Protein protein binding is the basis of virus and host cell interactions. With the application of technology of studying of protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) could be acquired. Non structure protein NS5A is one of the important regulatory factors in virus replication , transcription and signal transduction, but there are controversy in effect on HCV pathogenesis and resistance to interferon ?(IFN?). In order to describe the relationship between NS5A and host proteins, we use yeast two hybrid system 3 to screen the gene encoding proteins that could interact with NS5A from human hepatocyte library. Thirty five clones were obtained including apo A1, apo A2, apo B100, haplotype mitochondrion complete genome, phosphatidic acid phosphatase type 2B, albumin similar to tumor endothelial marker 5 precursor, matrix metalloproteinase 14, and three of the Homo sapiens hypothetical proteins. The study paved a way for further studies on the pathogenesis of HCV NS5A

To clone the genes of hepatocyte protein interacting with hepatitis C virus NS3 protein, "bait" plasmids of hepatitis C virus NS3 were constructed. After verifying that hepatitis C virus NS3 protein could be steadily expressed in AH109 yeast strain, yeast two hybrid assay was berformed by mating AH109 with Y187 that pre transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X ? gal activity. Nineteen yeast colonies that could grow on QDO and had ? gal activity were obtained, then the library plasmids were extracted and sequenced. The gene sequences from the 19 positive colonies were aligned with the genes deposited in GenBank. It was found hepatitis C virus NS3 protein could interact with some proteins which have different functions.

To investigate the potential role of hepatitis B virus(HBV) preS1 protein in mediating HBV adhesion to liver cell, we prepared recombinant proteins of HBV preS1 in yeast. PCR was performed to amplify the gene of HBV preS1 from the plasmid pCP10/HBV ayw subtype containing the whole fragment of HBV and the PCR product was cloned into pGEM T vector. The gene of HBV preS1 was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, the pGBKT7 plamids containing preSl were transformed into yeast cell AH109. The yeast protein was isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. The results showed that the presence of HBV presl proteins in yeast cells was confirmed by Western slot analysis. the molecular weight of the expressed product was about 30000 Da. The findings indicated that HBV preS1 was successfully expressed in yeast system.

OBJECTIVE: To determine imperatorin as the active ingredient of Angelica dahuricae in Jiedu tongqiao pills by HPLC. METHODS: The separation was performed on Waters Symmetryshielb33 C18(250 mm?4.6 mm,5 ?m) column with detection wavelength of 248 nm. The mobile phase consisted of methanol-water (65 ∶ 35) with flow rate of 1.0 mL?min-1.RESULTS:The linear range of imperatirin were 0.15～1.35 ?g. The average recovery was 100.4%(RSD=1.89%,n=9).CONCLUSION: The method is simple, accurate and reproducible for quality control of Jiedu tongqiao pills.

