Effects of Ixeris Chinensis (Thunb.) Nakai boiling water extract on hepatitis B viral activity and hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) infection and hepatocellular carcinoma are major diseases that affect the Taiwanese population. Therefore, the development of an alternative herbal medicine that can effectively treat these diseases is a research target. In this study, we tested Ixeris Chinensis (Thunb.) Nakai boiling water extract (ICTN BWE) in vitro and analysed its effects on the HBV and liver cancer. MATERIALS AND METHODS: We used a human liver cancer cell line (Hep3B, a cell line that continuously secretes HBV particles into a medium) as an experimental model for the screening of various ICTN BWE concentrations and their effects on the HBV in vitro. RESULTS: Our results showed that 75 µg/mL ICTN BWE downregulated the relative expression of the hepatitis B virus surface antigens (HBsAg) to 77.1%. Using the human liver cancer cell lines HuH-7 and HepG2, and 3-(4,5-dimethylthiazol-zyl)-2,5-diphenyl tetrazolium bromide (MTT) and tumour clonogenic assays, we then showed that ICTN BWE inhibits hepatocellular carcinoma growth. CONCLUSION: Fluorescent microscopy of DAPI(4',6-Diamidino-2-phenylindole)-stained nuclei and DNA fragmentation assays confirmed the inhibitory effects of ICTN BWE on liver tumour cell growth through induction of apoptosis.

Predictors of long-term outcomes in patients after elective stent implantation for unprotected left main coronary artery disease.

The purpose of this study was to investigate the predictor of long-term outcomes in patients after stent implantation for unprotected left main coronary artery (LMCA) disease. Coronary stenting has recently been advocated as an alternative procedure for LMCA disease. Information on the predictors of long-term outcomes in patients after stent implantation for unprotected LMCA disease is not clear. Seventy six patients (51 men and 25 women, age 68 +/- 10 years) with medically refractory angina received coronary stenting for unprotected LMCA disease. During a follow-up period of 40 +/- 26 months, 7 patients (9%) died because of cardiovascular disease in 5 (7%) and noncardiovascular disease in 2 (3%). In the other 69 patients, 19 patients (25%) needed repeated percutaneous coronary intervention (PCI) and/or coronary artery bypass grafting (CABG). In a univariate analysis, only female sex was related to the repeated PCI and/or CABG (P = 0.04). A history of cerebral vascular attack (CVA) (P = 0.005), anemia (P = 0.03) and lower left ventricular ejection fraction (LVEF) (P = 0.008) were related to the cardiovascular mortality. A history of myocardial infarction (P = 0.03), a history of CVA (P = 0.02), anemia (P = 0.02), and lower LVEF (P = 0.002) were related to the total mortality. In a multivariate analysis, female sex (P = 0.007; odds ratio 5.29, 95% confidence interval [CI] 1.57-17.80) and young age (P = 0.025; odds ratio 3.92, 95% CI 1.19-12.98) could predict the repeated PCI and/or CABG. Only a history of CVA could predict the cardiovascular mortality (P = 0.027; odds ratio 34.18, 95% CI 1.49-783) and only lower LVEF could predict the total mortality (P = 0.027; odds ratio 13.26, 95% CI 1.34-131). Female sex and young age could predict the repeated PCI and/or CABG in patients after stent implantation for unprotected LMCA disease. Furthermore, a history of CVA could predict the cardiovascular mortality and lower LVEF could predict the total mortality.

Ser-123 of the large antigen of hepatitis delta virus modulates its cellular localization to the nucleolus, SC-35 speckles or the cytoplasm.

Hepatitis delta virus (HDV) is a defective virus and requires hepatitis B virus (HBV) to supply envelope proteins (HBsAg) for maturation and secretion. It is known that two proteins produced by HDV, the small (SDAg) and large (LDAg) antigens, are located in the nucleolus, speckles and the cytoplasm and are involved in genome replication and virion packaging. However, little is known about how they are targeted to the specific sites where they act. A green fluorescence protein fused to LDAg (GFP-LD) has been shown previously to translocate from the nucleolus to SC-35 speckles in the presence of the casein kinase II inhibitor dichlororibofuranosyl benzimidazole. In this study, we determined which amino acids of GFP-LD were responsible for the translocation from the nucleolus to SC-35 speckles and created three GFP-LD derivatives, GFP-LDS2A, GFP-LDS123A and GFP-LDS2/123A. Fluorescence microscopy studies showed that Ser-123 mutants had a high tendency to target SC-35 speckles in both transfected HeLa and HuH-7 cells and suggested that Ser-123, but not Ser-2, plays a role in modulating LDAg translocation to the nucleolus or to SC-35 speckles. This study also demonstrated that HBsAg plays a role in facilitating the transportation of LDAg from the nucleus to cytoplasm. Compared with GFP-LD and GFP-LDS2A, mutants of Ser-123 were less efficiently transported to the cytoplasm and resulted in a lower level of secretion. In contrast, little or no isoprenylation mutant was observed in the cytoplasm of HuH-7 cells expressing HbsAg, suggesting that the isoprenylation of LDAg plays a role in export from the nucleus. Thus, the current study demonstrated that both cis and trans elements modulate HDAg translocation to various subcellular sites.

Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen.

During its life cycle, hepatitis D virus (HDV) produces two forms of delta antigen (HDAg), small delta antigen (SDAg) and large delta antigen (LDAg), which differ in their C-terminal 19 amino acids. Host enzymes termed ADARs (adenosine deaminases that act on double-stranded RNA) are required for LDAg production. These enzymes change the stop codon (UAG) of SDAg to a tryptophan codon (UGG). However, the temporal and spatial regulation of HDV RNA editing is largely unknown. In this study, we constructed three GFP fusion proteins containing different lengths of SDAg and characterized their cellular localization and effects on HDV replication. One of these fusion proteins, designated D(1-88)-GFP, inhibited LDAg but not SDAg production, suggesting that D(1-88)-GFP inhibits HDV RNA editing. Two experiments further supported this supposition: (i). RT-PCR analysis combined with NcoI restriction enzyme digestion revealed that HDV RNA editing was reduced by 42% in HeLa-D(1-88)-GFP when compared with HeLa cells; and (ii). the ratio of SDAg/LDAg production from the reporter RNAs was reduced in cells co-transfected with ADAR-expressing and reporter plasmids in the presence of D(1-88)-GFP. Double fluorescence microscopy found that D(1-88)-GFP was either associated with SC-35 or was adjacent to PML (premyelocytic leukaemia antigen) at nuclear speckles, but D(1-88)-GFP was not co-localized with ADAR, which was mainly located in the nucleolus. In situ hybridization showing co-localization of HDV RNA with D(1-88)-GFP at nuclear speckles suggested that HDV RNA editing might occur in the nuclear speckles and require other nuclear factor(s), in addition to ADAR.