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 .

The culture medium and cultural conditions of solid-state fermentation of Streptomyces Menmyco-93-63 were tested in this study. The suitable medium which contains rice, sorghum, millet bran, and rice hull with the proportion of 2:2:3:3 was developed for the spore production of Streptomyces Men-myco-93-63 using single substrate screening, mixture substrate screening and orthogonal experiments, and the sporulation was up to 2.52?109 CFU/g. And then, initial charge, initial ratio of water to solid, inoculating quantity, and culture temperature impact to sporulation of Streptomyces Men-myco-93-63 were tested. The favorite cultural conditions are developed as the following: the initial charge is 15 g in 500 mL Erlenmeyer flask; initial ratio of water to solid is 1.7:1.0 (V/W, rice hull excluding), inoculating quantity is 7 mL, culture temperature is 28℃.

Objective To evaluate the clinical value of 6 h 99mTc-diethyl iminodiacetic acid (99mTc-EHIDA) planar hepatobiliary scintigraphy (HBS),6 h tomographic HBS and 24 h planar HBS in diagnosis on biliary atresia(BA).Methods Seventy cases(32 male,38 female) with continuous jaundice received planar and tomographic HBS in Beijing Friendship Hospital from Jan.2005 to Dec.2007.The mean age was 48.7 d (29 d-4 months).According to final diagnosis,all cases were divided into BA group (45 cases) and non-BA group (25 cases).All cases fasted at least 4 hours before HBS.The equipment was 3 head IRIX from Philips company with low energy high resolution collimator.The tracer was 99mTc-EHIDA and the radiochemistry purity was more than 95 percent.The dosage was 7.4 MBq/kg.All diagnosis demonstrated by operation pathology and clinical follow-up.All cases received HBS at 5,10,15,20,30 min and 1,6 h after tracer injection.HBS would ended if radioactivity appeared in gallbladder or intestine.These cases would received tomographic HBS and 24 h HBS if radioactivity did not appear in gallbladder or intestine at 6 h post injection.All these images were analyzed by 2 or more nuclear medicine physicians.Results There were not radioactivity appearing in gallbladder and intestine on planar and tomographic HBS of 27 cases,which suggested the BA.There were radioactivity appearing in gallbladder and intestine on planar and tomographic HBS of 30 cases,which suggested the non-BA.Positive rate of 6 h tomographic HBS was significantly higher than that of 6 h planar HBS and there was significantly difference between the 2 methods.Positive rate of 6 h tomographic HBS was significantly higher than that of 24 h planar HBS and there was significant difference between the 2 methods.Conclusions 99mTc-EHIDA HBS is a noninvasive,safety,valuable examing method and has definitely clinical value in the diagnosis on BA.The clinical value of 6 h tomographic HBS is significantly higher than that of 6 h planar HBS and 24 h planar HBS.

Animals ; Apolipoproteins E ; deficiency ; genetics ; Blood Vessels ; anatomy & histology ; Fluorescence ; Lipofuscin ; Mice ; Mice, Knockout

Liver is regarded as one of the most important organs for drug clearance in the body, which mediates both the metabolism and biliary excretion of drugs. Transporters are a class of functional membrane proteins and control the movement of substances into or out of cells. Transporters, which are extensively expressed in the liver, play important roles in the drug hepatic disposition by regulating the uptake of drugs from blood into hepatocytes or the efflux of drugs and their metabolites into bile. In this review, the localization, functions and substrate selectivity of the major transporters in the liver will be summarized, and the impacts of these transporters on drug hepatic disposition, the potential drug-drug interactions as well as their genetic polymorphisms will also be reviewed.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Bile ; metabolism ; Biological Transport ; Drug Interactions ; Humans ; Liver ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; Metabolic Clearance Rate ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Anion Transporters, Sodium-Dependent ; metabolism ; Organic Anion Transporters, Sodium-Independent ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Pharmacokinetics ; Polymorphism, Genetic ; Symporters ; metabolism

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.

BACKGROUNDVertebrobasilar artery stenosis is an important cause of ischemic posterior circulation strokes. This study aimed at evaluating the safety and efficacy of treatment including conservative therapy alone and conservative plus endovascular therapy for elderly patients with symptomatic vertebrobasilar artery stenosis.

METHODSPatients older than 60 years with symptomatic vertebrobasilar artery stenosis (> or = 50%) confirmed by cerebral angiography were enrolled. All of them were treated with medical therapy and some with additional stent-assisted angioplasty (the stenting subgroup). Their clinical, imaging, intervention and follow-up data were analyzed.

RESULTSOne hundred and seventeen consecutive elderly patients (100 men, mean age (68.1 +/- 5.1) years) were enrolled and followed up for a mean time of 28.4 months; 81.7% of them were symptomatically resolved or improved; a stroke rate of 5.1% and a stroke-related death rate of 1.7% were found among them during the hospitalization and follow-up. In the stenting subgroup, 78 balloon expandable stents were employed in the 70 patients with a technical success rate of 98.7% and the mean degree of stenosis was significantly reduced from (81.7 +/- 14.3)% before stenting to (8.3 +/- 4.2)% after stenting (P < 0.001). Four (5.7%) periprocedural strokes occurred, of whom two led to death within 30 days after the procedure. During the follow-up (mean 27.7 months), sixty of the surviving 68 patients in the stenting subgroup were symptomatically resolved or improved. Only one (1.5%) posterior circulation stroke occurred, while duplex ultrasound scan of 34 patients demonstrated 10 (29.4%) in-stent restenosis.

CONCLUSIONSAppropriate utilization of conservative therapy alone and conservative plus endovascular therapy may improve short-term clinical outcomes for elderly patients with symptomatic vertebrobasilar artery stenosis. Furthermore, stent-assisted angioplasty is technically feasible and relatively safe in elderly patients.

Aged ; Aged, 80 and over ; Angioplasty, Balloon ; methods ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Prospective Studies ; Stents ; Treatment Outcome ; Vertebrobasilar Insufficiency ; therapy

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.
Absorption ; Aflatoxin B1 ; metabolism ; toxicity ; Animals ; Chickens ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Multienzyme Complexes ; chemistry ; metabolism ; Rats ; Temperature ; Toxicity Tests