm were as followings: glycerine-water-ethanol-PEG400-dihydroxyaluminum aminoacetate-citric acid-sodium carboxymethylcellulose-sodium carboxymethylcellulose 2 : 8 : 0.8 : 0.4 : 0.07: 0.15 : 0.1 : 0.5. The active ingredients of white mustard seed, corydalis, and gansui root were extracted by alcohol extraction method. Asiasarum volatile oil was extracted by oil extractor. The optimized drug loading amount was 11% with initial adhesive force, peeling strength and human sense as the evaluation index. Asthma rats model were established by sensitized with ovalbumin and nose-scratching time as the evaluation index. High dose (17%) group of drug-loaded cataplasm had the obvious inhibition effect on nose-scratching time of rats (P = 0.037 < 0.05). In comparison, middle dose (11%), low dose (4%) and positive-control groups had no obvious inhibitive effect on rats. White mustard seed cataplasm supplied a novel choice for anti-asthma therapy. And the overall pharmacodynamics assessment will be carried out on molecular level in near future.
Animals ; Anti-Asthmatic Agents ; administration & dosage ; chemistry ; Asthma ; drug therapy ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Female ; Humans ; Male ; Mustard Plant ; chemistry ; Rats ; Rats, Sprague-Dawley ; Seeds ; chemistry

Objective To explore the relationship between free fatty acid(FFA)composition and other glucose and lipid metabolic parameters in patients with metabolic syndrome(MS)in community. Methods Serum FFA profile was measured with gas chromatography and mass spectrometry in 158 patients with varied metabolic syndrome components(MSC),including 61 with MS and 97 at high-risk for MS,and 43 control subjects,with diagnostic criteria by the International Diabetes Federation(IDF).Results Patients with MS had higher parameters of polyunsaturated fatty acid(PUFA)and n6PUFA,as compared to the patients at high risk and normal subjects(P0.05). Among the high-risk group,those with diabetes had increased linoleic acid,n6PUFA and total fatty acid (TFA),and decreased saturated fatty acid(SFA)/TFA,as compared to those without diabetes(P

OBJECTIVETo study the mechanism of sensitivity variation to cisplatin caused by nm23-H1.

METHODSThe samles was divided into two groups: Tca8113 group and Tca8113/nm23-H1 group. Using MTT and flow cytometer, the changes of cell mortality rate, apoptosis and mitochondrial membrane potential were detected. By VG PQ Excell, the changes of the intracellular platinum were detected.

RESULTSIn vitro the cell mortality rate and apoptosis were increased in Tca8113/nm23-H1 group, comparing with Tea8113 group. Mitochondrial membrane potential was decreased in Tca8113/nm23-H1 group. The intracellular platinum was increased significantly in Tca8113/nm23-H1 group. This effect could be inhibited by oubain which was an inhibitor of Na+/K+-ATP.

CONCLUSIONnm23-H1 can increase the sensitivity of cisplatin on Tca8113 cell line. The mitochondrial membrane potential was decreased by nm23-H1 so that intracellular platinum was increased and finally increased the apoptosis or necrosis.

Apoptosis ; Cell Line ; Cell Line, Tumor ; Cisplatin ; Humans ; In Vitro Techniques ; NM23 Nucleoside Diphosphate Kinases ; Transfection