SMART-RACE was performed after isolating the total RNA of Armillariella tabescens to amplify the full-length cDNA of arabinosidase (GenBank Accession No. AJ620046). Bioinformatics analysis was used to analyze the code frame of arabinosidase, to predict its structure and function. Recombinant plasmid pPIC9-AF was constructed and then electroporated into methylotrophic yeast Pichia pastoris GS115. The secreted 6 ? His fusion protein was purified to analyze its enzymology property. This arabinosidase had high activity at 30-35℃ under acid condition, and was stable within wide range of pH and temperature. It maintained about 80% activity at the range of pH4. 0-8.0 and 20-40℃,wider than many other cloned arabinosidase. So it was worthy to go step further to study this enzyme, and recombinant expression provided a chance of highly expressing arabinosidase.

Armillariella tabescens EJLY2098 was capable of secreting p-mannanase by konjac inducement. A 34 orthogonal design was applied to determine the optimum medium of inducing mannanase by Armillariella tabescens EJLY2098. The results suggested that Armillariella tabescens EJLY2098 secreted the high-activity enzyme in the optimum medium, which was composed of 2% konjac, 1% peptone, 25% potato juice,0.3% KH2PO4,15% MgSO4?7H2O, 0.01% VitB1. Purified by DEAE-anion exchange chromatography, two eluting peaks (P1 and P2) with the p-mannanase activity were obtained, and one of them (named?-mannanase P2) was a single band by the SDS-PAGE, and the molecular weight of?-mannanase P2 was 78. 9kDa. The isoelectric point of?-mannanase P2 was estimated to be 4.0-4. 1. The optimum activity for the enzyme was found at 60℃and pH4. 0 - 6. 0, and the enzyme was stable between pH4. 5 - 6. 0. The activity of?-mannanase P2 were enhanced by Na+ and Ba2+ . This?-mannanase can be used in feed industy. a new fungi secreting?-mannanase was obtained, providing an important base for cloning mannanase gene and constructing recombin microbe expressing?-mannanase .

AIM:To evaluate effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene and clarify the involved mechanisms in DLD1-TRAIL/R colon cancer cells.METHODS: Human colon cancer cell line DLD1-TRAIL/R cells that were resistant to TRAIL-expressing adenovector(Ad/gTRAIL) were treated with Ad/gTRAIL combined with different chemotherapeutic agents.Then,the cell viability was measured by MTT method,and apoptotic signaling conditions,including activation of caspase-3 and caspase-8,expression of Bax and Bcl-XL,were measured by Western blotting analysis.RESULTS: In vitro data showed that several chemotherapeutic agents,including 5-fluorouracil(5-FU) and mitomycin c(MMC),overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R colon cancer cells.The combination of Ad/gTRAIL and 5-FU effectively suppressed tumor growth in vivo in subcutaneous tumors established from DLD1-TRAIL/R cells.Further data showed that treatment with the combination of Ad/gTRAIL and 5-FU or MMC led to enhance the activation of caspase-3.Moreover,MMC but not 5-FU induced overexpression of Bax gene that was sufficient to overcome the resistance to TRAIL gene in DLD1-TRAIL/R cells.CONCLUSION: Chemotherapeutic agents,such as 5-FU and MMC,overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R cells.The candidate mechanisms for MMC but not 5-FU to overcome this resistance might involve the induction of over-expressed Bax protein in DLD1-TRAIL/R cells.

Objective To establish a RP-HPLC method for determining indigo and indirubin in Baphicacanthus cusia from different producing areas and medicinal parts. Methods The separation was achieved by an Agilent TC-C18 Column (4.6 mm×250 mm, 5 μm) at 25 ℃ using methanol-water (75??25) as mobile phase at a flow rate of 1 mL??min-1.The detection wavelength was 290 nm. Results Indigo had a good linear relationship with peak area at range of 0. 051 3-0.820 8 μg (r=0.999 3).The recovery rate was 99.00% and RSD was 1.30% (n=6).Indirubin had a good linear relationship with peak area at range of 0.049 5-0.792 0 μg (r=0.999 9).The recovery rate was 98.88% and RSD was 1.51% (n=6). Conclusion The contents of the two components are obviously different in Baphicacanthus cusia because of different places or medicinal parts. The proposed method is simple, rapid and reliable. This method for determination of indigo and indirubin in Baphicacanthus cusia by RP-HPLC provides a basis for quality control of Baphicacanthus cusia.

AIMTo identify the drug-metabolizing enzymes involved in the hydroxylation of the new anti-inflammatory and anodyne imrecoxib.

METHODSImrecoxib was incubated with heterologous expression human cytochrome P450 (rCYPs) in vitro, and metabolites and remained parent drug were detected with liquid chromatography-multistage mass spectrometry. The contribution of 4 CYPs in the hydroxylation metabolism of imrecoxib was evaluated by total normalized rate (TNR) method.

