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Direct-acting antivirals (DAAs) have recently been developed to treat hepatitis C virus (HCV) infection, and interferon-free DAA treatment has improved liver function of HCV patients. The risk of hepatocellular carcinoma (HCC) occurrence following HCV eradication has been previously reported, but HCC may have been missed following imaging diagnosis before DAA administration in previous studies. Therefore, the present study aimed to identify definite predictors of HCC occurrence ≥1 year after DAA treatment. Among 956 patients receiving DAAs for HCV infection, 567 patients who achieved sustained virologic response with no history of HCC treatment were enrolled in this study between September 2014 and July 2021. The incidence of HCC in HCV-infected patients ≥1 year following DAA treatment, and the predictors contributing to HCC occurrence were identified using clinical characteristics and blood test results. In the present study, 25 patients developed HCC. The incidence of HCC was 1.4%, 3.2%, 4.9% and 6.8% at 2, 3, 4 and 5 years, respectively, from the end of treatment with DAAs. Multivariate logistic analysis revealed serum α-fetoprotein level at end of treatment (EOT-AFP) >3.8 ng/ml ≥1 year following treatment with DAAs (HR, 9.7; p

Objective Multiple therapeutic agents exist for advanced hepatocellular carcinoma (HCC), but prognostic factors in second-line and subsequent therapies are unclear. Ramucirumab is a molecular-targeted agent effective against hepatocytes with alpha-fetoprotein (AFP)400 ng/mL after sorafenib failure. We examined the prognostic factors and efficacy of ramucirumab with prior therapy other than sorafenib. Methods In our retrospective multicenter study, 33 patients were treated with ramucirumab for HCC with prior therapy other than sorafenib, including 1 patient who received 2 lines of ramucirumab. We analyzed background factors, liver reserve, the prognosis, and treatment duration and efficacy. Results The median albumin-bilirubin (ALBI) value showed little change during ramucirumab treatment. The ALBI value improved in 32% of patients, and their prognoses were better than in those who did not improve. Response and efficacy rates were not as high as those in the REACH-2 study but were similar when limited to patients with 2,500 ng/mL AFP. Thirteen patients received further treatment after ramucirumab failure and they had a significantly better prognosis from ramucirumab administration and also had a significantly better prognosis from the start of the first tyrosine kinase inhibitor than who did not received further treatment. In univariate and multivariate analyses of prognostic factors, the continuation of treatment with another drug after ramucirumab failure and a good ALBI value at initiation were significant. The presence of a ramucirumab response and treatment duration were not associated with the prognosis. A good ALBI value at initiation and ALBI value improvement during treatment were also identified as independent factors associated with eligibility for further treatment after ramucirumab failure. The treatment line did not correlate with the availability of treatment with another drug after treatment failure. Conclusions ALBI value improvement with ramucirumab treatment allows for subsequent treatment after failure and an improved overall prognosis.

A 61-year-old man was admitted due to alcoholic liver cirrhosis, portal vein thrombosis, hepatocellular carcinoma, and chronic pancreatitis. The patient's portal vein thrombosis improved with anticoagulant therapy. Serum amylase gradually increased, but there was no abdominal pain. The patient was placed under observation. The pain in both ankle and knee joints appeared on nine days after admission. Multiple osteonecrotic lesions of both elbows, both knees and both ankle joints were examined using

The risk of hepatocellular carcinoma (HCC) occurrence following hepatitis C virus (HCV) eradication has been previously reported, but the impact of HCV eradication on advanced HCC patient survival remains unclear. Therefore, the present study aimed to evaluate the effect of HCV eradication on the survival outcome of patients with advanced HCC treated with sorafenib. One hundred and three HCV-related advanced HCC patients who were treated with sorafenib were enrolled in this study. Of these, 43 patients were administered antiviral therapy before sorafenib treatment (HCV eradication group), while 60 patients remained HCV-infected (HCV non-eradication group). We analysed the impact of HCV eradication on survival in advanced HCC treated with sorafenib. Median overall survival (OS) was significantly longer in the HCV eradication group than in the HCV non-eradication group (24.0 months vs. 14.1 months; p = 0.001). Although there was no significant difference in the albumin-bilirubin (ALBI) score at the start of treatment between the HCV eradication group and the non-eradication group (p = 0.065), the ALBI score at 2 months after initiation of sorafenib treatment was significantly decreased in the HCV non-eradication group (p 0.001), but not in the HCV eradication group (p = 0.121). Multivariate logistic analysis revealed HCV eradication (hazard ratio [HR], 0.5; p = 0.006) and ALBI score at the start of treatment (HR, 2.47; p = 0.002) as factors that may contribute to OS. HCV eradication may serve an important role in the survival outcome of advanced HCC patients treated with sorafenib.

