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Article in Chinese | WPRIM | ID: wpr-486283


Objective To establish an HPLC method for the content determination of protodioscin and diosgenin in Dioscoreae Hypoglaucae Rhizoma from different habitats.Methods The chromatographic separation was performed on an Agilent C18 column (4.6 mm×150 mm, 5μm) with mobile phase of acetonitrile-water solution with gradient elution at the flow rate of 0.8 mL/min; the detection wavelength was 208 nm; the column temperature was 30℃; the injection volume was 20μL.Results Protodioscin showed a good linear relationship among the range of 1.73–8.64 μg (r=0.999 6), with the average recovery of 101.98% (RSD=1.53%); Diosgenin showed a good linear relationship among the range of 1.03–8.20μg (r=0.999 1), with the average recovery of 101.60% (RSD=2.41%). The contents of protodioscin and diosgenin in Dioscoreae Hypoglaucae Rhizoma from 10 different habitats were in the range of 0.89%–2.24% and 0.75%–3.22%, respectively.Conclusion The method is simple, accurate and with repeatability, which can be used as quality control method of Dioscoreae Hypoglaucae Rhizoma.

Chinese Journal of Hematology ; (12): 995-999, 2014.
Article in Chinese | WPRIM | ID: wpr-278961


<p><b>OBJECTIVE</b>To disclose the impact of Trp1707Ser mutation on the binding mechanism of rFVIII light chain (rFVIII LC) with VWF.</p><p><b>METHODS</b>Using long-chain PCR technique, we constructed rFVIII LC plasmids of both wild type and Trp1707Ser mutant type. BL21 competent cells were used for protein expression. Gradient renaturation was employed to refold protein. SDS-PAGE and Western blot were performed to identify the molecular weight of expressed protein. GST-Sefinose was used for protein purification and surface plasmon resonance (SPR) was employed to detect binding of B-domain-deleted rFVIII (BDD-rFVIII), wild and mutant rFVIII LC with VWF, respectively.</p><p><b>RESULTS</b>The results of SDS-PAGE and Western blot showed a molecular weight of 110×10(3) of expressed proteins, which were consistent with objective proteins. The expression quantity of wild type was higher than that of mutant type. A concentration-dependent combination of the 3 testing proteins with VWF was found. The KD value of BDDrFVIII (12.2) was lower than that of both rFVIII LCs (wild type 48.9 and mutant type 46.3), whereas there was no discrepancy between wild rFVIII LC and mutant rFVIII LC.</p><p><b>CONCLUSION</b>Trp1707Ser mutation didn't impact the binding of rFVIII LC expressed by BL21 competent cells with VWF. The heavy chain played a more important role in impacting the binding of FVIII with VWF.</p>

Mutation , von Willebrand Factor , Genetics
Article in Chinese | WPRIM | ID: wpr-385372


Objective To elucidate the function and regulatory mechanism of LyGDI involved delayed cell death in the human K562 cells and HL-60 cells induced by 60Co γ-rays. Methods Erythrosine B cells staining was used to count the apoptosis rate. PI staining and flow cytometry were applied to check the cell cycle. The expression of LYGDI and Rac1 was resolved by Western blot by using monoclonal antibody of LyGDI and Racl. The distribution of Racl protein in cells was observed with immunofluorescence by using the confocal microscope. Results The K562 cells showed G2/M phase arrest and the percent age was 71.3%. The apoptosis rate was very low at early post-irradiation stage in the K562 cells. The apoptosis rate was 14% in the K562 cells at 24 h post-irradiation with 8 Gy of γ-rays, and delayed cell apoptosis was present. LyGDI was cleaved in the K562 cells irradiated by 4 Gy 60 Co γ-rays after 24 hours post-irradiation. The expression of Racl protein was not altered at all, but the distribution was changed in the irradiated cells while the Racl protein moved to cell membrane and a little in cell nucleus. The Racl was activated with the losing the binding affinity with the LyGDI. Conclusion LyGDI could promote the delayed cell apoptosis, which is through the activation of the Rac1.