Bile acid regulates c-Jun expression through the orphan nuclear receptor SHP induction in gastric cells.

Bile reflux is considered to be one of the most important causative factors in gastric carcinogenesis, due to the attendant inflammatory changes in the gastric mucosa. In this study, we have assessed the molecular mechanisms inherent to the contribution of bile acid to the transcriptional regulation of inflammatory-related genes. In this study, we demonstrated that bile acid induced the expression of the SHP orphan nuclear receptor at the transcriptional level via c-Jun activation. Bile acid also enhanced the protein interaction of NF-kappaB and SHP, thereby resulting in an increase in c-Jun expression and the production of the inflammatory cytokine, TNFalpha. These results indicate that bile acid performs a critical function in the regulation of the induction of inflammatory-related genes in gastric cells, and that bile acid-mediated gene expression provides a pre-clue for the development of gastric cellular malformation.

H pylori eradication: a randomized prospective study of triple therapy with or without ecabet sodium.

AIM: To investigate whether adding ecabet sodium to the standard triple therapy for H pylori infection improve eradication rate. METHODS: Two hundred and fifty-seven H pylori-infected patients were randomly assigned to standard triple therapy (group A, n = 129) or triple therapy plus ecabet sodium (group B, n = 128). Successful eradication was defined as a negative (13)C-urea breath test 6-8 wk after completion of treatment. RESULTS: After completion of therapy, 194/257 patients showed negative (13)C-urea breath test results. According to intention-to-treat analysis, the infection was eradicated in 93/129 (72.1%) patients in group A and 101/128 (78.9%) in group B (P = 0.204). Per-protocol analysis showed successful eradication in 93/118 (78.8%) patients from group A and 101/114 (88.6%) from group B (P = 0.044). There were no significant differences in the side effects experienced by the patients in the two treatment groups. CONCLUSION: Our results suggest that the addition of ecabet sodium improves the efficacy of the standard triple therapy for H pylori.

Hepatitis B virus X protein induces hepatic steatosis via transcriptional activation of SREBP1 and PPARgamma.

BACKGROUND & AIMS: Hepatic steatosis occurs frequently in patients with chronic hepatitis B virus (HBV) or chronic hepatitis C virus (HCV) infection. Recently, several studies suggested that steatosis plays an important role as a cofactor in other liver diseases such as hepatic fibrosis, hepatitis, and liver cancer. In contrast to HCV, however, the molecular mechanism by which HBV mediates hepatic steatosis has not been clearly studied. Here, we show the molecular mechanism by which hepatitis B virus X protein (HBx) induces hepatic steatosis. METHODS: Lipid accumulation and the expression of various lipid metabolic genes were investigated in HBx-transfected Chang liver cells, HepG2-HBx stable cells, and HBx-transgenic mice. RESULTS: Overexpression of HBx induced hepatic lipid accumulation in HepG2-HBx stable cells and HBx-transgenic mice. It also up-regulated the messenger RNA and protein levels of sterol regulatory element binding protein 1, but not peroxisome proliferator-activated receptor alpha (PPARalpha). Moreover, we also determined that the expression of HBx increases PPARgamma gene expression as well as its transcriptional activity in hepatic cells, mediated by CCAAT enhancer binding protein alpha activation. Finally, we showed that HBx expression is able to up-regulate the gene expressions of various lipogenic and adipogenic enzymes in hepatic cells. CONCLUSIONS: We showed that the increased HBx expression causes lipid accumulation in hepatic cells mediated by sterol regulatory element binding protein 1 and PPARgamma, which could be a putative molecular mechanism mediating the pathophysiology of HBV infection.

Phosphorylation of the large subunit of replication factor C is associated with adipocyte differentiation.

Adipocyte differentiation is an ordered multistep process requiring the sequential activation of several groups of adipogenic transcription factors, including CCAAT/enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma, and coactivators. Here we show that replication factor C 140, which was known to act as a coactivator for CCAAT/enhancer-binding protein-alpha in our previous study, was phosphorylated on the proliferating cell nuclear antigen-bindng domain during the adipocyte differentiation process. Calmodulin-dependent protein kinase II was responsible for phosphorylating replication factor C 140 in the process of adipocyte differentiation. Ser518 of replication factor C 140 was identified as a major target of calmodulin-dependent protein kinase II phosphorylation in vitro. Calmodulin-dependent protein kinase II inhibitor attenuated phosphorylation of replication factor C 140 by differentiation inducers and blocked replication factor C 140-derived transcriptional activation. Taken together, these findings demonstrate that calmodulin-dependent protein kinase II signaling leads the cooperative transactivation of CCAAT/enhancer-binding protein-alpha and replication factor C 140 through an increase in replication factor C 140 phosphorylation, and subsequently enhances the transcriptional activation of target genes involved in adipocyte differentiation.

