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.

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

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

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 summarize our hospital's experience in the diagnosis and treatment of Candida infection in patients with acute necrotizing pancreatitis (ANP).

METHODSSeventy-eight cases with ANP were reviewed. There were diagnoses either by operative finding or by CT scanning. Sixty-two cases received prophylactic antibiotic treatment, other sixteen did not. For cultivation of Candida, blood, urine, stool, sputum and wound drainage fluid culture, and swabs were examined microbiologically for fungi.

RESULTSThe incidence of Candida infection in all patients with ANP was 17.9% (14/78) and mortality was 28.6% (4/14). The incidence of prophylactic antibiotic group was 19.4% (12/62) and mortality was 25.0% (3/12). Non prophylactic group was 12.5% (2/16) and 50.0%.

CONCLUSIONSOur data provide evidence for the clinical significance of Candida infection in patients with ANP. The current prophylactic antibiotic treatment can prevent a septic course of the ANP, but might lead to the evolution of Candida infection.

Adult ; Aged ; Aged, 80 and over ; Candidiasis ; drug therapy ; etiology ; Female ; Humans ; Male ; Middle Aged ; Pancreatitis, Acute Necrotizing ; complications ; drug therapy