Molecular shape under far-red light and red light-induced association of Arabidopsis phytochrome B.

Phytochrome B (phyB) is a plant photoreceptor protein that regulates various photomorphogenic responses to optimize plant growth and development. PhyB exists in two photoconvertible forms: a red light-absorbing (Pr) and a far-red light-absorbing (Pfr) form. Therefore, to understand the mechanism of phototransformation, the structural characterization of full-length phyB in these two forms is necessary. Here, we report the molecular structure of Arabidopsis thaliana phyB in Pr form and the molecular properties of the Pfr form determined by small-angle X-ray scattering coupled with size-exclusion chromatography. In solution, the Pr form associated as a dimer with a radius of gyration of 50 Å. The molecular shape was a crossed shape, in which the orientation of the photosensory modules differed from that in the crystal structure of dimeric photosensory module. PhyB exhibited structural reversibility in the Pfr-to-Pr phototransformation and thermal reversion from Pfr to Pr in the dark. In addition, Pfr only exhibited nonspecific association, which distinguished molecular properties of Pfr form from those of the inactive Pr form.

Systemic gemcitabine combined with hepatic arterial infusion chemotherapy with cisplatin, 5-fluorouracil, and isovorin for the treatment of advanced intrahepatic cholangiocarcinoma: a pilot study.

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) has a poor prognosis and usually presents as advanced disease. Hepatic arterial infusion chemotherapy (HAIC) is a promising option for advanced hepatocellular carcinoma; however, there have been few reports on the use of HAIC in patients with ICC. In the present study, we investigated the efficacy of treatment with systemic gemcitabine (GEM) combined with HAIC with cisplatin (CDDP), 5-fluorouracil (5-FU), and isovorin in patients with advanced ICC. METHODOLOGY: Seven patients with advanced ICC, who received systemic GEM combined with HAIC with CDDP, 5-FU, and isovorin were studied. RESULTS: The response rate after the first chemotherapy cycle was 57.1% (partial response, 4; stable disease, 2; progressive disease, 1). The cumulative survival rates at 1 and 2 years were 85.7% and 28.6%, respectively, and the median survival time was 22.3 months. With regard to grade 3 or 4 adverse reactions, the percentages of patients developing leukopenia, neutropenia, thrombocytopenia, anemia, and anorexia were 28.6%, 28.6%, 42.9%, 14.3%, and 14.3%, respectively. Na treatment-related deaths were encountered. CONCLUSIONS: Although this is a pilot study, we suggest that systemic GEM combined with HAIC with CDDP, 5-FU, and isovorin, may be a useful therapy for patients with advanced ICC.

Effect of transcatheter arterial infusion chemotherapy using iodized oil and degradable starch microspheres for hepatocellular carcinoma.

BACKGROUND: Transcatheter arterial infusion chemotherapy (TAI) using a combination of iodized oil (lipiodol) and degradable starch microspheres (DSMs) has been reported to be superior to TAI with either lipiodol or DSMs separately for the treatment of hepatocellular carcinoma (HCC), based on the results of a prospective randomized study. In the study reported here, we investigated the predictors influencing response and survival in HCC patients receiving TAI using lipiodol and DSMs. METHODS: A total of 50 HCC patients [Child-Pugh A/B, 34/16 patients; maximum tumor size 2.9 cm (mean); tumor number <5/≥5 = 29/21 patients] were administered a mixture of cisplatin and lipiodol, followed by the injection of DSMs. RESULTS: According to the criteria of the Liver Cancer Study Group of Japan, the response [complete response (CR) + partial response (PR)] rate and CR rate were 72 and 38%, respectively [CR, 19 patients; PR, 17; stable disease, 9; progressive disease, 5]. The 1-, 2-, 3-, and 4-year cumulative survival rates were 85, 67, 41, and 35%, respectively, and the median survival time was 32.6 months. Multivariate analysis identified tumor number <5 nodules [odds ratio 10.651, 95% confidence interval (CI) 2.168-52.317; P = 0.004] as an independent predictor of response and des-γ-carboxyprothrombin level <100 mAU/mL [hazard ratio (HR), 0.268, 95% CI 0.091-0.786, P = 0.017] and therapeutic effect CR or PR (HR 0.255, 95% CI 0.099-0.659; P = 0.005) as independent predictors of survival. CONCLUSION: Transcatheter arterial infusion chemotherapy using lipiodol and DSMs might be considered as a potential intervention in HCC patients, especially those with tumors of <5 nodules.

A novel transcatheter arterial infusion chemotherapy using iodized oil and degradable starch microspheres for hepatocellular carcinoma: a prospective randomized trial.

