Hypodermin C (HC) cDNA was amplified from recombinant pGEM - T/HC, cloned in frame with the signal sequence in yeast vector pPIC9k. The plasmid was linerarized and transformed into Pichia pastoris GS115 strain by electroporation method. Recombinant strain was screened by G418 resistant, and further confirmed by PCR. The recombinant strain which contains insert was induced in the medium containing 0.5% methanol. The supernatant was collected and then purified by anion exchange chromatography. SDS-PAGE indicated that the target protein is around 28kD. Western-blot showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed it had enzyme activity. Provided a method to produce enough antigens for carrying out extensive immunological analyses.
Animals ; Blotting, Western ; Cell Line ; Chromatography, Ion Exchange ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Gelatin ; metabolism ; Gene Expression ; Immune Sera ; immunology ; Pichia ; genetics ; Plasmids ; genetics ; Rabbits ; Recombinant Proteins ; isolation & purification ; metabolism ; Serine Endopeptidases ; genetics ; immunology ; metabolism ; Substrate Specificity ; Transformation, Genetic

BACKGROUNDPeroxisome proliferator-activated receptor-gamma (PPARgamma) is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element (PPRE), a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-gamma ligands on the PPRE-mediated pathway regulatory system.

METHODSTwo plasmids were constructed: pXOE-PPARgamma, in which the human PPARgamma gene was in the downstream of TFIIIA gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein (EGFP) gene was subcloned into PPRE. The xenopus oocytes were injected with these two plasmids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE-PPARgamma plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase (LPL) in the cells was determined by enzyme labeling linked immunosorbent assay (ELISA).

RESULTSThe expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flavonoids. The levels of LPL in both Hawthorn flavonoids groups and PPARgamma ligand prostagalandin E1 group injected with pXOE-PPARgamma plasmid increased significantly (< 0.001) compared with controls, and a concentration-response relationship was observed between LPL mass and Hawthorn flavonoids.

CONCLUSIONSIt is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARgamma ligand. Hawthorn flavonoids can increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

Alprostadil ; pharmacology ; Animals ; Crataegus ; Female ; Hypolipidemic Agents ; pharmacology ; Lipoprotein Lipase ; biosynthesis ; Medicine, Chinese Traditional ; Oocytes ; metabolism ; PPAR gamma ; physiology ; Peroxisome Proliferators ; pharmacology ; Plasmids ; Response Elements ; physiology ; Xenopus

Carcinoma, Hepatocellular ; genetics ; Genetic Predisposition to Disease ; Liver Neoplasms ; genetics ; Polymorphism, Single Nucleotide

BACKGROUND: Platelet endothelial cell adhesion molecule-1 (PECAM-1), also known as CD31, is mainly distributed in vascular endothelial cells. Studies have shown that PECAM-1 is a very significant indicator of angiogenesis, and has been used as an indicator for vascular endothelial cells. The present study aimed to explore the relationship between the expression of PECAM-1 and the degree of acute lung injury (ALI) and fibrosis in paraquat (PQ) induced lung injury in rabbits. METHODS: Thirty-six adult New Zealand rabbits were randomly divided into three groups (12 rabbits in each group) according to PQ dosage: 8 mg/kg (group A), 16 mg/kg (group B), and 32 mg/kg (group C). After PQ infusion, the rabbits were monitored for 7 days and then euthanized. The lungs were removed for histological evaluation. Masson staining was used to determine the degree of lung fibrosis (LF), and semi-quantitative immune-histochemistry analysis to determine the expression of PECAM-1. Pearson's product-moment correlation analysis was performed to evaluate the relationship between the expression of PECAM-1 and the extent of lung injuries expressed by ALI score and degree of LF. RESULTS: Rabbits in the three groups showed apparent poisoning. The rabbits survived longer in group A than in groups B and C (6.47±0.99 days vs. 6.09±1.04 days vs. 4.77±2.04 days) (P<0.05). ALI score was lower in group A than in groups B and C (8.33±1.03 vs. 9.83±1.17 vs. 11.50±1.38) (P<0.05), and there was statistically significant difference between group B and group C (P=0.03). LF was slighter in group A than in groups B and C (31.09％±2.05 ％ vs. 34.37％±1.62％ vs. 36.54％±0.44％) (P<0.05), and there was statistically significant difference between group B and group C (P=0.026). The PEACAM-1 expression was higher in group A than in groups B and C (20.31％±0.70％ vs. 19.34％±0.68％ vs. 18.37％±0.46％) (P<0.05), and there was statistically significant difference between group B and group C (P=0.017). Pearson's correlation analysis showed that the expression of PECAM-1 was negatively correlated to both ALI score (Coe=–0.732, P=0.001) and degree of LF (Coe=–0.779, P<0.001). CONCLUSIONS: The PECAM-1 expression significantly decreases in New Zealand rabbits after PQ poisoning, and the decrease is dose-dependent. The PECAM-1 expression is negatively correlated with ALI score and LF, showing a significant role in the development of lung injuries induced by PQ.

