Objective To clone, express and purify human PIF1 protein and analyze its ATPase activity. Methods The PIF1 cDNA was amplified by PCR from HeLa cell cDNA library and inserted to pET24b with histidine tag at its terminus to form pET24b-PIF1 plasmid. The recombinant pET24b-PIF1 plasmid was transformed to RosettaTM 2 (DE3) and the expression of PIF1 protein was monitored by SDS-PAGE analysis. By using fast protein liquid chromatograph (FPLC) system, the PIF1 protein was purified by affinity chromatograph and gel filtration. The ATPase activity of PIF1 was checked by thin layer chromatograph(TLC). Results The PIF1 protein was successfully cloned and expressed in E.coli. Conclusions The purification procedure of PIF1 protein was established using FPLC. The overexpressed and the purified PIF1 helicase has DNA and Mg2+ dependent ATPase activity.

Wip1 is a nuclear protein and a member of serine/threonine specific protein phosphatase type 2C(PP2C)family.It was initially identified as a gene whose expression was induced in response to ? or UV radiation in a p53-dependent manner and a negative feedback regulation of p38MAPK-p53 signaling.Then,Wip1 gene was confirmed a proto-oncogene and amplified or overexpressed in several human tumor types.This review will introduce the structures and functions of Wip1 and details on the signaling process of cancer progression.

Objective To investigate the role of NF-κB in aldosterone-1％NaCl-induced renal injury in uninephrectimized SD rats and the potential mechanisms.Methods Thirty-teo male SD rats were uninephrectomized and treated for 4 weeks.Rats were divided into four groups randomly:control group (n=8),1％NaCl group (1％NaCl in chow,n=8),aldosterone group (1％NaCl in chow,0.75 μg/h aldosterone delayed relase by osmotic mini-pump,SC,n=8),PDTC group (1％NaCl in chow,0.75 μg/h aldosterone,SC,100 mg/kg PDTC,IG,n=8).Systolic blood pressure (SBP),urinary protein,renal function and renal morphologic were observed.The expression of intercellular cell adhesion molecule 1 (ICAM-1) and connective tissue growth factor (CTGF) were measured respectively by Western blotting and real-time PCR.The activity and location of NF-κB in renal cortex were detected by electrophoretic mobility shift assay (EMSA) and immunohistochemisty.Results Rats of aldosterone group exhibited higher blood pressure and more serious renal injury characterized by proteinuria,glomerular sclerosis compared with rats of the 1％ NaCl group.Protein and mRNA levels of ICAM-1 and CTGF were significantly increased inaldosterone group rats than those in 1％NaCl group (all P＜0.05).Moreover,all these changes were associated with an increase in NF-κB activity.Treatment with PDTC which is a specific inhibitor of NF-κB notably alleviated SBP,proteinuria and renal injury in aldosterone-infused rats.Furthermore,PDTC markedly reduced the expression of ICAM-1 and CTGF (all P＜0.05).Conclusion PDTC can alleviate aldosterone-1％NaCl-induced renal injury in uninephrectimized SD rats by preventing the expression of ICAM-1 and CTGF.

Objective To assess the protective effect of remifentanil on cultured human hepatocytes against anoxia-reoxygenation injury. Methods Cultured hepatocytes were divided into 5 groups: group C receiving normoxia as control; groups AR, R, CH, R+CH receiving 15-hour xypoxia followed by 5-hour reoxygenation (group R receiving 5 ng/ml remifentanil, group CH 10 ?mol/L chelerythrine, group R+CH 5 ng/ml remifentanil and 10 ?mol/L chelerythrine before reoxygenation). The content of MDA in the hepatocyte mitochondria were measured. The rate of apoptotic cells was measured by flow cytometry. The expression of protein kinase C mRNA was measured by RT-PCR. Results Anoxia-reoxygenation caused dramatic increase in the content of MDA, the rate of apoptotic cells and the expression of protein kinase C mRNA. The three indexes mentioned above of groups R and CH were between that of groups C and AR (P

Objective To understand the situation of oxidative stress among diabetic nephropathy (DN) patients and observe the antioxidative effect of losartan at increasing dose in DN patients. Methods Thirty type 2 DN patients who neither smoked and nor took antioxidants were selected. The study began with an initial 4-6 weeks screening-treatment. Eligible patients then received losartan 50 mg/d daily for 8 weeks followed by losartan 100 mg/day daily for an additional 8 weeks. Blood glucose and blood pressure were closely monitored over the whole study period. All patients were followed up every other weeks, their 24-hour urine samples,fresh urine and venous blood sample were collected to measure urinary protein and creatinine excretion, urinary 8-OHdG, SOD, TAOC and MDA excretion , serum SOD, TAOC , MDA and other blood biochemistry parameters. Urinary 8-OHdG was determined by capillary electrophoresis and liquid phase chromatography. Results The total 24 hours urinary 8-OHdG excretion and the serum MDA concentration were higher than the normal values. The serum and urine SOD concentrations were lower than the normal values. There was an improvement in urinary 8-OHdG,serum and urine SOD, serum and urine MDA levels with losartan therapy. Compared with losartan 50 mg/d, the antioxidative effect of losartan 100 mg/d was more noticeable. Obvious decrease in 24-hour proteinuria on exposure to losartan was found, without severe adverse effect. Conclusions Oxidative stress damage is active in DN patients. Losartan has antioxidative effect on DN patients. Compared with losartan 50 mg/d, the antioxidative effect of losartan 100 mg/d is more marked, without increasing side effect. Losartan's antioxidative effect may be involved in its beneficial mechanisms on DN.