BACKGROUND: We designed a novel transcatheter arterial infusion chemotherapy (TAI) using iodized oil (lipiodol) and degradable starch microspheres (DSM) for hepatocellular carcinoma (HCC) patients. In this study, we investigated the efficacy of TAI using lipiodol and DSM in a prospective randomized trial. METHODS: We randomly divided 45 patients with HCC into 3 groups: TAI using lipiodol (lipiodol group, n = 15), TAI using DSM (DSM group, n = 15), and TAI using lipiodol and DSM (lipiodol + DSM group, n = 15). In the lipiodol group, a mixture of cisplatin and lipiodol was administered. In the DSM group, a mixture of cisplatin and DSM was administered. In the lipiodol + DSM group, a mixture of cisplatin and lipiodol was administered, followed by DSM. RESULTS: The response rates were 40% in the lipiodol group, 53.4% in the DSM group, and 80% in the lipiodol + DSM group, respectively. The response rate tended to improve in the lipiodol + DSM group (lipiodol group vs. lipiodol + DSM group, P = 0.07). The median progression-free survival time was 177 days in the lipiodol group, 287 days in the DSM group, and 377 days in the lipiodol + DSM group. The progression-free survival in the lipiodol + DSM group was significantly better than those in the DSM group (P = 0.020) and the lipiodol group (P = 0.035). There were no serious adverse effects among the 3 groups. CONCLUSIONS: TAI using lipiodol and DSM was superior to TAI using lipiodol only and TAI using DSM only because of improvements in therapeutic effects and progression-free survival.

Pilot study of combination therapy with transcatheter arterial infusion chemotherapy using iodized oil and percutaneous radiofrequency ablation during occlusion of hepatic blood flow for hepatocellular carcinoma.

OBJECTIVE: We have reported that radiofrequency (RF) ablation with balloon occlusion of the hepatic artery (balloon-occluded RF ablation) increases the coagulation area compared with standard RF ablation. In this study, we evaluated the efficacy and safety of combination therapy with transcatheter arterial infusion chemotherapy (TAI) using iodized oil and balloon-occluded RF ablation in patients with hepatocellular carcinoma. PATIENTS AND METHODS: We studied 12 patients with 12 HCC nodules (mean tumor diameter, 27.3 mm). All patients were classified as Child-Pugh Class A. Immediately after TAI using iodized oil, we performed balloon-occluded RF ablation. RESULTS: One treatment session of the combination therapy was done for 10 of 12 nodules (83%). The greatest long-axis and short-axis dimensions of the area coagulated after the combination therapy were 48.8+/- 5.5 mm and 41.9 +/- 4.1 mm, respectively. During follow-up (mean, 33.4 months), there was no local recurrence. The 1, 2, and 3-year survival rates were 100%, 92%, and 83%, respectively. No fatal complications were observed. CONCLUSIONS: The combination therapy is an effective and safe treatment under favorable liver reserve capacity. Using the combination therapy, it is possible to finish one treatment session for patients with HCC nodules measuring less than 3 cm in greatest dimension.

Alpha-tocopherol [corrected] and ascorbic acid attenuates the ribavirin [corrected] induced decrease of eicosapentaenoic acid in erythrocyte membrane in chronic hepatitis C patients.

BACKGROUND: Oxidative damage of the erythrocyte membrane plays an important role in ribavirin-induced anemia. The purpose of the present paper was to assess whether supplementation of alpha-tocopherol and ascorbic acid (vitamins) causes changes in the erythrocyte membrane fatty acid composition during interferon and ribavirin combination therapy for chronic hepatitis C patients. METHODS: Fatty acid compositions in erythrocyte membrane phospholipids were determined by gas chromatography at 0, 2, 4, 8 weeks, and at the end of combination therapy (26 weeks) for interferon with ribavirin in 32 patients with chronic hepatitis C who were randomized to receive vitamins or not (controls). RESULTS: Good compliance with orally administered vitamins and ribavirin were confirmed by their concentrations in erythrocytes or plasma. The hemoglobin level was negatively correlated with the ribavirin concentration at 8 weeks (r = 0.59, P = 0.01) after initiation of therapy in controls, but not in the vitamin group. Among the 26 kinds of fatty acids analyzed, only eicosapentaenoic acid (EPA) significantly decreased at 8 weeks after initiation of therapy (P = 0.03) and at the end of therapy (P = 0.004) in controls. Vitamins did not inhibit ribavirin-induced anemia, but attenuated the decrease of EPA in erythrocytes. The EPA level was negatively correlated with the drop in hemoglobin levels at 8 weeks after initiation of therapy in controls (r = 0.58, P = 0.015), but not in the vitamin group. CONCLUSIONS: Supplementation of alpha-tocopherol and ascorbic acid attenuates the ribavirin-induced decrease of EPA in erythrocyte membrane phospholipids in chronic hepatitis C patients.

