Effects of salt and drought stresses on antioxidant system and RbohC and RbohF genes expression in Brassica campestris.

To investigate the effects of salt stress and PEG-6000 simulating drought stress on the active oxygen and antioxidant enzyme activities, as well as the expression level of RbohC and RbohF genes, the seedlings of two Brassica campestris, Longyou 6 and Tianyou 2, were treated with U0126 (a MAPKK inhibitor), DMTU (a H2O2 scavenger), as well as DPI and IMD (NADPH oxidase inhibitors). The results showed that under both stresses, H2O2 accumulation as well as antioxidant enzyme (SOD, CAT, APX, GR) activities and the expression of RbohC and RbohF genes increased, while O2-· accumulation decreased. The O2-· accumulation, antioxidant enzyme activity and RbohC and RbohF genes expression in both varieties all significantly decreased. Compared to seedlings with on pretreatment before salt and PEG-6000 simulating drought stress, the accumulation of H2O2 decreased in seedlings pretreated with DMTU, DPI and IMD. However, the accumulation of H2O2 increased in those pretreated with U0126. Those results indicated that the NADPH oxidase, MAP kinase cascade and H2O2 were involved in the regulation of active oxygen production and antioxidant enzyme activity, as well as the expression of RbohC and RbohF under salt stress and drought stress.

[Hepatitis B virus P22(e) inhibit hepatocyte apoptosis via nuclear factor kappa B].

OBJECTIVE: To test whether nuclear factor kappa B plays an important role in the apoptosis-inhibitory effect of hepatitis B virus (HBV) P22(e) protein. METHODS: HepG2 cells were transfected with recombination plasmid pEGFP-HBVP22(e). The Act-D and TNF alpha were used to induce apoptosis. NF-kappa B inhibitor ALLN were used to inhibit the signaling pathway. The activation of NF-kappa B was EMSA, and the nulear translocation of NF-kappa B was determined by immuno-staining. RESULTS: Laser scanning confocal microscopy and EMSA indicated that HBV P22(e) protein enhanced the nuclear translocation of NF-kappa B after apoptosis induction. ALLN treatment inhibited the nuclear translocation of NF-kappa B, and blocked the apoptosis-inhibiting effect of HBV P22(e) protein. CONCLUSION: This study indicates that HBV P22(e) protein inhibits apoptosis of hepatocyte via the NF-kappa B signaling pathway.

[The influence of HBV/P22 protein on the apoptosis of HepG2 cells: an experimental study].

OBJECTIVE: To study the influence of HBV/P22 protein on the induced apoptosis of HepG2 cells. METHODS: In vitro, two HepG2 strains were transfected with pcDNA3.1+ and pcDNA3.1+HBV/P22 respectively and the cells were exposed to Act D and TNF alpha for 6h and then the induced apoptosis was detected by flow cytometry (FCM) and TUNEL technique. Supernatant HBeAg was detected by Abbott reagent. The intracellular expression of HBV/P22 protein was measured by Western blot and immunochemistry. In vivo, three cell groups were inoculated into nude mice by subcutaneous injections. After two weeks, Act D and TNF alpha were injected into the tumors and then the induced apoptosis in the tissues was detected by TUNEL technique. The expression of HBV/P22 protein in the tumor tissues was detected by immunochemistry. RESULTS: In vitro, in HepG2- pcDNA3.1+HBV/P22 cells, supernatant HBeAg was positive and intracellular HBV/P22 protein was positively expressed. The apoptosis proportion of HepG2-pCDNA3.1+HBV/P22 cells was markedly lower than HepG2 and HepG2-pcDNA3.1+ cells (P < 0.05). In vivo, HBV/P22 protein was expressed in the tumor tissues, and the apoptosis proportion in the group injected with HepG2-pcDNA3.1+HBV/P22 cells was markedly lower than those injected with HepG2 and HepG2-pcDNA3.1+cells (P < 0.05). CONCLUSION: HBV/P22 protein could inhibit the induced apoptosis of HepG2 cells both in vitro and in vivo.

[Hepatitis B virus P22e protein inhibits human hepatocellular carcinoma HepG2 cell apoptosis in vitro].

OBJECTIVE: To investigate the effects of the hepatitis B virus (HBV) P22e protein on the apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS: HepG2 cells were transfected with recombinant plasmid pEGFP-HBVP22e and exposed to Act-D and tumor necrosis factor alpha (TNFalpha) treatment to induce cell apoptosis. Flow cytometry was performed to determine the proportion of cells containing sub-G1 DNA to represent the number of apoptotic cells. Laser scanning confocal microscopy was used to observe the nuclear alterations in the apoptotic cells. RESULTS: HepG2EGFP-C2HBVP22e cell strain showed a much delayed apoptosis as well as obviously lowered apoptotic rate in comparison with the HepG2 strain (P<0.01). CONCLUSION: The introduction and expression of extraneous gene HBVP22e significantly inhibits the apoptosis of HepG2 cells.

[Stable expression of HBV C gene mutants in immortalized human B-cell lines].

OBJECTIVE: To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. METHODS: V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. RESULTS: DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant. CONCLUSION: Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.

[Establishment of immortalized B lymphoblast cell line from patients with chronic hepatitis B].

AIM: To establish immortalized B lymphoblast cell line (BLCL) from patients with chronic hepatitis B (CHB) in vitro. METHODS: The peripheral blood mononuclear cells (PBMC) were isolated from the patients with CHB by routine method and incubated with EB virus (EBV) in the presence of CpG DNA motifs and cyclosporin A (CysA) for about 28 days. Morphological characteristic of the established immortalized BLCL was observed by microscope and the expression of CD19 and CD23 on cellular surface was determined by flow cytometry. RESULTS: BLCL was successfully established and could be cultured and propagated for a long time in vitro. CONCLUSION: EBV-immortalized BLCL provides a resource of target cells for further research on the cellular immunity of patients with CHB.

[Investigation of single nucleotide polymorphisms in the promoter region of mannan-binding lectin gene in a Han population from Guangdong].

OBJECTIVE: To investigate the single nucleotide polymorphisms (SNP) in the promoter region of mannan-binding lectin (MBL) gene in a Han population in Guangdong Province. METHODS: A total of 167 blood samples were obtained from this Han population to isolate the genomic DNA from the leucocytes. The polymorphism alleles -550(G/C, named H/L alleles), -220(G/C, X/Y alleles) and +4(C/Tr, P/Q alleles) in the promoter region of MBL gene were detected by PCR with sequence-specific primers and molecular beacon real-time fluorescent PCR, and the frequencies of haplotypes and genotypes were analyzed. RESULTS: The frequencies of several genotypes in the 167 samples were: LYP/LYP, 10(5.9%); HYP/LYQ, 7(4.2%); LYP/LYQ, 94(56.3%); LXP/LXP, 6(3.6%); LYQ/LYQ, 4(2.4%); LXP/LYQ, 29(17.4%); HYP/LYP, 3(1.8%); HYP/LXP, 2(1.2%); HYP/HYP, 12(7.2%). CONCLUSION: The polymorphism genotypes in the promoter region of MBL gene in this chosen population are mostly LYP/LYQ and LXP/LYQ.