Objective: This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. Methods: Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed. Results: The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pl) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °c) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s−1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain. Conclusions: The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of l(+)-2,3-dihydrobutanedioic acid.

OBJECTIVETo explore the effect of arteriosclerosis obliterans (ASO) blood stasis syndrome (BSS) serum on vascular endothelial cell injury and to study the regulation of Taohong Siwu Decoction (TSD) on it.

METHODSUmbilical vein endothelial cell culture system was established. The serum endothelial cell injury model with ASO BSS was prepared. Low, medium, and high concentrations TSD containing serums were respectively added. The endothelial cell proliferation activity was observed by MTT method. Ultrastructures of endothelial cells were observed under transmission electron microscope. Changes of intracellular calcium ion concentration and the cytoskeleton were observed under laser confocal microscope. Contents of ET, NO, and transforming growth factor beta1 (TGF-beta1) in endothelial cell culture supernatant were detected by ELISA.

RESULTSIn ASO BSS serum group endothelial cell proliferation activities decreased, the cell structure was obviously destroyed, calcium ion concentration increased, contents of ET, NO and TGF-beta1 increased significantly (P < 0.01), and ET/NO ratio was imbalanced. After incubating with TSD drug containing serum, endothelial cell proliferation activities and injured cell structures were obviously improved; ET, NO and TGF-beta1 levels decreased (P < 0.05, P < 0.01), ET/NO ratios approximated to the normal level.

CONCLUSIONThe main mechanism of TSD for treating ASO ASS lied in improving injured vascular endothelial cells and endocrine disorder.

Arteriosclerosis Obliterans ; Cell Proliferation ; Drugs, Chinese Herbal ; therapeutic use ; Endothelial Cells ; Humans ; Medicine, Chinese Traditional ; Serum ; Transforming Growth Factor beta1 ; metabolism ; Umbilical Veins

OBJECTIVETo observe the early bone integration of oral implants after injection of exogenous nerve growth factor (NGF) and investigate the effects of NGF on peri-implant osseointegration.

METHODSTwelve New Zealand white rabbits were used in this study to establish bi-mandible implant model. Then local injection of 1 µg NGF was given on the right side of the mandible as experimental group and normal saline only was injected on the left side as control group once a day for seven days. The rabbits were respectively sacrificed at 2, 4 and 8 weeks after surgery. The implant-bone grinding samples were prepared and stained by toluidine blue for general observation, X-ray, histology and bone histomorphometry analysis.

RESULTSThe density of the new bone around implants at 2 and 4 weeks was lower than normal bone. Compared with the control group, the quantity of new bone and bone-implant contact ratio significantly increased in the experimental group. At 8 weeks, the new bone density in both groups was similar to the normal bone. In the experimental group, the haversian system was observed. Bone contact ratio was significantly different between experimental and control group at 2 and 4 weeks, but similar at 8 weeks.[control group at 2 weeks (26.67 ± 3.88)%, 4 weeks (52.59 ± 5.07)% and 8 weeks (97.33 ± 6.75)%, experimental group at 2 weeks (42.24 ± 6.67)%, 4 weeks (72.25 ± 6.30)% and 8 weeks (99.15 ± 4.68)%].

CONCLUSIONSApplying exogenous NGF in the early phase could accelerate the formation and maturation of trabecular bone around the implants and shorten the period of osseointegration. Nerve growth factor could promote osseointegration in the early stage of oral implantation.

Animals ; Bone Density ; Bone and Bones ; Dental Implants ; Mandible ; Nerve Growth Factor ; pharmacology ; Osseointegration ; Prostheses and Implants ; Rabbits

OBJECTIVETo investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro.

METHODSThe expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR.

RESULTSFactor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies.

CONCLUSIONFactor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.

Caspase 7 ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; Interleukin-8 ; biosynthesis ; genetics ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; agonists ; Thromboplastin ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To investigate the effect of lentivirus-mediated shRNA silencing SFRP5 on proliferation,in-vasion and migration of pancreatic cancer cell line PANC-1. Methods A SFRP5-knockdown recombinant plasmid was constructed and successfully transfected it into pancreatic cancer cell line PANC-1,blank plasmid transfection was treated as negative control and untreated cells as blank control group. The expression of SFRP5 at RNA and protein level in cell were detected by real-time PCR and Western blot, CCK-8 assay was applied to examine the effect of SFRP5 silencing on the proliferation, the cell migration of pancreatic cancer cell line PANC-1 was ana-lyzed by Transwell migration assay and cell scratch test was used to examine the cell invasion in PANC-1 cell. Results Stable transfected shRNA-SFRP5 cell of pancreatic cancer line was established successfully.The prolifera-tion capacity of SFRP5 group was significantly higher as compared to the negative control and blank control group by CCK8 assay(P<0.01).Similarly, cell invasion and migration of SFRP5 group were significantly higher compared to the negative control and blank control group(P<0.01). Conclusions SFRP5 lentiviral interference vectors can effectively decrease SFRP5 gene expression in PANC-1 cell of pancreatic cancer, thereby promoting cell proliferation,invasion and migration.