Molecular mechanism of hypoxia-mediated hepatic gluconeogenesis by transcriptional regulation.

Until now, it is known that hypoxia increases the glycolytic enzyme expression at the transcriptional level. Here, we show evidence that hypoxia increases hepatic glucose output and HIF-1 and ATF-2-mediated transactivation of phosphoenolpyruvate carboxykinase (PEPCK), which plays a critical role as a rate-limiting enzyme in gluconeogenesis, gene in liver. HIF-1 directly bound to the specific PEPCK promoter region through its cognate binding element and found as an active complex with coactivator CBP. Additionally, ATF-2 was also involved to regulate hypoxia-dependent PEPCK transcription in the transcriptional complex with HIF-1 and CBP. Interestingly, retinoic acid (RA) signaling induced the recruitment of HIF-1 on the PEPCK promoter, resulting from the functional interaction of HIF-1 and ATF-2 with coactivator CBP. Taken together, these results suggest that hypoxia signaling leads the hepatic glucose production and release via the increased gene expression of gluconeogenic enzymes, possibly playing a role in providing glucose to other tissues, such as endothelial, brain and muscle cells.

Relationships between the expressions of CDX1 and CDX2 mRNA and clinicopathologic features in colorectal cancers.

BACKGROUND: CDX1 and CDX2 are members of the caudal-type homeobox gene family and control the proliferation and differentiation of intestinal mucosal cells. Their expressions are commonly reduced in colorectal cancer, but reports about the relationships between their expressions and clinicopathologic features are rare. The aim of this study was to examine the expressions of CDX1 and CDX2 mRNAs in colorectal cancers and to assess the relationships between their expressions and clinicopathologic features. METHODS: CDX1 and CDX2 mRNA expressions were analyzed by real-time polymerase chain reaction in 48 colorectal cancers and in adjacent non-tumorous normal mucosal tissue. RESULTS: CDX1 and CDX2 mRNA expressions were significantly reduced in colorectal cancer tissues versus normal mucosal tissues (p=0.001, p=0.042, respectively). As compared with paired normal mucosal tissues, colorectal tissues showed reduced CDX1 mRNA expression in 64.6% (31/48) and reduced CDX2 mRNA expression in 66.7% (32/48) of cases. A statistically significant positive correlation was found between the expressions of CDX1 mRNA and CDX2 mRNA in colorectal cancer (r=0.543, p<0.001). However, the expressions of CDX1 and CDX2 mRNAs were not related to age, sex, cancer location, differentiation, lymphatic or vascular invasion, lymph node metastasis, stage or serum carcinoembryonic antigen level. CONCLUSIONS: CDX1 and CDX2 mRNA expressions were found to be significantly reduced in colorectal cancers but these expressional changes were not found to be related to clinicopathologic features.

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection.

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.

Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma.

Hepatocellular carcinomas (HCCs) show genomic alterations, including DNA rearrangements associated with HBV DNA integration, loss of heterozygosity, and chromosomal amplification. The genes most frequently involved are those encoding tumor suppressors. The p16INK4A tumor suppressor gene frequently displays genetic alteration in HCC tissues. The present study was performed to examine the incidence of methylated p16INK4A in the sera of liver cirrhosis (LC) and HCC patients, and to evaluate its role as a tumor marker of HCC. The sera of 23 LC patients and 46 HCC patients were examined in this study. The methylation status of p16INK4A was evaluated by methylation-specific PCR of serum samples. Methylated p16INK4A was detected in 17.4% (4/23) of LC patients and in 47.8% (22/46) of HCC patients. No association was demonstrated between p16INK4A methylation and serum AFP level. As the status of p16INK4A methylation was not associated with serum AFP level, it may have a role as a tumor marker of HCC.

[The effect of endotoxin on the expression of tissue factor in the CCl4-treated rat liver].

