Purpose:To construct replication-defective adenovirus which was recombinated with Egr-1 promoter and to Smad7, and to study whether it can express exogenous Smad7 protein in cytoplasm.Methods:Based on Adeno-X~(TM) expression system, the CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-X~(TM) genome, which was transformed into E.coli to get recombinant Adeno-X~(TM) plasmid DNA. The recombinant adenovirus was made and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. Then the Smad7’s location in cells was determined by immunocytochemistry.Results:Identified by restriction endonuclease analysis and PCR, recombinant adenovirus with Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0?10~(11) TCID50/ml. Both endogenous and exogenous Smad7 protein was found to be located in cytoplasm of fibroblast cells.Conclusions:With Adeno-X~(TM) expression system, utilizing the techniques of molecular cloning, recombinant adenoviral vector could be quickly and efficiently constructed which could be packaged into replication-defective Adenovirus and amplified in HEK293 cells. The recombinant adenovirus could express exogenous Smad7 protein in fibroblast cells.[

OBJECTIVETo observe the effect of external application of Zhuangshenling Recipe (ZR) combined with Western medicine (WM) on the heart function of chronic heart failure (CHF) patients of Xin-Shen yang deficiency, interior retention of water-fluid syndrome (XSYDIRWFS).

METHODSTotally 140 CHF patients of XSYDIRWFS were randomly assigned to two groups, the treatment group and the control group, 70 in each group. All patients received WM therapy. Those in the treatment group were applied with ZR at Xinshu (BL15) and Shenshu (BL23), while those in the control group were applied with placebos at Xinshu (BL15) and Shenshu (BL23). The therapeutic course for all was 12 weeks. The integrals of TCM syndrome, grading of cardiac function, brain natriuretic polypeptide (BNP), and 6 min walking distance were observed before and after treatment.

RESULTSAfter twelve weeks of treatment, the effective rate of improved grading of cardiac function, the total effective rate of TCM syndrome efficacy, and the BNP level were obviously better in the treatment group (P < 0.05). There was no statistical difference in 6 min walking distance between the two groups (P > 0.05).

CONCLUSIONExternal application of ZR combined with WM could improve the heart function of CHF patients of XSYDIRWFS.

Aged ; Chronic Disease ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Heart Failure ; drug therapy ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Yang Deficiency ; drug therapy

Cephalosporins are widely used antibiotics owing to their broad activity spectra and low toxicity. Many of these medically important compounds are made chemically from 7-aminodeacetoxycephalosporanic acid. At present, this intermediate is made by synthetic ring-expansion of the inexpensive penicillin G to form G-7-ADCA, followed by enzymatic removal of the side chain to obtain 7-ADCA. The chemical synthetic process is expensive, complicated and environmentally unfriendly. Environmentally compatible enzymatic process is favorable compared with chemical synthesis. In our previous research, metabolic engineered Escherichia coli strain (H7/PG15) was constructed and used as whole-cell biocatalyst for the production of G-7-ADC with penicillin G as substrate. The whole-cell biocatalysis was studied by single factor experiment, including the composition of substrates and the conversion conditions (OD600, pH, concentration of penicillin G, MOPS, glucose, time and FeSO4). After optimization, 15 mmol/L of G-7-ADCA was obtained. The process is convenient, efficient and economic. This work would facilitate the industrial manufacturing and further product research.
Anti-Bacterial Agents ; biosynthesis ; Biocatalysis ; Cephalosporins ; biosynthesis ; Escherichia coli ; metabolism ; Metabolic Engineering

Objective To compare coagulation data measured with domestic produced and imported coagulation testing solutions on SD rat and human by testing prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time(TT), fibrinogen(FIB).Methods Blood samples were obtained from SPF SD rat and human .Domestic produced and imported coagulation testing solutions were applied to test PT , APTT, TT, and FIB.Results Compared to rat data measured with imported coagulation testing solution , data measured with domestic produced coagulation testing solution of PT, APTT, FIB was significantly higher (P<0.05), while, data of TT was statistically lower(P<0.05), and there was no obvious difference in human blood coagulation .Conclusion The data measured with different coagulation testing solution varies on SD rat , so the laboratories are required to establish reference data according to different products .

OBJECTIVE To analyze the major reasons of wound infection after external fixator application and then introduce management measures to prevent following wound infections. METHODS Totally 542 patients adopting external fixators between May 2005 and May 2007 were retrospectively reviewed. All the external fixator-related infections were inspected and the excretions from these infected wounds were collected to perform bacterial culturing. RESULTS The total infection rate of these 542 patients after external fixator application was 2.77%. Among them, six were infected with the bacteria in distraction osteogenesis group and the infection rate was 8.82%; three were infected in bone un-union and bone defect group and the infection rate was 5.36%; whilest the common fracture-fixing group got the lowest infection rate of 0.39%. CONCLUSIONS Wire-crossing positions are the most frequently infected sites after external fixation and the drug-resisted bacteria are the most commonly detected pathogens. Thus, increasing the stability of fixators, enhancing the infection supervision of operation environment, draining the wound thoroughly and using antibiotics rationally are the most effective managing measures to prevent external fixator-related infections in orthopedics.

Objective To explore the relationships between erythropoietin( EPO), endothelin - 1 (ET - 1) and perinatal anoxia. Method ELISA was used to test cord blood EPO and ET-1 in 54 high risk neonates as subjects and 14 healthy neonates as controls.Results The cord blood EPO levels in amniotic fluids turbid Ⅲ degree group and group eclampsia/pre - eclampsia were higher than those in control group (t= 4.0842,3 680 allP

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.

Objective To study the influence of different routes of keyhole limpet hemocyanin ( KLH) immuniza-tion on the T-cell-dependent antibody response in mice.Methods SPF Kunming mice were divided into four groups: the intravenous injection group, subcutaneous injection group, intraperitoneal injection group and control group.Each mouse was injected 200 μg KLH intravenously, subcutaneously or intraperitoneally daily for consecutive 10 days, respectively. Mice in the control group were given solvent injection only.Serum concentration of IgG stimulated by KLH antigen was measured 7 days after the last dosing.Spleen was isolated to calculate the organ coefficient and examined by pathology u-sing hematoxylin and eosin staining.Results Intravenously, subcutaneously and intraperitoneally administered KLH stimu-lated the generation of secondary lymphoid follicles and germinal center to varying degrees, B cell apoptosis, increased a-mount of cells in the marginal zone and other pathological changes were observed in the spleen.Intravenous and intraperito-neal administration of KLH led to more pronounced pathological changes compared with that in the subcutaneous injection group.All of the three administration routes of KLH induced generation of IgG antibody, significantly higher than that in the control group (P<0.05).Intravenous injection of KLH generated the highest concentration of IgG and organ coefficient among the three administration routes ( P<0.05) .Conclusions Different immunization routes do affect the production of IgG antibody, organ coefficient and pathological changes in the spleen, and these differences should be taken into consider-ation when analyzing the T cell dependent antibody response in mice.

OBJECTIVE:To establish an UV-spectrophotometry method for determination of glutaraldehyde in solution METHODS:The concentration of glutaraldehyde in solution was measured at 233nm wavelength which was characteristic absorption peak of glutaraldehyde RESULTS:The calibration curve was linear within the range of 100～1 600?g/ml with r=0 9 998(n=5) The relative standard deviation of the measured results was≤2 13%(n=6) CONCLUSION:The method is simple,rapid and accurate