Objective To evaluate the effect of mechanical stretch preconditioning on pathological stretch-induced activation of γ-aminobutyric acid (GABA) signaling pathway in human type Ⅱ alveolar epithelial cells (AEC Ⅱ).Methods AEC Ⅱ cell line (A549 cells) culturedin vitro were divided into control group (group C), pathological stretch group (group P1) and mechanical stretch preconditioning group (group P2). In group C, A549 cells were cultured routinely. In group P1, A549 cells were exposed to 20% cyclic stretch for 6 hours. In group P2, A549 cells were exposed to 5% cyclic stretch for 60 minutes, and then exposed to 20% cyclic stretch for 6 hours. The cells were harvested for determination of the cell viability by methyl thiazolyl tetrazolium assay, lactate dehydrogeuase (LDH) release was determined by colorimetric method, the levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA), the mRNA expressions of IL-1β, IL-6 and TNF-α were determined by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expressions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid A receptor (GABAAR) were determined by Western Blot.Results Compared with group C, the cell viability of group P1 was significantlydecreased (A value: 0.196± 0.071 vs. 0.886±0.107), the release rate of LDH was significantly increased [(12.3±2.4)% vs. (1.9±0.5)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly increased [IL-1β (ng/L): 138.6±19.7 vs. 32.7±7.4, IL-6 (ng/L): 196.5±31.7 vs. 55.4±13.8, TNF-α (ng/L): 111.3±21.8 vs. 20.8±7.6; IL-1β mRNA (2-ΔΔCT): 2.79±0.44 vs. 0.83±0.12, IL-6 mRNA (2-ΔΔCT): 1.99±0.25 vs. 0.56±0.11, TNF-α mRNA (2-ΔΔCT): 2.54±0.37 vs. 0.72±0.09]; the protein expressions of GAD and GABAAR were significantly decreased [GAD (gray value): 0.38±0.12 vs. 1.75±0.45, GABAAR (gray value): 0.29±0.09 vs. 1.68±0.39; allP < 0.05]. Compared with group P1, the cell viability of group P2 was significantly increased (A value: 0.523±0.132 vs. 0.196±0.071),the release rate of LDH was significantly decreased [(6.9±1.7)% vs. (12.3±2.4)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly decreased [IL-1β (ng/L): 79.2±11.6 vs. 138.6±19.7, IL-6 (ng/L): 89.6±15.6 vs. 196.5±31.7, TNF-α (ng/L): 55.9±11.4 vs. 111.3±21.8; IL-1β mRNA (2-ΔΔCT): 1.92±0.36 vs. 2.79±0.44, IL-6 mRNA (2-ΔΔCT): 1.09±0.18 vs. 1.99±0.25, TNF-α mRNA (2-ΔΔCT): 1.77±0.25 vs. 2.54±0.37]; the protein expressions of GAD and GABAAR were significantly increased [GAD (gray value): 1.26±0.33 vs. 0.38±0.12, GABAAR (gray value): 1.04±0.15 vs. 0.29±0.09; allP < 0.05]. Conclusion The mechanism by which mechanical stretch preconditioning attenuates pathological stretch-induced injury in human AECⅡ is related to the activation of GABA signaling pathway.

Humans ; Hypopharyngeal Neoplasms ; Hypopharynx ; pathology ; Male ; Middle Aged ; Neoplasms, Muscle Tissue

Objective To establish and test the reliability and validity of the disease related psychological pressure scale of pregnant women with chronic HBV infection,so as to provide mental stress assessment tool for the pregnant women.Methods Item pool was developed after the interview and comprehensively retrieved literatures,then after expert review and rebuilt,the scale items were fixed.Totals of 368 pregnant women with chronic HBV infection disease were investigated and the reliability and validity of scale were test.Results The scale had 5 factors and 24 items,and the correlation coefficient between each factor score and total score was 0.712 - 0.894.The correlation coefficient among each factor was 0.409 - 0.631 which was significantly lower than that between each factor score and total score ( P ＜ 0.01 ).The cumulative contribution rate of 5 factors was 64.055％,and tes-retest reliability of 5 factors was 0.856,0.887,0.828,0.813.The Cronbach' s α of internal consistency of 5 factors was 0.788 - 0.865,and the Cronbach' s α of scale was 0.932.Conclusions The scale has good reliability and validity,and can be used for measuring the disease related pregnancy psychological pressure for chronic HBV infection pregnant women.