BACKGROUND/AIMS: Endotoxemia is known to cause hepatic microcirculation disturbance and hepatic injury through blood coagulation and thrombosis in the cirrhotic patients. This study was to investigate the effect of endotoxin on the expression of tissue factor in rat liver with persistent injury. METHODS: Male Sprague-Dawley rats were used for this experiment. Persistent hepatic injury was induced by injecting 0.1 mL of CCl4 and 0.1 mL of mineral oil per 100 grams of body weight, intraperitoneally. Twenty-four rats were divided into 4 groups and lipopolysaccharide (LPS, E. coli O111:B4) was injected intraperitoneally in 3 groups in a dose of 200 microgram/kg, 400 microgram/kg, and 800 microgram/kg, respectively. Expression of tissue factor was evaluated by real time PCR method. SYBR Green I, a DNA binding fluorophore was used for the detection of PCR product. RESULTS: Expression of hepatic tissue factor tended to increase according to the amounts of LPS. Specially, the expression of tissue factor was significantly increased in the group into which 400 microgram/kg of LPS was injected, compared with control group (p<0.05). CONCLUSIONS: Expression of tissue factor tends to increase according to the amounts of LPS in the persistent hepatic injury. LPS may play an important role in the expression of tissue factor in the CCl4-treated rat liver.

[Relationship between serum HBV DNA levels and liver histology in chronic hepatitis B].

BACKGROUND/AIMS: Serum HBV DNA levels are correlated with hepatic histologic activity in chronic HBV infection based on HBeAg. Liver injury may persist, even though HBV DNA are not detected by hybridization assay. This study was to investigate whether serum HBV DNA levels determined by more sensitive quantitative method are correlated with histologic activities in chronic HBV infections. METHODS: This study included 66 chronic HBV infected patients. HBV DNA level was quantified by Cobas Amplicor HBV Monitor. RESULTS: Serum HBV DNA levels in HBeAg-positive patients were significantly higher than HBeAg-negative patients. In HBeAg-positive patients, serum HBV DNA levels showed a significant negative correlation with portal-periportal activity and fibrosis (r=-0.451, -0.446 respectively). AST levels were correlated with lobular, portal-periportal activity and fibrosis (r=0.432, 0.365, 0.301 respectively), whereas ALT levels were related to lobular activity (r=0.294). Elevated AST levels predicted lobular activity, portal-periportal activity, and fibrosis with moderate to severe degree (OR 1.733, 95% CI 1.083-2.775; OR 1.518, 95% 1.028-2.243, p=0.336; OR 17.897, 95% CI 1.517-211.208, p=0.022, respectively). CONCLUSIONS: In HBeAg-positive patients, serum HBV DNA level correlates inversely with histologic activity. On the other hands AST level correlates with histologic activity and the stage of moderate or severe degree.

Serum hepatitis B virus (HBV) DNA levels at different stages of clinical course in patients with chronic HBV infection in an endemic area.

The aims of this study were to investigate serum hepatitis B virus (HBV) DNA levels at different clinical stages in patients with chronic HBV infection, and to determine the serum HBV DNA level that discriminated HBeAg-negative chronic hepatitis B(CHB) cases from inactive HBsAg carriers. In all, 222 patients, encompassing 68 HBeAg-positive CHB patients (HBeAg-positive, ALT-elevation), 89 HBeAg-negative CHB patients (HBeAg-negative, ALT-elevation), and 65 inactive HBsAg carriers (HBeAg-negative, ALT-normal), were tested. The ALT levels had been tested more than twice during the previous six months, and the serum HBV DNA levels were quantified by a polymerase chain reaction-based assay. The serum HBV DNA levels of the HBeAg-negative patients were significantly lower than those of the HBeAg-positive patients (median 2.7 x 10(4) vs. 1.6 x 10(8) copies/mL; p=0.000). In addition, the HBV DNA levels of the HBeAg-negative CHB patients were significantly higher than those of the inactive HBsAg carriers (median 2.2 x 10(5) vs. 3.2 x 10(3) copies/ mL; p=0.000). The optimal HBV DNA level for discriminating HBeAg-negative CHB cases from inactive HBsAg carriers was 2.0 x 10(4) copies/mL. The serum HBV DNA levels were lower than the cutoff value in 72.3% (47/65) of the inactive HBsAg carriers, and in 31.5% (28/89) of the HBeAg-negative CHB patients. The serum HBV DNA levels differed significantly between these two groups. However, the levels in the two groups overlapped extensively, preventing the definition of a differentiation cut-off value.