Here, we report a rare case of intrahepatic sarcomatoid cholangiocarcinoma that expressed granulocyte colony-stimulating factor (G-CSF). An 87-year-old man who presented with a continuous high-grade fever and cough over two weeks, and increased inflammatory response was admitted to our hospital. Laboratory tests revealed marked granulocytosis and high serum levels of G-CSF and interlukin-6. Imaging studies showed a huge liver mass in his right lobe and intrahepatic metastasis. He died of progressive disease. Pathological findings of the primary liver tumor at autopsy showed sarcomatous change; the specimen was positive for cytokeratins (AE1/AE3, cytokeratin-7, cytokeratin-19) and G-CSF. Few cases of G-CSF-producing intrahepatic sarcomatoid cholangiocarcinoma have been reported.

AIM To investigate the real-world efficacy and safety of sofosbuvir/ribavirin (SOF/RBV) therapy for Japanese patients with genotype 2 hepatitis C virus (GT2-HCV). METHODS A total of 182 patients with GT2-HCV infection who received SOF/RBV therapy for 12 wk at our hospital were enrolled. The patients comprised 122 men and 60 women (age range: 17-84 years; mean age ± SD: 60.1 ± 12.1 years). Relationships between virological response and clinical data were examined by logistic regression analyses. RESULTS The proportions of patients with liver cirrhosis and history of hepatocellular carcinoma (HCC) were 29.0% and 17.3%, respectively. The proportion of patients with prior interferon (IFN)-based therapy was 25.6%. SOF/RBV therapy rapidly decreased HCV RNA levels. Several patients required RBV dose reduction because of anemia or fatigue. Four patients discontinued the therapy. The rates of sustained virological response at 12 wk after the end of treatment were 87.9% (intention to treat: 160/182) and 94.1% (per protocol: 159/169). Multivariate analyses showed that history of HCC or IFN-based therapy independently reduced the efficacy of SOF/RBV therapy. CONCLUSION SOF/RBV therapy for GT2-HCV is safe, highly tolerated, and effective. History of HCC or IFN-based therapy independently reduces the efficacy of this treatment.

A 49-year-old man with chronic hepatitis B receiving treatment with entecavir visited a hospital with a complaint of abdominal pain. Computed tomography (CT) showed 2 liver tumors, each measuring 1cm in diameter, 1 in segment 7 and 1 in segment 4. Magnetic resonance imaging (MRI) showed a hypervascular tumor in segment 7 that appeared in a site different from that seen on CT. The liver tumor in segment 4 was not detected by MRI. Two months later, MRI showed a new liver tumor in segment 7/6 and that the liver tumor in segment 7 had increased to 2cm in diameter;blood tests showed eosinophilia. Enzyme-linked immunosorbent assay showed a high serum Toxocara antibody. The patient was diagnosed as having hepatic toxocariasis and was treated with albendazole for 8 weeks. After treatment, MRI showed that the liver tumors disappeared. Eosinophilia, multiple lesions, and the disappearance of the tumors were characteristic findings of visceral larva migrans.

Despite the use of pegylated-interferon (peg-IFN) plus ribavirin combination therapy, many patients infected with hepatitis C virus (HCV)-1b remain HCV-positive. To determine whether addition of pitavastatin and eicosapentaenoic acid (EPA) is beneficial, the "add-on" therapy option (add-on group) was compared retrospectively with unmodified peg-IFN/ribavirin therapy (standard group). Association of host- or virus-related factors with sustained virological response was assessed. In HCV replicon cells, the effects of pitavastatin and/or EPA on HCV replication and expression of innate-immunity- and lipid-metabolism-associated genes were investigated. In patients infected with HCV-1b, sustained virological response rates were significantly higher in the add-on than standard group. In both groups, sustained virological response rates were significantly higher in patients with genotype TT of IL-28B (rs8099917) than in those with non-TT genotype. Among the patients with non-TT genotype, sustained virological response rates were markedly higher in the add-on than standard group. By multivariate analysis, genome variation of IL28B but not add-on therapy remained as a predictive factor of sustained virological response. In replicon cells, pitavastatin and EPA suppressed HCV replication. Activation of innate immunity was obvious in pitavastatin-treated cells and EPA suppressed the expression of sterol regulatory element binding protein-1c and low-density lipoprotein receptor. Addition of pitavastatin and EPA to peg-IFN/ribavirin treatment improved sustained virological response in patients infected with HCV-1b. Genotype variation of IL-28B is a strong predictive factor in add-on therapy.

