Regulation of heparin-binding EGF-like growth factor expression by phorbol ester in a human hepatoma-derived cell line

AbstractHeparin-binding EGF-like growth factor (HB-EGF) is a recently identified potent mitogen for smooth muscle cells and fibroblasts. HB-EGF has been shown to be an EGF receptor ligand, and also to stimulate epithelial cell growth. A human hepatoma-derived cell line, Mahlavu, was analyzed for the production of HB-EGF mRNA and active HB-EGF protein. It was found that the cell line synthesized very low or undetectable basal level of HB-EGF mRNA. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a rapid and transient rise in HB-EGF mRNA level. HB-EGF in Mahlavu cells appears to be regulated by a protein kinase C (PKC) pathway, since PKC inhibitors, H7, staurosporin, and calphostin C, abrogated the induction of HB-EGF mRNA by TPA. Unlike vascular smooth muscle cells, induction of HB-EGF gene transcription by TPA was blocked completely by incubation with cycloheximide, suggesting that protein synthesis may be a prerequisite for HB-EGF gene transcription in Mahlavu cells. Mahlavu cells were also found to release a bioactive HB-EGF-like protein into conditioned medium which stimulates DNA synthesis in EP170.7 cells. This activity was neutralized by an anti-HB-EGF antibody. These results indicate that HB-EGF gene transcription is regulated via a PKC pathway, resulting in secretion of active HB-EGF into the culture medium of hepatoma-derived Mahlavu cells

Increased Plasma Levels of 8-Hydroxydeoxyguanosine Are Associated with Development of Colorectal Tumors

Increased oxidative stress is generally thought to be associated with tumorigenesis. In this cross-sectional study, we evaluated plasma 8-hydroxydeoxyguanosine (8-OHdG) levels in patients with colorectal adenoma and cancer, as a surrogate marker of oxidative damage to deoxyribonucleic acid (DNA). We collected blood samples from 58 patients with adenoma, 32 with early cancer, 25 with advanced cancer, and 36 without polyps or cancer (as controls), and measured plasma levels of 8-OHdG by enzyme-linked immunosorbent assay. Univariate analysis by logistic regression showed that an increased level of 8-OHdG was a significant risk for adenoma [odds ratio (OR) 1.393, 95% confidence interval (CI) 1.008–1.926, p = 0.045]. In patients with early cancer, univariate analysis revealed significant differences for age, body mass index (BMI), systolic blood pressure, and 8-OHdG level. Subsequent multivariate analysis revealed that 8-OHdG [OR 1.627, 95% CI 1.079–2.453, p = 0.020] and BMI [OR 1.283, 95% CI 1.038–1.585, p = 0.021] were significant risk factors for early cancer. However, 8-OHdG was not a significant risk factor for advanced cancer. Our results suggest that an increased plasma level of 8-OHdG is associated with development of colorectal adenoma and cancer

Genetic Polymorphisms of the Human PNPLA3 Gene Are Strongly Associated with Severity of Non-Alcoholic Fatty Liver Disease in Japanese

Nonalcoholic fatty liver disease (NAFLD) includes a broad range of liver pathologies from simple steatosis to cirrhosis and fibrosis, in which a subtype accompanying hepatocyte degeneration and fibrosis is classified as nonalcoholic steatohepatitis (NASH). NASH accounts for approximately 10-30% of NAFLD and causes a higher frequency of liver-related death, and its progression of NASH has been considered to be complex involving multiple genetic factors interacting with the environment and lifestyle.To identify genetic factors related to NAFLD in the Japanese, we performed a genome-wide association study recruiting 529 histologically diagnosed NAFLD patients and 932 population controls. A significant association was observed for a cluster of SNPs in PNPLA3 on chromosome 22q13 with the strongest p-value of 1.4 × 10(-10) (OR = 1.66, 95%CI: 1.43-1.94) for rs738409. Rs738409 also showed the strongest association (p = 3.6 × 10(-6)) with the histological classifications proposed by Matteoni and colleagues based on the degree of inflammation, ballooning degeneration, fibrosis and Mallory-Denk body. In addition, there were marked differences in rs738409 genotype distributions between type4 subgroup corresponding to NASH and the other three subgroups (p = 4.8 × 10(-6), OR = 1.96, 95%CI: 1.47-2.62). Moreover, a subgroup analysis of NAFLD patients against controls showed a significant association of rs738409 with type4 (p = 1.7 × 10(-16), OR = 2.18, 95%CI: 1.81-2.63) whereas no association was obtained for type1 to type3 (p = 0.41). Rs738409 also showed strong associations with three clinical traits related to the prognosis of NAFLD, namely, levels of hyaluronic acid (p = 4.6 × 10(-4)), HbA1c (p = 0.0011) and iron deposition in the liver (p = 5.6 × 10(-4)).With these results we clearly demonstrated that Matteoni type4 NAFLD is both a genetically and clinically different subset from the other spectrums of the disease and that the PNPLA3 gene is strongly associated with the progression of NASH in Japanese population

THE JOURNAL OF BIOLOGICAL CHEMISTRY Microsomal Enzymes of Cholesterol Biosynthesis from Lanosterol PURIFICATION AND CHARACTERIZATION OF A7-STEROL 5-DESATURASE OF RAT LIVER MICROSOMES*

Microsomal A7-sterol 5-desaturase of cholesterol biosynthesis is a multienzyme system which catalyzes the introduction of the A6-bond into A'-cholestenol to form 7-dehydrocholesterol. The detergent-solubilized 5-desaturase has been purified more than 70-fold and resolved from electron carriers and other rat liver microsomal enzymes of sterol biosynthesis by chromatography on DEAE-Sephacel, CM-Sepharose, and immobilized cytochrome b,; the 5-desaturase had not been fully resolved from cytochrom b6 reductase in earlier work. A functional electron transport system for the 5-desaturase has been reconstituted by combining the purified 5-desaturase and electron carriers with egg phosphatidylcholine liposomes. Optimizations of conditions for reconsitution have been obtained; both cytochrome b, and NADH-cytochrome b, reductase serve as electron carriers. A pyridine nucleotide-dependent flavoprotein is required and the requirement can be satisfied with either purified cytochrome bK reductase or cytochrome P-450 reductase. Cyanide and ironchelators strikingly inhibit the 5-desaturase activity, thus suggesting that 5-desaturase is a metalloenzyme as are other well-characterized cytochrome bK-dependent oxidases. 5-Desaturase is resolved from 4-methyl sterol oxidase activity of cholesterol biosynthesis by chromatography on the immobilized cytochrome b,. This resolution of the two oxidases not only indicates that introduction of the A6-bond and oxidation of 4a-methyl groups are catalyzed by different terminal oxidases, but resolution affords enzymes of sufficient purity to carry out reconstitution experiments. A novel assay based on substrate-dependent increments of oxidation of a-NADH has been developed for measurement of 5-desaturase activity. Measurement of stoichiometry of 5-desaturase demonstrates that for each equivalent of cis-desaturation of A7-cholestenol, 1 eq of NADH is consumed. Along with strict dependence upon oxygen, this observation confirms, as suggested by previous workers, that the 5-desaturation is catalyzed by a mixed function oxidase rather than a dehydrogenase. The biotransformation of lanosterol to cholesterol proceeds in a multiple process involving reduction of the AZ4-bond in the side chain, removal of three methyl groups from C-4 and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact