The template-independent teminal transferase activity of Taq DNA polymerase results in an overhanging dA at the 3′end of its PCR products. The pGEMX vector constructed in this experiment forms a single overhanging dT at its 3′end as the result of cleavage with Xcm I restriction enzyme. This vector is very efficient for direct cloning of PCR product obtained by using Taq DNA polymerase.Recombinant colonies can be selected by Blue/white screening. Moreover,insertion fragment can be easily released from the vector simply with either BamH I or Hind III digestion.

Some problems are arisen in teaching diagnostic pathology in our college for postgradu-ates of Pathology and Pathophysiology, the students' basic knowledge is weak, the cycle is short, the teaching team and resources are relatively insufficient. To promote the cultivation of graduate students, we should carry out the CBL teaching method, add the clinical practice and training, strengthen the teaching staff, and apply digital section and network platform.

To prepare sagittatoside B with epimedin B Hydrolyzed from cellulase. With the conversion ratio as the index, the effects of pH value, temperature, reaction time, dosage of enzyme and concentration of substrates on the conversion ratio were detected. L9 (3(4)) orthogonal design was adopted to optimize the preparation process. Hydrolyzed products were identified by MS, 1H-NMR, and 13C-NMR. The results showed that the optimum reaction conditions for the enzymatic hydrolysis were that the temperature was 50 degrees C, the reaction medium was pH 5.6 acetic acid-sodium acetate buffer solution, the concentration of substrates was 20 g x L(-1), the mass ratio between enzyme and substrate was 3: 5, and the relative molecular mass of the reaction product was 646.23. NMR data proved that the product was sagittatoside B. The process is simple and reliable under mild reaction conditions, thus suitable for industrial production.
Cellulase ; metabolism ; Drug Compounding ; methods ; Flavonoids ; chemistry ; Hydrogen-Ion Concentration ; Hydrolysis ; Temperature ; Time Factors

Objective To develop a new foldable test tube rack to solve the inconvenience and safety issues arising from transferring the specimens to test all together.Methods 482 urine specimens of 69 daily routines collected in the morning from various sections of the hospital were randomly divided into experimental group and control group.The experimental group was placed in the foldable test tube rack; however,the control group was managed with traditional methods.Results The index of stability in the experimental group was 98.3％,95.0％ in the control group,and there was significant difference in both groups ( x2 =4.06,P ＜ 0.05).Environmental pollution did not occur in the experimental group,while it happened six times in the experimental group,there was significant difference between both groups( x2 =4.06,P ＜ 0.05 ).The time for transferring specimens was (18.72 ± 2.92) minutes in the experimental group,(27.28 ± 3.87 ) minutes for the control group,there was significant difference between them( t =3.04,P ＜ 0.01 ).Conclusions Foldable test tube rack for transferring specimen is time-saving,as well as avoids re-sorting and places specimens,improves work efficiency; enhances the specimen placement stability,avoiding environmental pollution arising from dumping,overflowing,and sprinkling.

Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
Cloning, Molecular ; Fungal Proteins ; chemistry ; genetics ; isolation & purification ; Muramidase ; chemistry ; Plasmids ; Protein Folding ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Spectrometry, Fluorescence

Objective:To investigate the heterogeneity of epitopes recognized by anti-GBM autoantibodies in sera from a large cohort of Chinese patients with anti-GBM disease and its clinical significance.Methods: The present study included 108 patients with anti-GBM disease who were diagnosed in our hospital, between Jan 1991 and May 2009, with complete clinical and renal pathological data. Sera or plasma exchange of the patients were used to incubate with cryostat section of normal human renal tissue for indirect immunofluorescence (IIF) assay. The cryostat sections of normal renal tissue were pre-treated by 6 mol/L urea to unmask cryptic epitopes, and untreated cryostat sections were used to detect natural exposed epitopes. The sera were diluted from 1:2 to 1:512 to determine titers of anti-GBM autoantibodies Patients with anti-GBM autoantibodies against cryptic or exposed epitopes were further stratified;their clinical and pathological associations were analyzed. Results: Sera from all the 108 patients could recognize cryptic epitopes on normal renal tissue ( urea treated section). IIF showed IgG linear staining along GBM. However, sera from 56/108 patients (group A) could also recognize exposed epitopes on normal renal tissue (untreated section) ; sera from the rest 52/108 patients (group B) could not recognize exposed epitopes. In urea treated condition, the average titer of anti-GBM autoantibodies from sera of patients in group A was significantly higher than that in group B (P＜0.01) , ANCA-positive patients in group A were significant less than that in group B (P＜0.01) . There was no significant difference between the two groups in regard to other clinical data (including serum creatinine) and renal histopathologic data. Conclusion: Anti-GBM autoantibodies from some patients with anti-GBM disease could recognize natural exposed epitopes, however, their anti-GBM titer for cryptic epitopes was higher than that of those recognizing cryptic epitopes only and the prevalence of serum ANCA was significantly less.

The real sanghuang is a new species belonging to the Inonotus, which is commonly used for cancer treatment and human immune system improvement. This review summarized the progress on the studies of Phellinus Quel in recent years, including its taxonomy status, bioactive components, pharmacodynamics, separation and purification technologies. In addition, some related problems and perspectives were also discussed.
Animals ; Basidiomycota ; chemistry ; classification ; Humans ; Medicine, Chinese Traditional ; methods

Objective To investigate the characteristics of maternal thyroid function of pregnant women with negative thyroid antibody in high water iodine area. Methods The investigation sites were selected,which were the Hospital for Women and Children of Jinghai county in the high water iodine area(drinking iodine > 200 μg/L) and the Hospital for Women and Children of Heping district in Tianjin in the adaptive iodine area (drinking iodine < 10μg/L,popularization rate of iodized salt > 90％,residents urinary iodine > 200μg/L). In the maternal and child hospitals,50 pregnant women of each stage from obstetric clinics in first,second,third term of pregnancy were selected,the blood samples were collected and the thyroid function were measured with chemiluminescence. Water,salt and diurnal optional urine samples were measured for iodine concentration. Iodine levels of urine,water,salt were determined respectively by As-Ce catalysis spoctrophotometry method,quantitative determining kit which use time-recorded determination by catalytic effect on the As-Ce reaction and sodium hyposulfite titration method. Results ①In pregnant women with negative thyroid antibody,serum TT_4,TT_3,FT_4 in first term of pregnaney and TT_4,TT_3 in second term of pregnancy were significantly lower in high water iodine area than low water iodine area(111.97 nmol/L vs 140.46 nmoL/L,Z = 3.56,P < 0.01 ; 1.86 nmol/L vs 2.26 nmol/L,Z = 2.35, P < 0.05; 14.13 pmol/L vs 16.32 pmol/L,Z = 5.14,P < 0.01,and 11.98 pmol/L vs 14.30 pmol/L,Z = 5.75,P < 0.01 ; 4.04 pmol/L vs 4.32 pmol/L,Z = 2.76,P < 0.01),while TT_3 and TSH in third term of pregnancy were significantly higher(2.88 nmoL/L vs 2.70 nmol/L,Z=-2.27,P< 0.05; 2.37 mU/L vs 1.75 mU/L,Z =-2.70, P < 0.01).②Concentration of water iodine and urine iodine were higher(205.57μg/L vs 8.26 μg/L,Z =-14.71,P < 0.01 ; 305.91 g/L vs 191.86 g/L,Z =-5.30,P < 0.01),while salt iodine was lower(26.5 mg/kg vs 31.7 mg/kg,Z =-5.86,P < 0.01) in high water iodine area. ③Among 290 selected healthy pregnant women without medical history of thyroid diseases,there was no significant difference in positive rate of thyroid antibody in each term of pregnancy between high water iodine area and low water iodine area(10.20％ vs 10.64％ ; 14％ vs 9.52％ ; 4％ vs 7.69％ ; all P > 0.05). Conclusions The thyroid function of pregnant women with negative thyroid antibody in high water iodine area is different from pregnant women in low water iodine area with universal salt iodization. Enhanced monitoring on thyroid function of pregnant women in high water iodine area should be performed,especially in first and second trimester.

Objective To described the prevalence of school physical violence behaviors and to explore its associated factors among middle school students in Beijing. Methods In 2009, a randomly selected cross- sectional survey was conducted among 5718 students in grades 7 to 12 in Beijing. A self-report anonymous questionnaire involving physical violence at school and sociodemographic variables, such as sex, grades, family economic status and family structure, peer relationships, and communication with their parents etc. were completed by students themselves.Logistic regression was used to estimate the association between physical violence and sociodemographic variables. Results Among the students, 14.3% reported that they had had physical violence behavior in school during the past 12 months. Male students had been more likely to have physical violence behaviors than female students (Male 25.2%, Female 5.1% ). For both male and female students, poor school cohesion were the risk factors of physical violence behaviors (Male OR=1.060, Female OR=1.065). For male students, factors as father' s lower education level (OR=1.653 ), remarried/single-parent families ( OR = 1.834 ), low-grade ( grade 7 OR = 5.291; grade 11 OR =1.526) , poor school performance (OR=1.470) etc were the risk factors of physical violence behaviors; while better-off family economic status (OR=0.546), good peer relationships (OR=0.618) , and easy to communicate with the father (OR=0.756) were the protective factors of physical violence behaviors. For female students, easy to communicate with her mother (OR = 0.358)were the protective factors of physical violence behaviors. Conclusion For male and female students, the prevalence of school physical violence and its related factors were different. Actions on prevention against physical violence behaviors should be fully considered, including factors as gender, personal characteristics, family, school and peers etc.

OBJECTIVERespiratory syncystial virus (RSV) is the most common cause of lower respiratory infections in infants worldwide. There is no reliable vaccine or antiviral drug against RSV at present. RNA interference (RNAi) technology is a potent method to degrade expression of the cognate mRNA. In order to inhibit the replication of RSV at gene level, the effects of specific RNAi against M2-1 gene of RSV on inhibition of viral replication in cell culture system was observed in this study.

METHODSRSV M2-1 gene, which plays a key role in RSV transcription, was chosen in this study and was used as target gene and recombinant plasmid pshRNA7816 targeting the mRNA of RSV M2-1 gene coding sequence was constructed. The pshRNA7816 was transfected into Hep2 cells. The effects of the pshRNA7816 on changes of cytopathogenic effect (CPE) of Hep2 cell induced by RSV infection were observed microscopically. Viral plaque forming assay and MTT assay were used to detect the viral titer change and protective function of the pshRNA7816 on RSV infected Hep2 cell.

RESULTSThe recombinant RNAi plasmid pshRNA7816 which targets the mRNA of RSV M2-1 gene was successfully constructed. The pshRNA7816 significantly reduced CPE of RSV infected Hep2 cells, reduced the viral titer of RSV in the cells (P < 0.001). The pshRNA7816 raised the survival rate of RSV infected Hep2 cells (P < 0.001). Non-specific pshRNA plasmid did not show anti-RSV effects (P > 0.05).

CONCLUSIONThe recombinant pshRNA7816 plasmid which targeted the mRNA of RSV M2-1 gene showed a significant and specific anti-RSV effect.

Hep G2 Cells ; Humans ; Plasmids ; biosynthesis ; RNA Interference ; RNA, Small Interfering ; biosynthesis ; RNA, Viral ; genetics ; Respiratory Syncytial Virus Vaccines ; biosynthesis ; Respiratory Syncytial Virus, Human ; drug effects ; physiology ; Viral Proteins ; genetics ; Virus Replication ; drug effects