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV-infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL-cholesterol (LDL-C) and HCV core antigen were also evaluated, and expression of claudin-1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin-1 genes was significantly suppressed (P

Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development.

Members of the U-box family of proteins constitute a class of ubiquitin-protein ligases (E3s) distinct from the HECT-type and RING finger-containing E3 families. Two representative mammalian U-box proteins, UFD2a and CHIP, interact with the molecular chaperones VCP and either Hsp90 or Hsc70, respectively, and are implicated in the degradation of damaged proteins. We have now investigated the roles of mammalian U-box proteins by performing a comprehensive screen for molecules that interact with these proteins in the yeast two-hybrid system. All mammalian U-box proteins tested were found to interact with molecular chaperones or cochaperones, including Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP. These observations suggest that the function of U box-type E3s is to mediate the degradation of unfolded or misfolded proteins in conjunction with molecular chaperones as receptors that recognize such abnormal proteins.

The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7−/− embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.

The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cul1 in the nucleus and that this interaction is mediated by a central region of Cul1 distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cul1 that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase.

The U box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.

AIM: To determine significant indicators for the efficacy of sorafenib in patients with advanced hepatocellular carcinoma (HCC). METHODS: A total of 46 patients with Barcelona Clinic Liver Cancer stage C who received sorafenib for more than 30 d at the Iizuka Hospital from June 2009 to December 2012 were enrolled in this study. Multivariate and univariate analyses were performed to evaluate the associations of hepatic function according to Child-Pugh grade, location and size of the largest tumor and adverse events of sorafenib treatment, such as hand-foot syndrome (HFS), hypertension, diarrhea, and alopecia, with the efficacy of treatment, as measured by overall survival (OS) and time to progression (TTP). RESULTS: Patients included 39 men and 7 women whose ages ranged from 48 to 85 years (70.6 ± 9.6 years). HCC was classified according to etiology as follows: hepatitis C virus (n = 26), hepatitis B virus (n = 9), and other (n = 11). Liver function in patients was categorized as Child-Pugh grade A (n = 30) or B (n = 16). Tumors were categorized by size [ 5 cm (n = 13)] and the location of the largest tumor was used to categorize patients with intrahepatic (n = 28) or extrahepatic (n = 18) HCC. HFS, hypertension, diarrhea, and alopecia were present in 22 (47.8%), 19 (41.3%), 15 (32.6%) and 7 patients (15.2%), respectively. The median OS of all patients was 373 d and the median TTP was 112 d. The etiology of HCC did not correlate with the median OS and TPP. The median OS of patients with tumors < 5 cm was significantly longer than those with larger tumors (496 vs 245 d; HR = 0.19, 95%CI: 0.07-0.48; P < 0.01). According to the results of a multivariate analysis, the size of the largest tumor affected OS (HR = 0.22, 95%CI: 0.08-0.59; P < 0.01). The median TTP was significantly longer in patients with extrahepatic compared to intrahepatic major HCC (224 vs 98 d; HR = 0.32; 95%CI: 0.14-0.67; P < 0.01). The median TTP of patients with HFS was significantly longer than those without it (195 d vs 83 d; HR = 0.41, 95%CI: 0.20-0.82; P < 0.05), and the median TTP was significantly longer in patients with hypertension (195 d vs 84 d; HR = 0.43, 95%CI: 0.21-0.84; P < 0.05). According to the results of the multivariate analysis, extrahepatic major HCC (HR = 0.36, P < 0.01) and HFS (HR = 0.44, P < 0.05) prolonged TTP. CONCLUSION: Extrahepatic major HCC and HFS are associated with prolonged TTP and are useful indicators for judging the efficacy of sorafenib treatment.