Hepatitis C virus core protein inhibits deoxycholic acid-mediated apoptosis despite generating mitochondrial reactive oxygen species.

BACKGROUND: Hepatitis C virus (HCV) core protein is known to cause oxidative stress and alter apoptosis pathways. However, the apoptosis results are inconsistent, and the real significance of oxidative stress is not well known. The aim of this study was twofold. First, we wanted to confirm whether core-induced oxidative stress was really significant enough to cause DNA damage, and whether it induced cellular antioxidant responses. Second, we wanted to evaluate whether this core-induced oxidative stress and the antioxidant response to it was responsible for apoptosis changes. METHODS: HCV core protein was expressed under control of the Tet-Off promoter in Huh-7 cells and HeLa cells. We chose to use deoxycholic acid (DCA) as a model because it is known to produce both reactive oxygen species (ROS) and apoptosis. RESULTS: Core expression uniformly increased ROS and 8-hydroxy-2'-deoxyguanosine (8-OHdG) under basal and DCA-stimulated conditions. Core protein expression also increased manganese superoxide dismutase levels. Core protein inhibited DCA-mediated mitochondrial membrane depolarization and DCA-mediated activation of caspase-9 and caspase-3, despite the increase in ROS by DCA. Core protein inhibited DCA-mediated apoptosis by increasing Bcl-x(L) protein and decreasing Bax protein, without affecting the proportion of Bax between mitochondria and cytosol, resulting in suppression of cytochrome c release from mitochondria into cytoplasm. CONCLUSIONS: HCV core protein induces oxidative DNA damage, whereas it inhibits apoptosis that is accompanied by enhancement of ROS production. Thus, oxidative stress and apoptosis modulation by core protein are independent of each other.

In situ detection of oxidized n-3 polyunsaturated fatty acids in chronic hepatitis C: correlation with hepatic steatosis.

BACKGROUND: Oxidative stress contributes to the pathogenesis of chronic hepatitis C. The aim of this study was to assess the peroxidation of n-3 polyunsaturated fatty acids (PUFAs) in the liver and its relation to hepatic steatosis in chronic hepatitis C. METHODS: We immunohistochemically detected malondialdehyde (MDA)-, 4-hydroxy-2-nonenal (HNE)-, and 4-hydroxy-2-hexenal (HHE)-protein adducts in liver biopsy specimens from 55 patients with chronic hepatitis C. Cells stained positively for HHE-protein adducts were quantified using computer-based image analysis. Fatty-acid composition was determined, by gas chromatography, for the noncancerous portions of resected livers, with or without steatosis, obtained from two patients with hepatitis C virus-associated hepatocellular carcinoma. RESULTS: The detection rate of HHE-protein adducts (63.6%) was significantly higher than that of MDA-protein adducts (21.8%; P < 0.001) or HNE-protein adducts (29.1%; P < 0.001). Areas positively stained for HHE-protein adducts (HHE-positive areas) were significantly larger in 18 patients with steatosis (6.2 +/- 3.6%) than in 17 patients without steatosis (3.4 +/- 2.6%; P = 0.01). Resected liver tissue with steatosis showed a larger HHE-positive area (18.6%) and higher ratio of n-6 PUFA content to n-3 PUFA content (3 : 1) than liver tissue without steatosis (7.2%; 2 : 3). On multivariate analysis, the HHE-positive area (odds ratio, 1.55; 95% confidence interval [CI], 1.08-2.23; P = 0.019) was a factor associated with the presence of hepatic steatosis. CONCLUSIONS: HHE-protein adducts, which are a good marker for oxidative stress, are associated with steatosis in chronic hepatitis C.

Cytokine-dependent anti-viral role of CD4-positive T cells in therapeutic vaccination against chronic hepatitis B viral infection.

There are several lines of evidence suggesting that specific vaccine therapy with a standard hepatitis B virus (HBV) vaccination reduces HBV replication. The aim of this study was to investigate the anti-viral mechanism of vaccine therapy in chronic hepatitis B patients. Nineteen patients were assigned to receive either vaccine therapy (n = 13) or no treatment as a control (n = 6). Vaccinated patients were analyzed for T cell proliferative responses specific for envelope antigen and cytokine production by antigen-specific T cells. ELISPOT and cytotoxicity assays also were carried out for limited blood samples. Serum HBV DNA levels decreased significantly at 3 months after completion of therapy and thereafter as compared to the baseline ones, and were significantly lower in vaccinated patients than in controls at 12 and 18 months after completion of therapy. Vaccination induced antigen-specific CD4+ T cell proliferative responses in four patients (30.8%). The production of high levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by antigen-specific T cells was found in six patients (46.0%) who showed significantly lower HBV DNA levels in serum at 6 (P = 0.04) and 18 months (P = 0.005) after completion of therapy than those without high levels of cytokine production. Vaccination did not induce antigen-specific CD8+ T cells or cytotoxic T cells. These results suggest that envelope-specific CD4+ T cells may control directly HBV replication by producing anti-viral cytokines rather than providing help for cytotoxic T cells in therapeutic vaccination against chronic HBV infection.