OBJECTIVETo observe anti-arrhythmic effect of acupuncture pretreatment in the rat of myocardial ischemia and reperfusion (MIR) and to explore the role of cAMP and Gsa protein in beta-adrenergic receptor signaling.

METHODSMIR was produced by ligation and reperfusion of the left anterior descending coronary artery in the rat. Arrhythmic score, content of cAMP and Gsalpha protein in ischemic myocardium were compared among the normal control (NC), ischemia and reperfusion (IR), electroacupuncture (EA) and EA plus propranolol (EAP) groups.

RESULTSThe arrhythmic score in the IR group at 10 min after reperfusion was higher than the NC group (P < 0.01); in the EA group the score was decreased (P < 0.01 vs the IR group); the score in the EAP group was similar to the IR group, much higher than the EA group (P < 0.01). The similar results for the contents of cAMP and Gsalpha protein were found in the ischemic myocardium. It is suggested that EA pretreatment significantly attenuates the arrhythmic incidence rate and the enhancement of the contents of myocardial cAMP and Gsalpha protein induced by MIR, and the attenuating effect is significantly inhibited by the intraperitoneal pretreatment of propranolol, a specific beta-adrenoceptor antagonist.

CONCLUSIONPretreatment of EA can produce anti-arrhythmic effect in the rat of MIR, which is mediated by the post-receptor signaling pathway of beta-adrenergic receptor.

Acupuncture Therapy ; Animals ; Arrhythmias, Cardiac ; prevention & control ; Calcium ; metabolism ; Cyclic AMP ; analysis ; GTP-Binding Protein alpha Subunits, Gs ; analysis ; Male ; Myocardial Ischemia ; metabolism ; therapy ; Rats ; Rats, Wistar ; Receptors, Adrenergic, beta ; physiology ; Signal Transduction ; physiology

Adult ; Carcinoma, Hepatocellular ; surgery ; Cryosurgery ; Female ; Humans ; Liver Neoplasms ; surgery ; Male ; Middle Aged ; Pneumothorax, Artificial

BACKGROUNDLaparoendoscopic single-site surgery (LESS) approaches have been reported for treating various kidney and pelvic procedures, and are feasible and effective in selected patients. In this study, we aimed to present the initial experience and evaluate the efficacy of laparoscopic radical prostatectomy performed through a single incision using a multichannel port.

METHODSBetween July 2010 and April 2011, six patients diagnosed with early stage prostate cancer underwent LESS radical prostatectomy (RP) in our institute. A multichannel port was inserted transperitoneally through a 2-cm umbilical incision. Specially articulating and flexible laparoscopic were used. Some technical tricks and points were applied during the operation to overcome the drawbacks and reduce the difficulties of this approach. Two continuous urethrovesical sutures in both sides were performed to complete both lateral aspects of anastomosis. The two ends of the suture threads were fixed by double Lapro-Clips, instead of the difficult knot-tying.