Aim To investigate the factors influenced the efficacy of tolvaptan (TLV) in liver cirrhosis. Methods We retrospectively enrolled 61 consecutive patients with refractory hepatic ascites. All of them had been treated with furosemide and spironolactone before admission, and treated with TLV for 7 d in our hospital. The effect of TLV was defined by the rate of body weight loss, and the factors that influenced TLV efficacy were analyzed using multiple regression. Results Coexistent hepatocellular carcinoma (HCC) was the only significant predictive variable that attenuated the efficacy of TLV. In stratified analysis, high doses of furosemide decreased the efficacy of TLV in patients with HCC, and increased efficacy in those without HCC. In the latter, a high Child-Pugh-Turcotte score had a positive influence and a high concentration of lactate dehydrogenase had a negative influence on the effectiveness of TLV. Conclusion Development of ascites may differ between patients with liver failure and those with HCC progression. A sufficient preceding dose of furosemide decreases diuretic effect of TLV.

Objective Bezafibrate (BF) has been used to treat biliary damage, particularly in patients with primary biliary cirrhosis (PBC), and its clinical efficacy has been demonstrated. The mechanism of action is thought to involve activation of the PPARalpha-MDR3-phospholipid (PL) secretion pathway. We tried to confirm this hypothesis in patients with hepatobiliary disease. Methods The levels of serum gamma-glutamyl transpeptidase and alkaline phosphatase, and those of bile components were examined before and after BF administration in patients with obstructive jaundice undergoing percutaneous transhepatic biliary drainage (PTBD). Hepatic expression of PPARalpha and MDR3 was quantified by real-time PCR in patients with PBC or non-alcoholic fatty liver disease (NAFLD). Results In patients with obstructive jaundice, BF decreased the serum levels of biliary enzymes and increased the bile concentration of PL. In patients with PBC or NAFLD, the expression levels of MDR3 were already up-regulated before starting the BF treatment. Although BF treatment did not further up-regulate MDR3 expression in NAFLD patients, PPARalpha expression was significantly increased. Conclusions BF enhanced the secretion of PL into bile in cholestatic patients undergoing PTBD. However, in patients with PBC or NAFLD, diseases that represent cholesterol overload, MDR3 was already expressed at a high level to compensate for bile acids overproduction, and its expression was hardly affected by BF. In patients with chronic liver diseases such as PBC, BF may induce clinical effects via mechanisms independent of PL secretion.

Recent investigations indicate that hepatitis C virus (HCV) infection is closely associated with hepatocytic lipid metabolism and induces hepatic steatosis. However, the actual lipid metabolism in HCV-infected liver has not been extensively investigated in humans. In this study, we evaluated the expression of lipid metabolism-associated genes in patients with HCV infection by real-time PCR. Sterol regulatory element-binding protein (SREBP)-2 expression was unchanged and low density lipoprotein receptor expression was markedly reduced by 90% in HCV-infected liver. The expression of apolipoprotein B100, microsomal triglyceride transfer protein and ATP-binding cassette G5 was significantly increased. Up-regulation of cholesterol synthesis-associated genes, including HMG-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase and squalene synthase, confirmed enhanced de novo cholesterol synthesis. The expression of cholesterol 7alpha-hydroxylase and farnesoid X receptor was enhanced, while bile salt export pump expression was unchanged. Fatty acid synthase expression was increased which was accompanied by increased expression of liver X receptor alpha and SREBP-1c. In summary, the regulation of lipid metabolism was impaired and cholesterol and fatty acid synthesis continued to increase without negative feedback in HCV-infected liver. These changes may be beneficial for HCV replication.

We previously studied fatty acid metabolism in the liver of nonalcoholic fatty liver disease (NAFLD) and reported the activation of the LXRalpha-SREBP-1c pathway in hepatocytes. LXRalpha regulates cholesterol metabolism as well as fatty acid metabolism, and its agonistic ligands are oxysterols. Moreover, there is some evidence that excess cholesterol intake is involved in the onset of NAFLD. Therefore, in this study, we examined the expression of cholesterol metabolism-associated genes in the NAFLD liver by real-time PCR. Expression of LXRalpha and ACAT1 was up-regulated in NAFLD and this was more noticeable in non-obese rather than in obese patients. Although the expression of the LDL receptor, which acts on cholesterol uptake, and of SREBP-2, a positive key regulator of cholesterol, was suppressed, the expression of enzymes that promote cholesterol synthesis was uniformly increased in NAFLD. Gene expression of apoB100 and microsomal triglyceride transfer protein, which are associated with VLDL secretion, and ABCG5, which is involved in cholesterol excretion, was significantly elevated in NAFLD. Because cholesterol accumulates in hepatocytes in NAFLD liver, cholesterol uptake and synthesis should be physiologically down-regulated. However, cholesterol synthesis was activated in NAFLD liver, meaning that cholesterol metabolism is dysregulated in NAFLD. Overproduction of cholesterol may lead to an increased level of oxysterols, activation of LXRalpha and SREBP-1c, and enhanced fatty acid synthesis.