Hepatitis C virus infection upregulates expression of the type I interferon receptor in human peripheral blood mononuclear cells.

To assess whether hepatitis C virus infection (HCV) affects type I interferon (IFN) receptor expression, the expression levels of two subunits of this receptor, IFNAR1 and IFNAR2, in peripheral blood mononuclear cells (PBMCs) were determined by flow-cytometric assay in 68 anti-HCV antibody-positive patients (47 positive for HCV RNA and 21 negative for HCV RNA) and 14 healthy controls. The percentages of IFNAR1- and IFNAR2-expressing cells were significantly higher in HCV RNA-positive patients than in HCV RNA-negative patients or healthy controls. In multiple regression analysis, the presence of HCV RNA in serum was independently associated with the expression of both IFNAR1 and IFNAR2 in PBMCs (P=0.007 for IFNAR1 and P=0.003 for IFNAR2). The frequency of IFN-gamma-producing peripheral CD4(+) and CD8(+) cells was also significantly higher in HCV RNA-positive patients than in HCV RNA-negative patients or healthy controls and there was a significant correlation between IFN receptor expression and the frequency of IFN-gamma-producing cells. These results suggest that HCV infection upregulates the expression of the type I IFN receptor in PBMCs through enhanced IFN-gamma production by peripheral CD4(+) and CD8(+) cells as one of mechanisms that regulate the expression of the type I IFN receptor.

Hepatic expression of type I interferon receptor for predicting response to interferon therapy in chronic hepatitis C patients: a comparison of immunohistochemical method vs. competitive polymerase chain reaction assay.

The focus of this study is to determine both mRNA and protein expression levels of the type I interferon (IFN) receptor subunits, IFNalpha receptor (IFNAR1) and IFNalpha/beta receptor (IFNAR2), in the liver, and to assess which quantification method is most useful in predicting response to IFN in chronic hepatitis C patients. Liver biopsy specimens from 41 chronic hepatitis C patients subsequently treated with IFN were immunostained with IFNAR1 and IFNAR2 antibodies, and also analyzed for both receptor subunit mRNA levels, using competitive polymerase chain reaction. Immunostaining of IFNAR1 and IFNAR2 was observed in the cell membrane and cytoplasm of hepatocytes in all liver specimens. Hepatic expression of IFNAR1 (r=0.45, P=0.012) or IFNAR2 mRNA (r=0.46, P=0.009) was weakly correlated with their protein expression in hepatocytes. The labeling indexes of IFNAR1 (136.7+/-94.1 vs. 77.4+/-76.8, P=0.032) and IFNAR2 (153.1+/-80.2 vs. 87.2+/-75.5, P=0.011) in liver specimens were significantly higher in sustained virologic responders (n=17) than in non-sustained virologic responders (n=24), while mRNA levels of either receptor subunit did not differ between response groups. These results suggest that protein expression of the type I IFN receptor in liver is a more useful measure than its mRNA expression in predicting response to IFN in chronic hepatitis C patients.

Expression of type I interferon receptor in liver and peripheral blood mononuclear cells in chronic hepatitis C patients.

To assess whether peripheral blood mononuclear cells (PBMCs) can be substituted for liver tissue in monitoring the hepatic expression of interferon receptors (IFNAR1 and/or IFNAR2) in chronic hepatitis C, we quantified the mRNA of both subunits, using the competitive polymerase chain reaction, in the liver specimens from 37 chronic hepatitis C patients and in PBMCs from 29 of 37 patients. PBMCs from 10 healthy subjects were also quantified for both subunits. Hepatic expression of IFNAR1 and IFNAR2 were more frequent than PBMCs: IFNAR1, 23/27 (85%) vs 7/19 (37%), P = 0.0021, IFNAR2, 34/37 (92%) vs 19/29 (66%), P = 0.0182. Neither subunits was detected in PBMCs from control subjects. The expression level of IFNAR2 in the liver was related to that in PBMCs (r = 0.613, P = 0.0052). These results suggest that hepatitis C virus infection may up-regulate the expression of the type I interferon receptor and that the measurement of IFNAR2 expression in PBMCs may be useful for monitoring its expression in liver.