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

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

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

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

OBJECTIVETo study the mechanism of plant growing promoted by Mycorrhizal fungus through the difference of proteomes.

METHODThe differential proteomes between uninoculated and inoculated endophytic fungi, Epulorhiza sp. on Anoectochilus roxburghii were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrum.

RESULT AND CONCLUSIONTwenty-seven protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Twenty-two candidate proteins were identified by database comparisons. The function of these proteins mostly involved in signal transduction, metabolic regulation, as well as photosynthesis and substance metabolism. The results indicate that the regulator control system of plant is influenced by fungi action, and the positive regulation improves substance metabolism and photosynthesis, which results in strong plant and higher resistance. It is also deduced that silent genes may exist in endosymbiosis plants.

Basidiomycota ; physiology ; Electrophoresis, Gel, Two-Dimensional ; Host-Pathogen Interactions ; Mycorrhizae ; physiology ; Orchidaceae ; growth & development ; metabolism ; microbiology ; Plant Proteins ; analysis ; Proteome ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUNDLower extremity bursae are very vulnerable to injury during strenuous physical exercises. Understanding the imaging characteristics of normal bursae is essential for early diagnosis of morphological abnormalities. Therefore, we evaluated the normal range of lower extremity bursae in healthy young men using high-resolution ultrasound (HR-US) imaging.

METHODSBursae in the lower extremities were examined by HR-US in 290 Chinese healthy young men with a median age of 18 years (range, 18-23 years). The bilateral suprapatellar bursa (SPB), deep infrapatellar bursa (DIPB), popliteal bursa (PB), and retrocalcaneal bursa (RCB) were imaged and measured for analysis.

RESULTSThe HR-US identification rates of the SPB, DIPB, PB, and RCB were 89.0% (517/580), 55.0% (319/580), 29.4% (171/580), and 49.5% (287/580), respectively. With the assumption that the bursae were normal in 95% of the study participants, the length and width values at the maximal cross-section of the SPB, DIPB, PB, and RCB were ≤18.00 and 6.09 mm, 8.10 and 2.11 mm, 7.67 and 3.93 mm, and 7.82 and 2.04 mm, respectively.

CONCLUSIONSUsing HR-US imaging, we were able to analyze lower extremity bursae with high detection rates in healthy young men. The normal ranges of lower extremity bursa dimensions in healthy young men measured by HR-US in this study could be used as reference values for evaluation of bursa abnormalities in the lower extremity.

Adolescent ; Adult ; Bursa, Synovial ; pathology ; Cross-Sectional Studies ; Humans ; Lower Extremity ; pathology ; Male ; Young Adult

OBJECTIVETo investigate the effects of a new anticoagulant, annexin V derivative (AND) on anticoagulation and antithrombosis.

METHODSHigh and low doses of AND were given to rabbits (groups 1 and 2 respectively) by intravenous (iv) bolus injections followed by half the respective AND doses by iv infusion over 2 hours. Control groups were iv given heparin (group 3) and saline (group 4) of the same volume and procedure as that in group 1 and 2. Blood cell count, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen level were examined before and 15, 30 and 60 min after iv bolus and 2 hours after the end of iv infusion. A 3.0 mm x 15 mm balloon was put into femoral artery to induce endothelial denudation 15 min after IV bolus and the blood pressure of femoral artery was monitored until the pulse pressure recorded 0 mm Hg when the vessel was occluded completely by a thrombus. The femoral arteries were collected and the thrombi were stripped off for measuring their lengths, wet and dry weights.

RESULTSAnticoagulation parameters: APTT at 15 min after iv bolus in AND group was significantly longer than that in group 4 (P < 0.05) but shorter than that in group 3 (P < 0.05); APTT and TT in group 3 were significantly longer than those in groups 1, 2 and 4. Fibrinogen: 0.70 mg/kg AND may decrease fibrinogen. Antithrombosis values: the wet and dry weights in AND groups were significantly lighter than those in group 3 and 4 (P < 0.05). The dry weight in high-dose AND group was remarkably lighter than that in low-dose group (P = 0.029). The length of thrombus in low-dose AND group was remarkably shorter than that in group 4 (P = 0.013), but not for group 3 (P > 0.05). It was remarkably shorter in high-dose AND group than in both group 3 (P < 0.001) and 4 (P = 0.015). The time when pulse pressure equaled to 0 was longer in AND group than in group 4 (P < 0.05), but not in 3.

CONCLUSIONAND is an effective anticoagulant and antithrombosis agent, the highest anticoagulation effect occurs at 15 min after IV bolus. Its anticoagulation effect is not more potent than that of standard heparin, while antithrombosis capacity is more effective. AND in treating thrombosis clinically might be promising.

Animals ; Annexin A5 ; administration & dosage ; pharmacology ; Anticoagulants ; administration & dosage ; pharmacology ; Blood Coagulation ; drug effects ; Disease Models, Animal ; Fibrinogen ; analysis ; Humans ; Injections, Intravenous ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Rabbits ; Random Allocation ; Thrombin Time ; Thrombosis ; prevention & control