Nonalcoholic fatty liver disease (NAFLD) is a common liver disease whose prevalence has increased markedly. We reported previously that fatty acid synthesis was enhanced in NAFLD with the accumulation of fatty acids. To clarify the disorder, we evaluated the expression of genes regulating fatty acid synthesis by real-time PCR using samples from NAFLD (n=22) and normal liver (control; n=10). A major regulator of fatty acids synthesis is sterol regulatory element-binding protein-1c (SREBP-1c). Its expression was significantly higher in NAFLD, nearly 5-fold greater than the controls. SREBP-1c is positively regulated by insulin signaling pathways, including insulin receptor substrate (IRS)-1 and -2. In NAFLD, IRS-1 expression was enhanced and correlated positively with SREBP-1c expression. In contrast, IRS-2 expression decreased by 50% and was not correlated with SREBP-1c. Forkhead box protein A2 (Foxa2) is a positive regulator of fatty acid oxidation and is itself negatively regulated by IRSs. Foxa2 expression increased in NAFLD and showed a negative correlation with IRS-2, but not with IRS-1, expression. It is known that SREBP-1c is negatively regulated by AMP-activated protein kinase (AMPK) but expression levels of AMPK in NAFLD were almost equal to those of the controls. These data indicate that, in NAFLD, insulin signaling via IRS-1 causes the up-regulation of SREBP1-c, leading to the increased synthesis of fatty acids by the hepatocytes; negative feedback regulation via AMPK does not occur and the activation of Foxa2, following a decrease of IRS-2, up-regulates fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased. We previously evaluated the expression of fatty acid metabolism-related genes in NAFLD and reported changes in expression that could contribute to increased fatty acid synthesis. In the present study, we evaluated the expression of additional fatty acid metabolism-related genes in larger groups of NAFLD (n=26) and normal liver (n=10) samples. The target genes for real-time PCR analysis were as follows: acetyl-CoA carboxylase (ACC) 1, ACC2, fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP-1c), and adipose differentiation-related protein (ADRP) for evaluation of de novo synthesis and uptake of fatty acids; carnitine palmitoyltransferase 1a; (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), long-chain L-3-hydroxyacylcoenzyme A dehydrogenase alpha (HADHalpha), uncoupling protein 2 (UCP2), straight-chain acyl-CoA oxidase (ACOX), branched-chain acyl-CoA oxidase (BOX), cytochrome P450 2E1 (CYP2E1), CYP4A11, and peroxisome proliferator-activated receptor (PPAR)alpha for oxidation in the mitochondria, peroxisomes and microsomes; superoxide dismutase (SOD), catalase, and glutathione synthetase (GSS) for antioxidant pathways; and diacylglycerol O-acyltransferase 1 (DGAT1), PPARgamma, and hormone-sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, although fatty acids accumulated in hepatocytes, their de novo synthesis and uptake were up-regulated in association with increased expression of ACC1, FAS, SREBP-1c, and ADRP. Fatty acid oxidation-related genes, LCAD, HADHalpha, UCP2, ACOX, BOX, CYP2E1, and CYP4A11, were all overexpressed, indicating that oxidation was enhanced in NAFLD, whereas the expression of CTP1a and PPARalpha was decreased. Furthermore, SOD and catalase were also overexpressed, indicating that antioxidant pathways are activated to neutralize reactive oxygen species (ROS), which are overproduced during oxidative processes. The expression of DGAT1 was up-regulated without increased PPARgamma expression, whereas the expression of HSL was decreased. Our data indicated the following regarding NAFLD: i) increased de novo synthesis and uptake of fatty acids lead to further fatty acid accumulation in hepatocytes; ii) mitochondrial fatty acid oxidation is decreased or fully activated; iii) in order to complement the function of mitochondria (beta-oxidation), peroxisomal (beta-oxidation) and microsomal (omega-oxidation) oxidation is up-regulated to decrease fatty acid accumulation; iv) antioxidant pathways including SOD and catalase are enhanced to neutralize ROS overproduced during mitochondrial, peroxisomal, and microsomal oxidation; and v) lipid droplet formation is enhanced due to increased DGAT expression and decreased HSL expression. Further studies will be needed to clarify how fatty acid synthesis is increased by SREBP-1c, which is under the control of insulin and AMP-activated protein kinase.