RESULTSImrecoxib is metabolized by CYP2C9, CYP2D6 and CYP3A4, with the rate of 62.5%, 21.1% and 16.4%, respectively.

CONCLUSIONCYP2C9 is the major enzyme involved in imrecoxib hydroxylation metabolism.

Aryl Hydrocarbon Hydroxylases ; metabolism ; Cyclooxygenase 2 Inhibitors ; metabolism ; Cytochrome P-450 CYP2C9 ; Cytochrome P-450 CYP2D6 ; metabolism ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; metabolism ; Hydroxylation ; Pyrroles ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Sulfides ; metabolism

AIMTo investigate the variation of CYP2C9 isoenzyme activity in the microbial model in response to inhibitors of CYP2C9.

METHODSUsing C. blakesleeana AS 3. 910 as a model strain, the impact of CYP2C9 inhibitors on the metabolites yields of CYP2C9 substrates was determined and the drug-drug interactions among CYP2C9 substrates were evaluated. Liquid chromatography-mass spectrometry was used to analyze biotransformation products.

RESULTSBenzbromarone decreased the yield of 4'-hydroxytolbutamide from 100% to 14.5%; sulfaphenazole decreased the yield of O-demethylindomethacin from 75.2% to 9.9%; valproic acid decreased the yield of 4'-hydroxydiclofenac from 98.6% to 2.7%, separately. Tolbutamide, indomethacin and diclofenac interacted with each other, resulting in the decreased formation of metabolites catalyzed by CYP2C9.

CONCLUSIONThree CYP2C9 inhibitors inhibit the activity of CYP2C9 isoenzyme in C. blakesleeana AS 3. 910 differently, and there are drug-drug interactions among CYP2C9 substrates.

Aryl Hydrocarbon Hydroxylases ; antagonists & inhibitors ; metabolism ; Benzbromarone ; pharmacology ; Biotransformation ; drug effects ; Catalysis ; drug effects ; Chromatography, High Pressure Liquid ; methods ; Cunninghamella ; enzymology ; metabolism ; Cytochrome P-450 CYP2C9 ; Diclofenac ; analogs & derivatives ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Drug Interactions ; Fungal Proteins ; antagonists & inhibitors ; metabolism ; Indomethacin ; pharmacology ; Isoenzymes ; antagonists & inhibitors ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; methods ; Substrate Specificity ; Sulfaphenazole ; pharmacology ; Tolbutamide ; analogs & derivatives ; metabolism ; pharmacology ; Valproic Acid ; pharmacology

The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.

Objective To explore the relationship between alcohol drinking and fatty liver disease and its influencing factors.Methods From October to December,2013,a total of 394 people who underwent a physical examination in the medical examination center of Wenzhou central hospital were selected for this study.Anthroposomatology measurement and biochemical tests were conducted.Results There were significantly statistical differences of triglyceride,uric acid and cholesterol in different drinking groups (P <0.05).The prevalence of high triglyceride and uric acid were increased with alcohol consumption (P <0.05).There was no significant difference of alcohol consumption between fatty liver and non -fatty liver group (P =0.42).Logistic regression showed that waist -hip ratio,hypertension,overweight,obesity and hypertriglyceridemia were risk factors of fatty liver,while daily alcohol consumption cannot be thought as risk factor yet. Conclusion Waist -hip ratio,hypertension,overweight -obesity and hypertriglyceridemia were the risk factors of fatty liver.Alcohol consumption could contribute to the prevalence of triglyceride and uric acid.

Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.

OBJECTIVETo explore the differences of metabolic footprint in the conditioned culture medium of placental explants between early-onset and late-onset severe preeclampsia.

METHODSIn 13 cases of early-onset severe preeclampsia and 14 cases of late-onset severe preeclampsia, the placentas were sampled at the surface of the maternal placenta. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the differences in the metabolites in the conditioned culture medium of the placental villous explants cultured in 6% atmospheric O(2) for 96 h. Standard samples were used to establish the tryptophan and kynurenine chromatography library by HPLC-MS to analyze the concentration of tryptophan and kynurenine in the conditioned culture medium.

RESULTSThirty-six metabolites showed statistically significant differences between early-onset and late-onset severe preeclampsia (P<0.05). The concentration of kynurenine was significantly higher in early-onset severe preeclampsia than in late-onset severe preeclampsia (P<0.05).

CONCLUSIONEarly-onset and late-onset severe preeclampsia may have different pathogeneses. By detecting the concentration of metabolites, metabolomic strategies provide a new means for predicting the onset time of severe preeclampsia.

Chorionic Villi ; metabolism ; Culture Media, Conditioned ; chemistry ; Female ; Humans ; In Vitro Techniques ; Kynurenine ; metabolism ; Ornithine ; metabolism ; Placenta ; metabolism ; Pre-Eclampsia ; metabolism ; Pregnancy ; Tryptophan ; metabolism