Long term epidemiological effects of vaccination on hemorrhagical fever with renal syndrome (HFRS) in Shaanxi provincial HFRS epidemic areas / 中华流行病学杂志

Objective To evaluate the protective rate and the variation of HFRS-IgG on hemorrhagical fever with renal syndrome (HFRS) vaccine.Methods Cluster,random sampling and cross-sectional study were used to assess the protective rate of HFRS vaccination.Level of HFRS-IgG was detected with ELISA in epidemic and non-epidemic areas of HFRS.Results Curve equation was obtained as Yprocective rate=(0.863+0.283/Xvaccination term) × 100％ by protective rate with vaccination term.Protective rates showed a reducing trend,90％ after 7-8 years of vaccination,88％ after 10 years,and 94％ on average.Absorbance (A) value of HFRS-IgG was 4 times higher in persons with vaccination than those without,in the epidemic area.Higher antibody level could be obtained after primary vaccination,but the level of antibody had a 50％ reduction after 5-10 years of vaccination,and a 60％ reduction after 10 years of vaccination.Conclusion HFRS antibody had a 50％ reduction after 5-10 years of vaccination.The protective rate of HFRS vaccination had a 90％ loss,after 7-8 years of vaccination.Booster dose was necessary after 7 years of vaccination.

Expression and clinical significance of metastasis-associated in colon cancer 1 (MACC1) in HBV-related hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To clarify the expression and clinical significance of metastasis-associated in colon cancer 1 (MACC1) mRNA in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The expression and distribution of MACC1 were assessed by quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemical staining (IHC) in a cohort of hepatitis B virus-related HCC, including 138 in early (A), 96 in intermediate (B) and 120 in advanced stages (C). The association of MACC1 mRNA with disease progression and outcomes was analyzed by univariate and multivariate Cox analysis.</p><p><b>RESULTS</b>The intratumoral expressions of MACC1 mRNA in HCC stage I (0.001 76, range: 0.000 54 - 0.002 47), stage II (0.002 49, range: 0.000 55 - 0.006 78) and stage III (0.008 35, range: 0.006 86 - 0.009 88) were about 3-, 4- and 14-fold higher than that in the normal liver tissue (0.000 59, range: 0.000 57 - 0.000 60), respectively. Intratumoral expression of MACC1 mRNA increased with disease progression from stage I to stage III. HCC clinical staging classification, age, portal vein invasion and tumor differentiation were significantly associated with intratumoral high expression of MACC1 mRNA (All P < 0.05). Immunohistochemical staining showed that there was an increased MACC1 expression in cytoplasm of HCC cells and positive nuclear staining in some cases. Increased MACC1 mRNA expression could predict poor outcome and recurrence in stage A and B HCC postoperatively. The median tumor-free survival and total survival of patients with high MACC1 mRNA expression were 34.0 and 40 months, respectively, significantly lower than that in those with low expression (48.0 and 48.0 months) (all P < 0.01). Cox analysis showed that Child-Pugh grading and high expression of MACC1 mRNA were independent predictive factors, and high expression of MACC1 was an independent predictive factor affecting the tumor-free survival.</p><p><b>CONCLUSIONS</b>MACC1 mRNA up-regulation is a feature of disease progression in HCC. MACC1 mRNA expression in the HCC may become an independent predictive factor for recurrence and survival in postoperative HCC patients.</p>

Hepatitis B virus X protein inhibits hepatoma cell growth in vitro through p14(ARF)-dependent and p14(ARF)-independent pathways / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of hepatitis B virus X protein (HBx) on hepatoma cell growth through p14(ARF)-dependent and p14(ARF)-independent pathways.</p><p><b>METHODS</b>HBx and p14(ARF) were transfected either separately or in combination into HepG2 cells containing wt-p53 but not expressing p14(ARF). The cells were divided into 4 groups, namely pcDNA3 (control), pcDNA3HBx, pcDNA3p14(ARF), and pcDNA3HBx + pcDNA3p14(ARF) groups. Flow cytometry was used to examine the apoptosis rates and cell cycle progression of HepG2 cells in different groups. The expression of p14(ARF), MDM2, p53, and p21(WAF1) proteins were investigated by detecting the activity of p21(WAF1) promoter-luciferase and using Western blotting.</p><p><b>RESULTS</b>The apoptosis rates of HepG2 cells in pcDNA3HBx and pcDNA3p14(ARF) groups were significantly higher than that in the control group (14.11%, 13.72% vs 10.66%). Compared with the control group, pcDNA3HBx and pcDNA3p14(ARF) groups also showed significantly higher cell percentages arrested at G(0)/G(1) phase (63.62%, 61.75% vs 57.42%), luciferase activity of p21 promoter (1.25-/+0.05, 1.09-/+0.06 vs 0.77-/+0.03) and expressions of p53 and p21(WAF1). The cell apoptosis rate, percentage of cells in G(0)/G(1) phase and expression level of p14(ARF) were even higher in pcDNA3HBx+pcDNA3p14(ARF) group (18.61%, 66.74%, and 3.53-/+0.43, respectively) than in either p14(ARF) or HBx group.</p><p><b>CONCLUSION</b>HBx induces p53 expression through p14(ARF)-dependent and independent pathways to activate p21(WAF1) promoter, leading to G(0)/G(1) arrest and apoptosis of HepG2 cells.</p>

Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.</p><p><b>METHODS</b>The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed.</p><p><b>RESULTS</b>Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured.</p><p><b>CONCLUSION</b>Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.</p>

Screening of binding proteins to interferon-alpha promoter DNA by phage display technique / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display.</p><p><b>METHODS</b>PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis.</p><p><b>RESULTS</b>Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP).</p><p><b>CONCLUSION</b>Many proteins with different functions could bind with interferon alpha promoter.</p>

Study of the down-regulating effect of hepatitis C virus NS5A protein on NACA promoter / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of hepatitis C virus (HCV) non-structure protein NS5A on the activity of calcium-regulating protein alpha subunit of nascent polypeptide-associated complex (NACA) promoter.</p><p><b>METHODS</b>HepG2 cell plasmid pCAT3-NACA, containing NACA promoter, was transfected alone or cotransfected with pcDNA3.1(-)-NS5A, and chloramphenicol acetyl transferase (CAT) enzyme activity was assayed by enzyme-linked immunoassay (ELISA).</p><p><b>RESULTS</b>The CAT activity in the pcDNA3.1(-)-NS5A cotransfection group was 20.7% of the CAT activity in the pCAT3-NACA group.</p><p><b>CONCLUSION</b>HCV non-structural protein NS5A has a down-regulating effect on the promoter of NACA gene.</p>

Hepatitis B virus and deletion of its COOH-terminal forty amino acids: proliferative impact on hepatoma cell line SMMC-7721 / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the biological impact of 40 amino acid deletion at the C-terminal of hepatitis B virus X on the proliferation of hepatoma cells.</p><p><b>METHODS</b>Cells of SMMC-7721 hepatoma cell line were transfected with HBx and its derivative HBx3'-40, harboring the 40 amino acid deletion at the distal C-terminal region. Cell growth curve, colony formation in soft agar plate and tumorigenesis assay in nude mice were used to observe the alterations induced by the transfection of HBx and HBx3'-40. The expression level of PCNA in tumor cells was also investigated.</p><p><b>RESULTS</b>The growth rates of the cells transfected with HBx and HBx3'-40 were markedly increased as compared with that of the control group. The colony formation rates were enhanced in the cells transfected with HBx(48.7 +/- 8.1) and HBx3'-40 (82.8+/-6.0), comparing with the control (26.9 +/- 3.5) %. In the tumorigenic assay, the size and weight of tumors were significantly increased in the cells transfected with HBx (0.412 +/- 0.212, 0.395 +/- 0.159) % and HBx3'-40 (1.476 +/- 0.232, 0.987 +/- 0.279) %, as compared with the control group (0.051 +/- 0.024, 0.033 +/-0.004) %. The expression level of PCNA in tumors was increased in both HBx (59.00 +/- 2.58) % and HBx3'-40 (69.25 +/- 3.77) % transfected cells, comparing with the control (37.67 +/- 2.52) %. Overall, the cells transfected with HBx3'-40 demonstrated the highest proliferative capacity.</p><p><b>CONCLUSION</b>The deletion of 40 amino acids in the C-terminal of HBx is correlated with an enhanced proliferation of hepatoma cells and may play an important role in the malignant transformation of the liver.</p>

Interaction effect of p53 with HBV in hepatoma cell line SMMC-7721 / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the interaction of hepatitis virus B (HBV) and tumor suppressor p53.</p><p><b>METHODS</b>Plasmid pCMVp53 was transfected or cotransfected with pCMVHBVa (wild type HBV) or PCMVHBVb (mutant type HBV) into the hepatoma cell line SMMC-7721 by lipofectamine. Apoptosis cells were labeled by annexin V-FITC and confirmed by flow cytometry. Reporter plasmids PG13-CAT or p21-luc were cotransfected respectively in each group to indicate transactivation activity of p53 and it's effect on p21 promoter. Western blot was performed to observe p53 expression in each group.</p><p><b>RESULTS</b>The group transfected by pCMVp53 alone exhibit higher luciferase activity and higher apoptosis rate, otherwise, p53 expression, enzyme activity of PG13-CAT or p21- luc and cell apoptosis rate were much higher in the group cotransfected by pCMVp53 and pCMVHBVa, but not in the other cotransfected group; HBV replication was enhanced in p53 cotransfected group.</p><p><b>CONCLUSION</b>p53 expression and effects could be enhanced by HBV and p53 had positive regulation effect on HBV replication.</p>

