Search

Objective: To investigate the effect of amino acids around two Leu-based motifs (Leu 7/Ile 8 and Met 16/Leu 17) in the cytosolic tail of porcine Ia-associated invariant chain (Ii) on endomembrane system positioning function., Methods: At first, two Leu-based motifs and amino acids around motifs were mutated through site-directed mutagenesis of the megaprimer PCR method, and the mutant genes were ligated to the vector pEGFP-C1, respectively. Twenty-one mutations of Ii recombinant plasmid were obtained and transiently transfected into the COS-7 cells by Lipofectamine(TM) 2000. Fluorescence microscopy was used to detect the intracellular localization of mutation Ii., Results: Leu 7/Ile 8 and Met 16/Leu 17 independently mediated the intracellular localization of mutation Ii. When one leucine motif was kept and the amino acid around the other leucine motif was mutated, fusion protein GFP-Ii was located in the endomembrane system or distributed in the whole cell., Conclusion: pig Ii comprises two independent sorting signals, and the functional Leu-based sorting signal requires specific neighboring residues.

Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.

Objective: To evaluate the efficacy and safety of adefovir dipivoxil in treatment of decompensated liver cirrhosis patients with YMDD motif mutation during lamivudine therapy., Methods: The disease relapsed in 14 hepatitis B patients with decompensated liver cirrhosis during lamivudine treatment due to the YMDD motif mutation. All 14 patients had positive HVBDNA and active hepatitis, and were evaluated as Child-Pugh Score C (CPS-C). The patients were treated with lamivudine 50 mg/d and adefovir dipivoxil 10 mg/d for 6 months., Results: One patient signed off due to non-hypoxemic hyperlactacidemia; other 13 patients showed decreased serum HBVDNA. All patients had serum HBVDNA < or =10(5) copies/ml and 7 patients had HBVDNA < or =10(4) copies/ml. Six patients regained normal serum ALT level and Child-Pugh scores decreased in all patients., Conclusion: Adefovir dipivoxil has satisfied efficacy and safety in treatment of decompensated liver cirrhosis patients with YMDD motif mutation during lamivudine treatment.

Objective: To study the incidence and the predictive factors of HBV polymerase YMDD variation among patients with chronic hepatitis B and liver cirrhosis during lamivudine therapy., Methods: The clinical data and serial sera of 313 chronic HBV infected patients (249 chronic hepatitis B and 64 liver cirrhosis) treated with lamivudine were collected. YMDD variations were determined by mispairing PCR-RFLP assay. The data were analyzed using SPSS software., Results: The cumulative rates of variation among patients with chronic hepatitis B and liver cirrhosis were 8.84% and 17.19%, 20.91% and 32.40%, 26.92% and 39.56%, 26.92% and 58.79% after 12, 24, 36 and 48 months of lamivudine treatment, respectively. The results of log-rank test and Cox's proportional hazard model analysis indicated that lamivudine monotherapy, low ALT level, high HBV DNA level, and the patients with liver cirrhosis at baseline were significantly related to an occurrence of YMDD variation., Conclusion: This study suggests that lower ALT and higher HBV DNA levels at baseline before lamivudine treatment, lamivudine monotherapy without combining alpha-interferon, and the patients with liver cirrhosis seem to be statistically significant for predicting the occurrence of YMDD variation.

Objective: To investigate the relationship between curative effect and incidence rate of the YMDD mutations of hepatitis B virus during lamivudine treatment for chronic hepatitis B patients twenty-four weeks., Methods: 120 patients with chronic hepatitis B were treated with lamivudine (100 mg/d) over 24 weeks. The levels of serum ALT and HBV-DNA and clinical symptoms were detected at baseline and the 24th weeks after treatment. HBV-DNA YMDD mutation in 17 patients was detected by gene chips at baseline and the 24th weeks., Results: (1) Beside I case of YMDD/YVDD mutation was found before lamivudine treatment, 7 mutation were found after 24 weeks treatment, 2 with pure mutation strain, one of them was YIDD mutation, and the other was YVDD mutation; 2 and 3 others were YVDD/YMDD mutations and YMDD/YVDD mutations respectively. (2) The serum ALT and HBV DNA levels of YMDD mutation group showed no significantly change compared with normal group at baseline and 24th week., Conclusion: The level of serum ALT and HBV DNA showed no significantly changes with the incidence of YMDD mutations at the 24 week treatment

Objective: To explore the point mutation in hepatitis B virus polymerase (HBV P) gene in HBV-infected patients resistant to lamivudine., Methods: HBV P gene was amplified by PCR and the products was sequenced to analyze the YMDD mutation. Then the variants were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the following restriction enzymes: Fok I, Ssp I, Alw441 and were separated by 8.0% polyacrylamide gel electrophoresis., Results: Comparing with the sequences of standard HBV genome, there were 16 patients with G743C mutation and 1 patient with G743A mutation, and the codon ATG turned to ATC and ATA, YMDD motif changed into YIDD. But this kind of YIDD mutation was not proved by PCR-RFLP assay in the 17 patients., Conclusions: The G743C and G743A mutations in HBV P gene, resulting in YMDD motif changed into YIDD, are detected only by direct sequencing, not by PCR-RFLP. The new kind of G743C and G743A point mutations in HBV P gene is important for the detection of HBV P gene YMDD mutation.

Objective: To observe the effect of combined application of Lamivudine and Bushen recipe (BSR) in treating patients with chronic hepatitis B (CHB) and its influence on tyrosine-methionine-aspartate-aspartate (YMDD) motif., Methods: Eighty-eight patients of CHB with positive HBV-DNA were divided into 3 groups, Group A treated with Lamivudine, Group B treated with Lamivudine plus BSR1 and Group C treated with Lamivudine plus BSR2, the serum HBV-DNA, hepatic biological parameters and YMDD motif mutation were observed., Results: The positive rate of HBV-DNA (6.45%) and YMDD motif mutation (3.23%) in Group C were lower than those in Group A respectively, after 52 weeks treatment., Conclusion: Combined application of Lamivudine and BSR could enhance the therapeutic effect in treating CHB to certain extent, and could reduce the mutation of YMDD motif.

Objective: To study the types and emergence time of YMDD motif mutation in hepatitis B virus (HBV) polymerase gene during lamivudine treatment., Methods: The serum samples were collected from 33 patients with HBV DNA rebounding and 2 non-responders after at least one year lamivudine treatment. HBV polymerase gene was amplificated by PCR, then the products were detected by restriction fragment length polymorphism (RFLP) and by direct sequence analysis., Results: The variants with YMDD mutation were 14 out of the 35 patients. Mutation patterns detected in these patients included four YIDD, six YVDD, three YI/VDD and one YI/MDD. The mean emergence time of YMDD variants was 11.07+/-3.65 months after the treatment, and the earliest one and the latest one occurred 5 months and 17 months after the treatment respectively. The emergence times of YIDD, YVDD, YI/VDD were (10.00 +/- 1.41) months, (11.67 +/- 4.41) months and (13.33 +/- 3.31) months respectively, which had no statistical significance (F = 0.543, P < 0.05). Three patients treated with lamivudine 200 mg every day after the mutation were followed up for 6 months, whose HBV variants had not vanished., Conclusions: There are many kinds of HBV variants after lamivudine treatment, including YIDD, YVDD, YI/VDD and YI/MDD. The emergence time of variants is quite variable between different types and the mean time is (11.07 +/- 3.65) months after treatment, and there is no relationship between the type of YMDD mutation and the time of lamivudine administration.