Alfimeprase(ALF)is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase.The target gene alf was obtained from the clone vector p43-alf and cloned into the Pichia pastoris expression vector pPICZ? A.Through high efficiency transformation and Zeocin selection,the recombinant strains of pPICZ?A-alf /GS115 were isolated.In order to achieve a high level expression of recombinant Alfimeprase(rALF),optimization of pH value,methanol daily addition concentration,cell density and methanol induction time points were carried out,and the production of rALF reached up to 425 mg/L.By His?Bind chromatography,the purity of secreted rALF was as high as 95 %.SDS-PAGE and Western blot analysis show that rALF has a molecular weight of about 24 kDa and is bound specifically to anti-His?tag monoclonal antibody.Activity identification results of the modified fibrin plate method demonstrate that the secreted rALF has high fibrinolytic activity.Thus sets up an optimized expression system for ALF,which will play an important role in its further studies and industrial production.

Functional constipation (FC) is a common functional bowel disorder disease that affects life quality of a large number of people. This study aimed to explore the impact of different intensities of electro-acupuncture (EA) treatment for FC patients. Totally, 111 patients with FC meeting the Rome III criteria were randomly assigned to different intensities of EA groups (low and high intensity of EA groups) and medicine-controlled (MC) group. In EA groups, patients were treated with EA at quchi (LI11) and shangjuxu (ST37) bilaterally for 4 weeks, 5 times/week in the first 2 weeks, and 3 times/week in the last 2 weeks. In MC group, 5 mg mosapride citrate was administered orally 3 times/day for 4 weeks. Spontaneous bowel movement frequency each day was recorded using a constipation diary. Self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were used to assess the patients' psychological state. Cortisol (CORT), substance P (SP), and vasoactive intestinal polypeptide (VIP) were evaluated at baseline and at the end of 4 weeks after treatment. As compared with the baseline, there was statistically significant increase in stool frequency every week (P<0.01), but there was no statistically significant difference among the three groups. As compared with the baseline, after 4 weeks of EA therapy, the scores of SDS and serum levels of CORT were decreased significantly in low intensity of EA group (P<0.01), and the serum levels of SP and VIP were increased significantly (P<0.05); the scores of SAS and SDS and serum levels of CORT were decreased significantly in high intensity of EA group (P<0.05), and the serum levels of SP and VIP were increased significantly (P<0.05); the serum levels of CORT and VIP were increased significantly in MC group (P<0.05). As compared with MC group, after 4 weeks of treatment, the serum levels of SP were signifcicantly increased in low intensity of EA group (P<0.01). Low and high intensities of EA could increase the stool frequency, improve the FC patient's anxiety and depression, reduce the serum levels of CORT, and increase the serum levels of SP and VIP effectively. It is concluded that both low and high intensities of EA are effective for FC patients, but there is no significant difference between the low and high intensities of EA.

Objective　In view of the fact that asbestos is not only a key occupational hazard, but also an important environmental pollutant, it is necessary to develop a proper method to decrease the carcinogenecity of asbestos fibers. This study was designed to determine if the surface modification of chrysotile asbestos fiber (CAF) with rare earth compounds (REC) can ameliorate CAF-induced DNA damages in human embryo lung (HEL) cells. Methods　After incubation with REC solution at different concentrations at room temperature for 1 h, natural and REC-pretreated CAF was added to cell culture at various doses. At the selected time as the experiment designed, DNA damages of the HEL cells were detected by Unscheduled DNA Synthesis (UDS) and Single Cell Gel Electrophoresis (SCGE) assays. Results　The UDS induced by natural CAF was elevated with the increase of CAF doses. There was a good dose-response relationship between the UDS and the amount of CAF in the medium and the coefficient of correlation (R) was 0.958 at P<0.05. In REC-pretreated CAF groups, the UDS declined with the increase of REC doses. Both catalase (CAT) and dimethylsulfoxide (DMSO) also reduced the CAF-induced enhancement of UDS. In SCGE assay, CAF induced DNA chain breakage and the magnitude of DNA chain breakage increased in a dose-dependent manner and the coefficient of correlation (R) was 0.992 at p<0.01, while REC-pretreated CAF significantly decreased the induction of DNA chain breakage in a dose-dependent manner(r=0.989, p<0.05). Conclusion　It can be concluded that CAF-induced DNA damages in HEL cells may be partly mediated by oxygen derivatives, and the surface modification of CAF with REC might hide critical sites on the fiber surface, thereby reducing the fiber-mediated production of oxygen derivation and lowering the CAF-induced UDS and DNA chain breakage in HEL cells.

Fourteen compounds were isolated from the n-butanol fraction of the 95% aqueous ethanol extract of the stems and twigs of Strychnos cathayensis by D101 macroporous resin, silica gel, ODS, Sephadex LH-20 column chromatography, and semipreparative RP-HPLC. Their structures were elucidated as ethyl 4-O-β-D-allopyranosyl-vanillate (1), n-butyl 4-O-β-D-allopyranosyl-vanillate (2), n-butyl 4-O-(6′-O-syringoyl)-β-D-allopyranosyl-vanillate (3), n-butyl 4-O-(6′-O-vanilloyl)-β-D-allopyranosyl-vanillate (4), n-butyl 4-O-(6′-O-syringoyl)-β-D-glucopyranosyl-vanillate (5), n-butyl 4-O-α-L-rhamnopyranosyl-syringate (6), methyl 3-methoxy-4-(β-D-allopyranosyloxy) benzoate (7), pseudolaroside B (8), butyl syringate (9), glucosyringic acid (10), methyl syringate (11), methyl 4-hydroxy-3-methoxybenzoate (12), clemochinenoside C (13), and clemoarmanoside A (14), respectively, on the basis of spectroscopic data interpretation and by comparison with literature information. Compounds 1-6 are artificial products of phenolic acid esterified by ethanol or n-butanol. It is noted that the precursors (4-O-(6′-O-syringoyl)-β-D-allopyranosyl-vanillic acid and 4-O-(6′-O-vanilloyl)-β-D-allopyranosyl-vanillic acid) of compounds 3 and 4 are new compounds. The hepatoprotective, anti-inflammatory, antioxidant and cytotoxic activities of compounds 1-13 were evaluated in vitro at a concentration of 10 μmol·L^-1. Compounds 1, 2 and 6-10 exhibited potential hepatic protection effects with cell survival rates ranging from 53.6% to 55.5% (acetaminophen, 45.4% at 8 mmol·L^-1). Compound 4 demonstrated anti-inflammatory activity with nitric oxide inhibitory rate of 74.6%. Compounds 3 and 5 showed potential antioxidant activities with malondialdehyde inhibitory rates of 53.2% and 56.1%, respectively.