[Natural YMDD motif mutations of HBV polymerase in the chronic hepatitis B virus infected patients].

BACKGROUND/AIMS: Lamivudine, a nucleoside analogue has been widely used as an effective antiviral agent for the treatment of patients with chronic hepatitis B infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine very frequently occurs after long-term use of lamivudine. It is well known that the mutation is selected by the lamivudine. We hypothesized that a few mutant strains of YMDD motif are present as quasispacies before the lamivudine treatment, are selected by the treatment, and breakthrough during treatment. We investigated the prevalence of the YMDD motif mutants in patients with chronic hepatitis B infection who had not been treated by antiviral agents before. METHODS: The study included the serums of 40 patients with chronic heptitis B infection, which stored at -70 degrees C. Thirty-four patients had chronic hepatitis and 6 patients had cirrhosis. Thirty-one patients were diagnosed by liver biopsy. The average age and range were 29 years and 13-57 years respectively. None had taken any antiviral agents before. To detect YMDD mutants, YVDD (M552V), and YIDD (M552I), we used direct sequencing and the restriction fragment length polymorphism (RFLP) method. RESULTS: The YMDD mutant was detected by RFLP method in 7.5% (3/40) of the patients with chronic hepatitis B infection, in two patients with chronic hepatitis and one with cirrhosis. All were YMDD+ YIDD mutants. CONCLUSIONS: The YMDD motif mutation occurs spontaneously without antiviral therapy in patients with chronic hepatitis B infection.

Normal structure, variations, and anomalies of the pancreaticobiliary ducts of Koreans: a nationwide cooperative prospective study.

BACKGROUND: This study evaluated the normal pancreaticobiliary ducts of Koreans and assessed the frequency and pattern of variations and anomalies of these structures. METHODS: A prospective, nationwide multicenter study was performed in which 10 university hospitals in Korea participated from March 1997 to June 1999. A total 10,243 patients undergoing ERCP were enrolled. RESULTS: The mean (SD) maximal and midportion diameters in millimeters of the common hepatic duct were, respectively, 6.1 (1.8) and 5.3 (1.6). The mean maximal and midportion diameters (mm) of the common bile duct were, respectively, 6.4 (1.8) and 5.5 (1.7). The mean maximal and midportion diameters (mm) of the main pancreatic duct in the head, body and tail were, respectively, 3.2 (1.1), 2.7 (1.0), and 2.5 (2.3); and, respectively, 2.2 (0.9), 1.6 (0.7) and 1.4 (0.6). Pancreaticobiliary duct diameters for patients above the age of 40 were greater than those of patients less than 40 years of age (p < 0.05). The frequency of choledochal cyst and anomalous union of the pancreaticobiliary ducts were, respectively, 0.32% and 4.1%. Pancreas divisum and annular pancreas were found, respectively, in 0.49% and 0.05%. CONCLUSIONS: A knowledge of normal pancreaticobiliary ductal structures as well as the frequency and pattern of variations including anomalies is essential for the diagnosis and treatment of pancreaticobiliary disorders.

NF-kappa B binding activity and cyclooxygenase-2 expression in persistent CCl(4)-treated rat liver injury.

The involvement of NF-kappa B binding activity is known to be important in the mechanism of acute liver injury and in the induction of cyclooxygenase (COX-2). This study was performed to evaluate NF-kappa B binding activity and the expression of COX-2 in chronic liver injury induced by carbon tetrachloride (CCl(4)). Liver tissues from Sprague-Dawley rats were collected at 1, 3, 5, and 7th week after intraperitoneal injection of 0.1 mL of CCl(4)/100 g body weight twice a week. Reactive oxy-gen species (ROS) were measured in the postmitochondrial fraction by dichlorofluorescein formation with a fluorescent probe. An electrophoretic mobility shift assay was performed for NF-kappa B binding activity. Western blot was performed to measure the level of COX-1, COX-2, p65, p50, and I B proteins. ROS and NF-kappa B activity increased during the CCl4-induced chronic liver injury. The expression of nuclear p65 protein and p50 protein increased compared with that of the control, while the cytoplasmic I B protein decreased as the inflammation persisted. The expression of COX-2 in CCl(4)-treated rat liver increased compared with that of the control. It could be suggested that ROS produced by CCl(4) treatment increased NF-kappa B binding activity and thereby COX-2 expression, and these might be implicated in the progress of chronic liver damage.