The aim of this study is to develop the Tripterygium glycosides nanoemulsion gels and investigate its pharmacodynamics. Oleic acid was used as oil phase, polyoxyethylene castor oil as surfaetant, and 1,2-propanediol as cosurfactant to screen the formula of Tripterygium glycoside nanoemulsion using the pseudo-temary phase diagrams. Then the nanoemulsion gels was prepared. The ICR mouse ears were sensitazated by 7% DNCB, and then were excited by 0.3% DNCB to stimulate the model of mouse chronic dermatitis and eczema. The concentrations of IFN-γ, IL-4 and IL-8 in mouse blood were determined by ELISA. The results showed that Tripterygium glycosides nanoemulsion gels could significantly inhibit the swelling of mouse ears(P < 0.01) and ameliorate the edama and erythema of model mouse ears skin. Also it could significantly decrease the expression of IFN-γ and IL-4 in model mouse blood. Tripterygium glycosides nanoemulsion gels had a good therapeutic effect on mouse model of dermatitis and eczema. It was expected to provide a new and long-acting exterernal preparation for the treatment of dermatitis and eczema.
Animals ; Chemistry, Pharmaceutical ; instrumentation ; methods ; Dermatitis ; drug therapy ; immunology ; Drug Carriers ; chemistry ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Emulsions ; chemistry ; Female ; Glycosides ; chemistry ; pharmacokinetics ; Humans ; Interleukin-4 ; immunology ; Interleukin-8 ; immunology ; Mice ; Mice, Inbred ICR ; Nanoparticles ; chemistry ; Tripterygium ; chemistry

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of clevidipine butyrate and its primary metabolite clevidipine acid in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm x 4.6 mm, 5 µm) with a gradient mobile phase consisting of methanol and 5 mmol · L(-1) ammonium formate. A chromatographic total run time of 13.0 min was achieved. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M-H]- --> m/z 234.1 for clevidipine butyrate, m/z 354.0 [M-H]- --> m/z 208.0 for clevidipine acid and m/z 256.1 [M-H]- --> m/z 227.1 for elofesalamide (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The linear calibration curves for clevidipine butyrate and clevidipine acid were obtained in the concentration ranges of 0.5-100 ng · mL and 1-200 ng · mL(-1), separately. The lower limit of quantification of clevidipine butyrate and clevidipine acid were 0.5 ng · mL(-1) and 1 ng · mL(-1). The intra and inter-assay precisions were all below 12.9%, the accuracies were all in standard ranges. Stability testing indicated that clevidipine butyrate and clevidipine acid in dog blood with the addition of denaturant methanol was stable under various processing and/or handling conditions. The validated method has been successfully applied to a pharmacokinetic study of clevidipine butyrate injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg · h(-1) for 0.5 h.

Adenoviridae ; genetics ; Calcium-Binding Proteins ; genetics ; metabolism ; Cells, Cultured ; Female ; Genetic Vectors ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Myometrium ; cytology ; metabolism ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

Objective To investigate the expression of nestin, a type Ⅵ intermediate filament protein in the glomeruli with foot process effacement and the potential relationship between nestin expression in the kidney and the degree of proteinuria. Method Immunohistochemistry was used to determine the localization of nestin in the kidney samples obtained from needle biopsies of normal human and patients with minimal change disease (MCD). Puromycin aminonucleoside (PAN) nephrosis rat models were established by a single intraperitoneal injection of PAN. Both real time quatitative reverse PCR and Western blot methods were applied to evaluate the levels of nestin expression at day 1, 4, 10 and 20 after PAN injection. Results Immunohistochemistry showed that the expression of nestin in glomeruli of MCD patients was significantly reduced compared with normal samples (0.93±0.08 vs 1.65±0.12, P<0.05) . The mRNA and protein expressions of nestin in the rat kidney were transitorily increased by 1.23 folds and 1.48 folds of control group (NC) after 1 day of PAN injection (P<0.05), then decreased quickly in the following days. The mRNA levels of nestin in the kidney were 35.8% and 12.1% of NC after 4 days and 10 days of PAN injection, respectively, (P<0.01) as determined by real time PCR. After 20 days of PAN injury, nestin mRNA expression partly recovered to 65.8% of NC (P< 0.05 ). The protein levels of nestin detected by Western blot presented the similar trend, which were 77.0%, 58.0% and 83.4% of NC after 4 days, 10 days and 20 days of PAN injection, respectively (P<0.05). The degree of proteinuria in puromycin aminonucleoside nephrosis rats was negatively correlated with both mRNA and protein levels of nestin in the kidney(r=-0.667,P<0.05 and r=-0.621 ,P<0.05, respectively). Conclusions The expression of intermediate filament protein nestin is down-regnlated in the kidney characterized with foot process effacement and negatively correlated with the degree of proteinuria in puromycin aminonucleoside nephrosis rats. Nestin may play a potential role in modulating the structure and function of podocyte.

Objective To evaluate the success rate, safety and efficacy of endoscopic retrograde cholangiopancreatography (ERCP) after Billroth Ⅱ gastrectomy. Methods Data of 75 patients with biliary disease after Billroth Ⅱ gastrectomy, who underwent ERCP from January 2007 to November 2009, were retrospectively analyzed. Results In 75 patients, afferent loop intubation was achieved in 69 (92%) and selective cannulation of bile duct were successful in 68 (68/69, 98. 5%). Diagnostic procedures were carried out in 3 patients, and therapeutic in 65 others, which included EST plus stone removal and ENBD in 16, ERBD in 19, EMBE in 18 and EBD plus stone removal and ENBD in 12. Afferent loop perforation occurred in 1 patient (1.3%) and was treated surgically, and 2 acute pancreatitis (2. 6%) were treated conservatively.There was no complication of bleeding. Conclusion ERCP after Billroth Ⅱ gastrectomy is safe and efficiency for biliary disease.