ABSTRACT
3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) is the key enzyme to synthesize eight-carbon sugar in plant and gram-negative bacterial cell wall. To analyze the polymerization and characterization in plant KDO8PS, the candidate gene was cloned from fresh Phyllostachys edulis seedling by RT-PCR. The open reading frame of PeKDO8PS is 876 bp deduced into 291 amino acid residues. The target protein was overexpressed in Escherichia coli induced by IPTG and then lager amount of fusion protein was purified through two-step methods with affinity chromatography and size-exclusion chromatography (SEC). SEC analysis shows that PeKDO8PS protein existed mainly in the form of dime in solution. Glutaraldehyde cross-linking experiments confirmed that the enzyme could form dimers. Further we identified that KDO8PS at high concentration two dimers could form tetramer in aqueous solution by analytical ultracentrifuge (AUC) analysis. The pH of the catalytic reaction was between 4.0 and 9.0, the optimum pH value was 8, the thermal stability range was between 25 and 65 ℃, and the optimum temperature was about 55 ℃. The enzyme activity was inhibited by some metal ions at lower concentrations, especially in the presence of Fe3+metal ion and activated by metal protease inhibitor EDTA at low concentration.
ABSTRACT
[ Objective ] To screen the duck model with congenital infection of duck hepatitis B virus (DHBV) by polymerase chain reaction (PCR) method. [Methods] Serum DHBV-DNA level in one-day-old ducklings was detected by PCR method and was compared with that by Dot-blot method. Ducklings with serum DHBV-DNA being negative confirmed by PCR method were inoculated DHBV-DNA positive serum to establish acquired infection models. Pathological features of liver tissues in the congenital infec tion model and the acquired infection model were also observed. [Results] Sensitivity and specificity of PCR detecting serum DHBV-DNA were superior to those of Dot-blot method. In the congenital infection model, viremia maintained long time, the titer of serum DHBV-DNA was high and the inflammatory af fection in liver tissues was slight as compared with those in the acquired infection model. [Conclusion] The duck model with congenital infection of DHBV screened by PCR method is more suitable for the phar macological and pharmacodynamic research of drugs for chronic hepatitis B.
ABSTRACT
Objective To investigate the effect of ligustrazine on the intracellular translocation of Smad protein in HSC-T6 cell line.Methods HSC-T6 cell was cultured with ligustrazine at the dose of 10-5 mol/L in the culturing dish for 2 hours.After the culturing,the translocation of Smad-2 and Smad-4 proteins was detected by immunofluorescence assay.Results There was no evidence of the translocation of Smad-2 and Smad-4 proteins in HSC-T6 cell after the culturing.Conclusion Ligustrazine can block the signal pathway of TGF-?/Smad,which may be one of its important mechanisms of inhibiting the proliferation of HSC-TS cell.
ABSTRACT
Objective To evaluate the protective action of Phyllanthus orinaria(PU)for immune liver injury in mice.Methods Forty-eight NIH mice were allocated randomly to high-dose(HG)PU group,low-dose(LG)PU group,bifenbate group(BG),normal control group(NCG),and model group(MG).From the first day of establishing the models,20 g ? kg-1 of PU solution was given for gastric perfusion(GP)to HG PU group,10 g ? kg-1 of PU solution for GP to LG PU group,0.15 g?kg-1 of bifenbate solution for GP to BG group,and the equal amount of normal saline to the normal control group and the model group,qd for 12 days.Serum ALT level and visceral parameters of liver and spleen were determined,and the liver pathological feature was also examined.Results The ALT level,parameters of liver and spleen were markedly decreased in BG group(P 0.05).Conclusion PU has a good effect on counteracting the immune live injury in mice by markedly decreasing the activity of serum transaminase and visceral parameters of liver and spleen,and improving the necrosis of hepatic cells.