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57 Kip2 , an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCF Skp2 has now been shown to be responsible for regulating the cellular level of p57 Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57 Kip2 was impaired in Skp2 -/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57 Kip2 in vivo . Overexpression of WT Skp2 promoted degradation of p57 Kip2 , whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57 Kip2 . Mutation of the threonine residue (Thr-310) of human p57 Kip2 that is conserved between the COOH-terminal QT domains of p57 Kip2 and p27 Kip1 prevented the effect of Skp2 on the stability of p57 Kip2 , suggesting that phosphorylation at this site is required for SCF Skp2 -mediated ubiquitylation. Finally, the purified recombinant SCF Skp2 complex mediated p57 Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCF Skp2 complex plays an important role in cell-cycle progression by determining the abundance of p57 Kip2 and that of the related CDK inhibitor p27 Kip1 .

Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3.

The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined.Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities.The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans.

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.

Non-alcoholic fatty liver disease (NAFLD) is considered to be associated with metabolic syndrome; however, a number of NAFLD patients are not obese. To explore any differences in lipid metabolism between obese and non-obese patients, we determined the expression of fatty acid metabolism-related genes. Expression levels of target genes were quantified by real-time PCR using liver biopsy samples from NAFLD patients and normal controls. Serum adipocytokine levels were also determined. The expression of genes related to fatty acid synthesis and uptake was generally up-regulated in NAFLD patients; however, no significant difference was seen between obese and non-obese groups. Most of the genes tested related to fatty acid and reactive oxygen species (ROS) elimination, were overexpressed in NAFLD and the levels were significantly higher in non-obese patients. As an exception, peroxisome proliferator-activated receptor alpha expression was suppressed in NAFLD and the levels were lower in the obese group. Triglyceride synthesis-related genes were up-regulated and lipolytic enzymes were decreased in NAFLD, but there was no significant difference between the obese and non-obese groups. In NAFLD, increased de novo synthesis and uptake of fatty acids led to further hepatocyte accumulation of fatty acids. The up-regulation of fatty acid oxidation and the antioxidant pathway and the suppression of lipolysis seemed to be involved in this process. Expression of genes related to fatty acid oxidation and ROS elimination were higher in the non-obese group than in the obese group, which contributes to the trend of more severe liver injury, insulin resistance and steatosis in obese patients.

To investigate the efficacy of short-term peg-interferon (PEG-IFN) monotherapy for chronic hepatitis C patients who achieved an immediate virological response.Defining an "immediate virological response (IVR)" as the loss of serum hepatitis C virus (HCV) RNA 7 d after the first administration of PEG-IFN alpha, we conducted a 12-wk course of PEG-IFN alpha2a monotherapy without the addition of ribavirin for 38 patients who had low pretreatment HCV RNA load and exhibited IVR. The patients included 21 men and 17 women, whose ages ranged from 22 to 77 years (mean +/- SD: 52.0 +/- 17.8 years). There were 4 patients with HCV genotype 1b, 23 patients with genotype 2a and 4 patients with genotype 2b. HCV genotype was not determined for the remaining 7 patients. Patients were categorized into a sustained virological response (SVR) group, if serum HCV RNA remained negative for 24 wk after the end of treatment, or into a relapse group.Based on the intention-to-treat analysis, 35 patients (92.1%) achieved SVR. One patient (2.6%) relapsed with serum HCV RNA 12 wk after the end of treatment. Two patients (5.3%) withdrew from the study during the 24-wk follow-up period. With regard to the HCV RNA genotype, the SVR rates were 100% (4/4) for genotype 1b, 95.7% (22/23) for genotype 2a and 100% (4/4) for genotype 2b. The SVR rate in 7 patients, whose HCV RNA genotypes were not determined, was 71.4% (5/7).Short-term PEG-IFN alpha2a monotherapy is highly effective for chronic hepatitis C patients who have low pretreatment HCV RNA load and exhibit IVR.