RESULTSTotal operative time was (265 ± 43) minutes. Mean blood loss was (230 ± 65) ml. All cases were completed successfully, without conversion to open surgery or adding additional abdomen ports. No patient required a blood transfusion and no intraoperative complications occurred. The Foley catheter was removed at the 14th day (range 12th - 16th) after surgery. At the 12th week of follow-up, all patients had an undetectable prostate-specific antigen level. Two patients used 2 or 1 pad for continence daily; other patients had achieved good continence.

CONCLUSIONIn selected cases, LESS-RP is feasible and effective; these technic points and the flexible-articulating instruments are helpful to reduce the operation difficulties.

Aged ; Humans ; Laparoscopy ; methods ; Male ; Prostatectomy ; methods

OBJECTIVETo analyze the relationship between pesticide exposure and adverse pregnancy outcomes in famers.

METHODSA search was conducted to collect the articles about the relationship between pesticide exposure and adverse pregnancy outcomes published worldwide from 1990 to February 2012. Meta-analysis was performed on the collected articles using RevMan 4.2 software.

RESULTSTwelve articles were collected. Compared with the controls, the pesticide-exposed famers showed a combined odds ratio (OR) for spontaneous abortion of 1.52 (95%CI: 1.04 ∼ 2.21; P = 0.03), a combined OR for premature birth of 1.33 (95%CI: 1.09 ∼ 1.61; P = 0.005), a combined OR for dead fetus of 1.22 (95%CI: 1.16 ∼ 1.29; P < 0.01), a combined OR for stillbirth of 1.90 (95%CI: 0.58 ∼ 6.28; P = 0.29), a combined OR for birth defect of 2.02 (95%CI: 0.84 - 4.69; P = 0.12), a combined OR for low birth weight of 1.62 (95%CI: 0.60 ∼ 4.39; P = 0.34), a combined OR for neonatal death of 2.18 (95%CI: 0.54 ∼ 8.88; P = 0.28), and a combined OR for delayed conception of 1.43 (95%CI: 0.93 ∼ 2.18; P = 0.1). Pesticide exposure increased the risks for spontaneous abortion, premature birth, and dead fetus, but was not significantly associated with stillbirth, birth defect, low birth weight, neonatal death, and delayed conception.

CONCLUSIONPesticide exposure can cause adverse pregnancy outcomes in farmers, increasing the risks of spontaneous abortion, premature birth, and dead fetus.

Agriculture ; Female ; Humans ; Maternal Exposure ; Pesticides ; adverse effects ; Pregnancy ; Pregnancy Outcome ; Rural Population

Objective To explore the inhabitants' knowledge about Keshan disease(KD) in endemic area in Heilongjiang Province and analyze its influencing factors. Methods Yongjin Village, a national KD monitoring site in Fanrong Township of Fuyu County in Heilongjiang Province,was selected as an investigating spot. Specially designed questionnaire was applied in the investigation, which included three aspects, demographical information (gender, age, educational level, etc.), KD-related knowledge survey (KD prevention measures including a healthy diet supplementing selenium, preventing microbial infection, inducers such as climate change, fatigue, cold, mental shock, overeating) and KD controlling measures (take prescribed medicine, dietetic regulation and regular exercise, etc.) Results Seventy-senven point six percent(272/352) knew the disease by the name in the endemic area, while only 29.7% (81/272)and 30.8% (84/272)were aware the preventive measures and inducers , respectively. The awareness rates of KD preventive measures and inducers among the KD patients were 6/17 and 9/17 respectively. The influencing factors of the awareness rate of KD name were gender, vocation and the state of education (χ2 value were 9.838,9.878,12.462, all P < 0.05). However, factors influencing the awareness rates of preventive measures were gender and the state of education(χ2 value were 7.400,20.251, both P < 0.05). Conclusions The awareness rates of KD preventive measures and inducers axe low in the endemic area. The major factors influencing the awareness rate of KD are gender and the educational degree.