The role of hepatitis B virus X gene and p53 on hepatocellular carcinoma cell growth / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth.</p><p><b>METHODS</b>Two kinds of plasmids containing sense and antisense human wild p53 gene respectively were constructed. SMMU-7721 cells were transfected with HBx, sense-wtp53 antisense-wtp53 separately or cotransfected with either HBx and sense-wtp53 or HBx and antisense-wtp53. Flow cytometry was adopted to measure the apoptosis rates and the effects of HBx on cell cycle progression. The activity of p21(Waf1) promoter-luciferase construct was detected. Growth curves for SMMU-7721 stably transfected with pcDNA3 and pcDNA3HBx were analyzed.</p><p><b>RESULTS</b>After doxorubicin administration, HBx was noticed able to initiate apoptosis of the liver cells. The apoptosis rate was 5.32% in the pcDNA3 transfected and 12.66% in the pcDNA3HBx transfected groups respectively. HBx could also abrogate p53-mediated apoptosis. The apoptosis rate in groups transfected with pcDNA3, pcDNA3wtp53 and pcDNA3HBx + pcDNA3wtp53 was 5.32%, 11.72% and 4.67% respectively. In compared with the normal group, the number of cells in transiently HBx-expressed group and HBx-transfected group decreased 4.79% and 10.25% respectively. HBx inhibited the activity of p21(Waf1) promoter-luciferase constructed (P < 0.05) and promoted cell growth. The growth rate of HBx expression cells was faster.</p><p><b>CONCLUSION</b>Under DNA damage, HBx reduced expression of p21(Waf1) by repressing the activity of p53 protein, followed by disturbing the regulation of G(0)-G(1) cell cycle checkpoint, and promoted the growth rate of hepatoma cells.</p>

Study on the interaction between hepatitis virus C nonstructural protein 4A and calcium modulating cyclophipin ligand by in vivo coimmunoprecipitation / 中华传染病杂志

Objective To prove the interaction between hepatitis virus C(HCV)nonstruetural protein 4A(HCV NS4A)and calcium modulating cyclophilin tigand(CAML)with yeast-two hybrid- ization and coimmunoprecipitation.Methods The gene encoding CAML was cloned,and subcloned into the yeast expression vector pGADT7 and eucell expression vector pcDNA3.1/His-A.The back- cross test between HCV NS4A and CAML was performed in yeast cells.After that,the pCMV-Myc/ NS4A plasmid and pcDNA3.1/His-A-CAML plasmid were co transfected into 293 cells and,then, coimmunoprecipitation and Western blot were performed.Results The gene encoding CAML was cloned sucessfully,and then the gene was subcloned into yeast expression vectors,pGADT7.After the interaction between NS4A and CAML was ensured in yeast cells,the eukaryotic expression vec- tors of NS4A and CAML were constructed and their interaction was ensured again by Co-immunopre- cipitation.Conclusions The interaction between HCV NS4A and CAML is proved.CAML is one of the proteins involved in Ca~(2+)signaling,which suggests that the interaction of HCV NS4A and CAML may be a new clue of the chronic mechanism of HCV infection.Future studies will be required to de- fine the physiologic significance of this interaction.

Screening of interferon?binding proteins by yeast-two hybrid technique / 中华传染病杂志

Objective To screen proteins binding with interferon?(IFN?)from human hepatic cDNA libraty by yeast-two hybrid technique.Methods The IFN?gene was amplified by polymerase chain reaction(PCR)and constructed into pGBKT7 vector as the bait plasmid in yeast-two hybrid system3,pGBKT7-IFN?was then transfected into yeast AH109.The transfected yeast were mated with yeast Y187 containing liver cDNA library plasmid in 2?YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp Leu-His-Ade)and synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-?-gal for selecting.After plasmid extracting and en- zyme cutting analysis,the blue colonies were performed sequence analysis,the results were analyzed by bioinformatics.Results IFN?gene was successfully cloned and expressed in yeast cells.Thirty- four positive colonies were obtained using yeast-two hybrid technique.After sequence analysis,eight clones were found may have a binding effect with IFN protein.Conclusions IFN?genes was success- ful cloned and eight proteins that could bind with IFN?protein were also screened.