Objective To evaluate the effect of ulinastatin on γ-aminobutyric acid (GABA) signal pathway in mice with ventilator-induced lung injury (VILI).Methods Thirty-six male Wister mice were randomly divided into 3 groups using a random number table:control group (group C), ventilator-induced lung injury group (group VILI),and ventilator-induced lung injury+ ulinastatin group (group UTI),n =12 in each group.VILI was induced by 4 h mechanical ventilation with tidal volume 40 ml/kg in groups VILI and UTI.Ulinastatin 1×10 5 U/kg was injected intraperitoneally 1 h before ventilation in group UTI,while the equal volume of normal saline was given in groups C and VILI.The mice were then sacrificed,the left lung was lavaged,and broncho-alveolar lavage fluid (BALF)was collected for determination of concentrations of protein,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and intercellular adhesion molecule-1 (ICAM-1).The lung tissues were re-moved for determination of the wet to dry lung weight (W/D)ratio,the mRNA expression level of IL-1β,TNF-αand ICAM-1.The pathological changes of the lungs were determined under light micro-scope and the lung injury scores were also determined.Immunohistochemistry and Western blot were used to detected the protein expression level of GAD and GABAA R.Results The W/D ratio (6.7 ± 2.4 vs.8.5±2.3)and lung scores [(6.9±2.3)scores vs.(1 1.8±2.7)scores]were significantly de-creased in group UTI than those in group VILI.The concentrations of IL-1β[(56±1 1)ng/L vs.(77 ±1 5)ng/L],TNF-α[(105±29)ng/L vs.(1 58±37)ng/L]and ICAM-1 [(205±46)ng/L vs.(293 ±61)ng/L]in BALF in group UTI were significantly decreased than those in group VILI.The mRNA ex-pression levels of IL-1β(1.81±0.26 vs.2.58±0.34),TNF-α(1.61±0.15 vs.2.94±0.27)and ICAM-1 (1.74±0.27 vs.2.79±0.31)were significantly decreased in group UTI than those in group VILI.The protein expression levels of GAD (0.44±0.08 vs.0.18 ±0.04)and GABAA R (0.30 ±0.09 vs.0.15 ± 0.04)were significantly increased in group UTI than those in group VILI.Conclusion Ulinastatin can at-tenuate VILI probably through activating GABA signaling pathway.

Female ; Gastroenteritis ; complications ; Humans ; Infant ; Male ; Nitric Oxide ; physiology ; Rotavirus Infections ; complications ; Seizures ; etiology

OBJECTIVETo study coronary artery stenosis, myocardial ischemia, and left ventricular dysfunction in dual source computed tomography (DSCT) coronary artery angiography.

METHODSTotally 32 patients underwent both DSCT and X-ray coronary angiography within one week to detect coronary artery stenosis separately. Meanwhile, the values of left ventricular myocardial mass (LVMM) , left ventricular ejection fraction (LVEF) , and left ventricular stroke volume (LVSV) were calculated using cardiac function software in DSCT. Electrocardiography was carried out to diagnose myocardial ischemia. The coronary artery stenosis, values of LVMM, LVEF, and LVSV, and myocardial ischemia were compared.

RESULTSThe results of DSCT and X-ray coronary angiography were not significantly different. LVMM, LVEF, LVSV, and myocardial ischemia were significantly different between two- or three-branch groups or between middle or severe groups (both P<0.05) . However, no such significant difference was observed between single and two branch groups and between mild and middle groups. There were no statistically different findings for LVEF and LVSV, but there was statistical deference between LVMM and myocardial ischemia (P<0.05) . For single branch and middle to severe stenosis in left anterior descending (LAD) coronary artery, right coronary artery, left circumflex coronary artery, only the values of LVMM,LVEF,and LVSV in LAD group showed significant difference (P<0.05) .

CONCLUSIONSMore stenotic branches and severer stenosis in coronary artery often are associated with higher incidence of myocardial ischemia and severer left ventricular dysfunction. The stenosis of LAD coronary artery has especially severe impact on cardiac functions. LVMM is a sensitive indicator for myocardial ischemia in coronary artery stenosis.

Aged ; Coronary Angiography ; methods ; Coronary Stenosis ; complications ; diagnostic imaging ; physiopathology ; Female ; Humans ; Male ; Middle Aged ; Myocardial Ischemia ; etiology ; Tomography, X-Ray Computed ; methods ; Ventricular Function, Left ; physiology