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Background and Aim:Visceral fat accumulation reportedly increases the risk of hepatocellular carcinoma (HCC) development in patients with chronic liver disease. However, it has not beeen fully elucidated whether visceral fat accumulation increases the risk of HCC recurrence after curative treatment in patients with suspected non-alcoholic steatohepatitis (NASH). Therefore this was investigated in the current study.Methods:62 patients with naïve HCC with suspected NASH were enrolled. All were curatively treated with percutaneous radiofrequency ablation between 1999 and 2006. The visceral fat area (VFA) was determined in each patient from CT images, taken at the time of HCC diagnosis. Patients were divided into two groups based on VFA: the high VFA group (>130 cm2 in males, >90 cm2 in females, n = 27) and the others (n = 35). The effects of VFA on HCC recurrence were analysed together with other factors including patients’ background, tumour-related factors and liver function-related factors.Results:The cumulative recurrence rates differed significantly between the two groups; 15.9, 56.5 and 75.1% at 1, 2 and 3 years, respectively, in the high VFA group, and 9.7, 31.1 and 43.1%, respectively, in the controls (p = 0.018). Multivariate analysis indicated visceral fat accumulation (risk ratio 1.08, per 10 cm2, p = 0.046) and older age (risk ratio 1.06 per 1 year, p = 0.04) as independent risk factors of HCC recurrence.Conclusions:Visceral fat accumulation is an independent risk factor of HCC recurrence after curative treatment in patients with suspected NASH.

A recent study revealed that the p110 α ( PIK3CA ), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110 α sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA . These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30–125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

Background —VacA and CagA proteins have been reported to be major virulence factors of Helicobacter pylori . However, antibodies against these proteins are frequently found in the sera of Japanese patients regardless of their gastroduodenal status. Aim —To evaluate the expression of VacA and CagA proteins by H pylori strains isolated in Japan. Methods —By using specific antibodies raised against recombinant VacA and CagA proteins, the expression of VacA and CagA was evaluated in 68 H pylori strains isolated from Japanese patients; a vacuolating assay and genotyping of the vacA gene were also used in the evaluation. The results were analysed in relation to the gastroduodenal diseases of the hosts. Results —VacA and CagA proteins were expressed in 59/68 (87%) and in 61/68 (90%) isolates respectively. The vacuolating assay was positive in 57/68 (84%) isolates, indicating that most immunologically VacA positive strains produced active cytotoxin. The prevalence of infection with strains expressing CagA and positive for vacuolating activity (Type I) was very high, 54/68 (79%), irrespective of the gastroduodenal status of the host. Conclusion —Most H pylori isolates in Japan are positive for vacuolating cytotoxin and CagA, and thus these virulence factors cannot be used as markers to discern the risk of developing serious gastroduodenal pathologies in the hosts. However, the high prevalence of infection with strains positive for vacuolating cytotoxin and CagA may contribute to the characteristics of H pylori infection in Japan.

cag pathogenicity island (PAI) is reported to be a major virulence factor of Helicobacter pylori. To characterise cagA and the cag PAI in Japanese H pylori strains. H pylori isolates from Japanese patients were evaluated for CagA by immunoblot, for cagA transcription by northern blot, and for cagA and 13 other cag PAI genes by Southern blot. cagA negative strains from Western countries were also studied. Induction of interleukin-8 secretion from gastric epithelial cells was also investigated. All Japanese strains retained cagA. Fifty nine of 63 (94%) strains had all the cag PAI genes. In the remaining four, cag PAI was partially deleted, lacking cagA transcripts and not producing CagA protein. Details of the PAI of these strains were checked; three lacked cagB to cagQ (cagI) and continuously cagS to cag13 (cagII), and the remaining one lacked cagB to cag8. Western cagA negative strains completely lacked cag PAI including cagA. Nucleotide sequence analysis in one strain in which the cag PAI was partially deleted showed that the partial deletion contained 25 kb of cag PAI and the cagA promoter. Interleukin-8 induction was lower with the cag PAI partial deletion strains than with the intact ones. All Japanese cag PAI deleted strains were derived from patients with non-ulcer dyspepsia, whereas 41 of 59 (70%) CagA-producing strains were from patients with peptic ulcers or gastric cancer (p<0.05). Most Japanese H pylori strains had the intact cag PAI. However, some lacked most of the cag PAI in spite of the presence of cagA. Thus the presence of the cagA gene is not an invariable marker of cag PAI related virulence in Japanese strains.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis and is highly amenable to the development of novel therapeutic strategy. The human alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in HCC. In order to destroy AFP-producing HCC specifically, replication defective adenoviral vectors containing the transcriptional control elements of the AFP gene were designed. Expression of suicide genes by the AFP promoter/enhancer induced prodrug sensitivity in AFP (+) cells but not AFP (-) cells. The expression of suicide genes by ubiquitous promoter, however, showed no selectivity after prodrug treatment. Adenoviral vector transduced genes efficiently not only in vitro but also in vivo, and AFP-producing HCC xenografts regressed by transduction with transcriptionally targeted vectors and subsequent systemic administration of prodrug in animal model. Utilization of the transcriptional regulatory element to drive drug sensitive genes can be a promising strategy for cancer specific therapy.

Background—Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5–10% of strains are reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin. Aims—To establish a polymerase chain reaction (PCR) system which amplifies a segment of the 23S rRNA gene containing the mutation points with primers specific forH pylori, so that H pylori infection and the mutation associated with clarithromycin resistance can be examined simultaneously. Methods—To detectH pylori infection and the mutation simultaneously, primers specific for the H pylori 23S rRNA gene were designed based on sequence conservation among H pylori strains and sequence specificity as compared with other bacteria. DNA from 57 cultured strains and from 39 gastric juice samples was amplified in the seminested 23S rRNA PCR. Clinical applicability was evaluated in 85 patients. Results—DNA samples from 57 cultured strains were all amplified. The novel assay and the urease A PCR agreed in 37/39 gastric juice samples with no false positives. The assay did not amplify the DNA of bacteria other thanH pylori. Eight of 85 samples had the mutation before treatment. In clarithromycin based treatment, eradication was achieved in 2/5 (40%) with the mutation and 29/34 (85%) without the mutation. Conclusion—The assay using gastric juice is quick (within 12 hours) and non-invasive (endoscopy not required), enabling rapid initiation of appropriate antibiotic treatment.

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ ( t ranscriptional co‐ a ctivator with PD Z ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

BACKGROUND/AIMS Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. METHODS The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. RESULTS p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. CONCLUSIONS Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.