Objective To investigate the effect of lentivirus-mediated shRNA silencing SFRP5 on proliferation,in-vasion and migration of pancreatic cancer cell line PANC-1. Methods A SFRP5-knockdown recombinant plasmid was constructed and successfully transfected it into pancreatic cancer cell line PANC-1,blank plasmid transfection was treated as negative control and untreated cells as blank control group. The expression of SFRP5 at RNA and protein level in cell were detected by real-time PCR and Western blot, CCK-8 assay was applied to examine the effect of SFRP5 silencing on the proliferation, the cell migration of pancreatic cancer cell line PANC-1 was ana-lyzed by Transwell migration assay and cell scratch test was used to examine the cell invasion in PANC-1 cell. Results Stable transfected shRNA-SFRP5 cell of pancreatic cancer line was established successfully.The prolifera-tion capacity of SFRP5 group was significantly higher as compared to the negative control and blank control group by CCK8 assay(P<0.01).Similarly, cell invasion and migration of SFRP5 group were significantly higher compared to the negative control and blank control group(P<0.01). Conclusions SFRP5 lentiviral interference vectors can effectively decrease SFRP5 gene expression in PANC-1 cell of pancreatic cancer, thereby promoting cell proliferation,invasion and migration.

Objective To understand epidemiological characteristics of chronic obstructive pulmonary disease(COPD)in people aged over 40 years in a rural area of Tianjin.Methods Using cluster sampling,1 508 subjects over 40 years old at five villages in Xinkaikou Township,Baodi District,Tianjin were investigated with respiratory questionnaire,lung function test and physical examination.Confirmed patients with COPD were examined by chest roentgenography and electrocardiography.Results One hundred and forty-two subjects in that area suffered from COPD,with prevalence of 9.4%,24 of them (16.9%)were diagnosed as cor pulmonale.Prevalence of COPD increased with age,higher in men (13.5%)than that in women(6.2%),higher in smokers(12.2%)than that in non-smokers(7.2%), higher in those with family history(21.4%)than that in those without it(8.45%),and higher in those with coughing history during their childhood(75.0%)than that in those without it(9.2%),all with a P-value of less than 0.01.Univariate analysis showed that out-door air pollution,cooking,time length of burning firewood during cooking,smoking,coughing history during childhood,gender,age,family history all were predisposing factors for COPD.Multivariate analysis with logistic regression model showed that gender,age, family history were independently predisposing factors for COPD.Quality of life was better in non-COPD subjects than in those with COPD,with statistically significant difference.Conclusions Prevalence of COPD was relatively higher in people of rural Tianjin,with gender,age,family history and outdoor air pollution as main risk factors.

OBJECTIVETo investigate the effect of IL-24 expression on the growth of glioma cells.

METHODSThe IL-24 gene was transfected into rat glioma C6 cells with a retroviral vector. The expression of IL-24 in C6/IL-24 glioma cells was confirmed by RT-PCR. MTT assay and flow cytometry were used to study tumor cell proliferation in vitro. Tumorigenicity in vivo was studied in inbred SD male rats by the growth of intracerebrally inoculated tumor.

RESULTSIt was confirmed by RT-PCR that the exogenous IL-24 gene expressed in C6/IL-24 cell. The C6/IL-24 cell proliferation in vitro and tumorigenicity in vivo were both inhibited compared with its parental C6 cell.

CONCLUSIONIL-24 expression in glioma cells somehow inhibits their growth in vitro and in vivo.

Animals ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Glioma ; metabolism ; pathology ; Interleukins ; biosynthesis ; genetics ; Male ; Neoplasm Transplantation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics

OBJECTIVETo establish the validation method and criteria for counting bacteria and fungi in microbial limit test which is described in the Pharmacopeia of China (ChP) 2005.

METHODAccording to the method set up for validation, the tested microorganisms with known counts were added to samples followed by the determination of the recovery.

RESULTWith different preparing method for testing samples, the recoveries for the tested microorganisms in testing samples were found to be over 70%.

CONCLUSIONValidation method for counting contaminated bacteria and fungi in drugs is recommended to follow the method established in this paper. The recovery for tested microorganisms should be not less than 70%.

Aspergillus niger ; isolation & purification ; Bacillus subtilis ; isolation & purification ; Bacteria ; isolation & purification ; Candida albicans ; isolation & purification ; Colony Count, Microbial ; methods ; Drug Contamination ; Drugs, Chinese Herbal ; standards ; Escherichia coli ; isolation & purification ; Fungi ; isolation & purification ; Staphylococcus aureus ; isolation & purification

OBJECTIVETo synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.

METHODSRecombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.

RESULTSThe purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.

CONCLUSIONSAg85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.

Acyltransferases ; administration & dosage ; isolation & purification ; therapeutic use ; Adjuvants, Immunologic ; therapeutic use ; Animals ; Antigens, Bacterial ; administration & dosage ; isolation & purification ; therapeutic use ; Bacterial Proteins ; administration & dosage ; isolation & purification ; therapeutic use ; Escherichia coli ; Immunity, Cellular ; Interferon-gamma ; Mice ; Mycobacterium tuberculosis ; immunology ; metabolism ; Recombinant Proteins ; administration & dosage ; isolation & purification ; therapeutic use