OBJECTIVETo study the effects of Yiqi Huayu Bushen Recipe (YHBR) and its disassembled recipes on mRNA expressions of collagen I, III, X, matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMP) in extracellular matrix of cervical disc in model rats of cervical vertebral disc degeneration.

METHODSThe mRNA expressions of collagens, MMP-13 and TIMP-1 were detected by RT-PCR. The strips were scanned by gel imaging system scanner, and the optical density was autocalculated by computer.

RESULTSCompared with those of the sham-operative group, the mRNA expressions of collagen I , Ill and X and MMP-13 of the model rats increased markedly (P < 0.01), which were lowered by YHBR and its disassembled recipes (P < 0.01 or P < 0.05), and the levels after YHBR treatment were significantly different to those after Western medicine treatment. However, no remarkable change was found in TIMP-1 mRNA expression in the model rats (P > 0.05).

CONCLUSIONIn the degenerated intervertebral disc the mRNA expressions of collagen I , III, X and MMP-13 increased, TIMP-1 mRNA expression decreased and the proportion of MMPs/TIMP was in imbalance. YHBR and its disassembled recipes could postpone the degeneration of intervertebral disc through regulating mRNA expressions of collagens and their correlated metabolic enzymes.

Animals ; Cervical Vertebrae ; Collagen ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Extracellular Matrix ; drug effects ; metabolism ; Intervertebral Disc ; drug effects ; metabolism ; pathology ; Intervertebral Disc Displacement ; drug therapy ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

OBJECTIVETo optimize formulation of tanshinone II(A)-loaded PLGA nanoparticles and compare the difference of two methods in preparation and quality of nanoparticles.

METHODThe two methods were nanoprecipitation method and emulsion-evaporation method. Single factor experiments and central composite design and response surface method were used to optimize the formulation of nanoparticles. The nanoparticles were characterized at size, morphology, entrapment efficiency, drug loading, drug recovery rate, crystallinity and drug release in vitro.

RESULTThe mean diameters were 225 nm and 183 nm, the entrapment efficiency were 95.49% and 87.99%, the drug loading were 2.03% and 0.16%, and the drug recovery rates were 38.42% and 17.59% respectively for nanoprecipitation method and emulsion-evaporation method.

CONCLUSIONNanoprecipitation method was better than emulsion-evaporation method for preparation of tanshinone II(A)-loaded PLGA nanoparticles.

Chemical Precipitation ; Crystallization ; Diterpenes, Abietane ; Emulsions ; Lactic Acid ; chemistry ; Nanoparticles ; chemistry ; Particle Size ; Phenanthrenes ; chemistry ; isolation & purification ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Quality Control ; Salvia miltiorrhiza ; chemistry ; Technology, Pharmaceutical ; methods ; Volatilization

Adult ; Aged ; Aged, 80 and over ; Colles' Fracture ; therapy ; Female ; Fracture Fixation ; Humans ; Male ; Middle Aged ; Splints

Objective To explore the significance of designing with monitoring-flap in massive com- pound bone grafts for repairing massive bone defects in extremities.Methods From January 2001 to De- cember 2004,large bone defects in 19 patients(11 men and 8 women,age:6 to 35 years,mean age:18.6 years)were repaired by vascularized free fibular transplant with a monitoring-flap combining with massive deep frozen bone allografts.Average length of the bone defects was 16.6 cm(range,12 to 25 cm).A 7 days' con- tinuously clinical examination including observing the color,turgor,temperature,capillary refill,and bleeding after a needle sticking of the monitoring-falps were used postoperatively,if any one of these were abnormal,the circulation of the compound bone grafts must be in danger and some measures such as re-operation should be taken immediately.Dynamic image analysis was used for evaluating the bone union.Results One monito- ring-flap was vascular artieulo,and the articulo was relieved after exploration and resection of vein thrombus; another one was marginal part necrosis;the remains were normal.All of monitoring-flaps healed normally after 23.2 months(range,6 to 54 months)follow-up.15 patients had the radiographic evidence of bone unions 3 months after surgery.11 patients had been removed intermal fixation,complete bone unios were found one year postoperatively.Conclusion Designing with monitoring-flap in massive compound bone grafts for repairing massive bone defects,and can clearly understand the circulatory statue of compound bone grafts and early pre- dict the final results of massive bone allografts.

OBJECTIVETo optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid.

METHODUsing the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA.

RESULTIncreasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose.

CONCLUSIONProduction of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.

Biomass ; Biotechnology ; methods ; Dose-Response Relationship, Drug ; Fermentation ; Glucose ; pharmacology ; Oleanolic Acid ; biosynthesis ; Saccharomyces cerevisiae ; cytology ; drug effects ; metabolism