Saburo Onishi

Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Angiotensin II type 1 receptor blocker inhibits fibrosis in rat nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is now the most frequent cause of chronic liver impairment in developed countries and is a suggested causative factor in the development of cryptogenic cirrhosis and hepatocellular carcinoma. At present there is no effective and accepted therapy for NASH. The renin‐angiotensin system is involved in hepatic fibrosis through activation of hepatic stellate cells, major fibrogenic cells in the liver. Hepatic stellate cells are activated by liver injury to express excessive matrix proteins and profibrogenic cytokines such as transforming growth factor–beta 1. Medicines that inhibit this pathway may be of therapeutic potential in NASH. Using a methionine‐choline–deficient rat model of NASH, we studied the potential utility of an angiotensin II type 1 receptor blocker (ARB), olmesartan, on biochemical, histologic, and antioxidant measures of disease activity. ARB significantly attenuated increases in aspartate aminotransferase, activation of hepatic stellate cells, oxidative stress, expression of transforming growth factor–beta 1, expression of collagen genes, and liver fibrosis. Conclusion: Our observations strongly suggest a potential preventive role for ARB in the progression of nonalcoholic steatohepatitis. (HEPATOLOGY 2007.)

The α and β Classes Carbonic Anhydrases from Helicobacter pylori as Novel Drug Targets

Helicobacter pylori (H. pylori) successfully resides in the human stomach in highly acidic conditions, causing a variety of gastroduodenal lesions, including gastric ulcer, gastric cancer and MALT lymphoma. For acid acclimation of H. pylori, two types of enzymes, urease and carbonic anhydrase (CA), play a central role. They cooperatively function to maintain neutral pH in the bacterial cytoplasm and periplasm. The genome project of H. pylori identified two different classes of CA with different subcellular localization: a periplasmic α-class CA (hpαCA) and a cytoplasmic β-class CA (hpβCA). These two CAs are catalytically efficient with almost identical activity to that of the human isoform CA I for the CO2 hydration reaction, and highly inhibited by many sulfonamides/sulfamates, including acetazolamide, ethoxzolamide, topiramate and sulpiride, all clinically used drugs. Furthermore, certain CA inhibitors, such as acetazolamide and methazolamide, were shown to inhibit the bacterial growth in vitro. Since the efficacy of eradication therapies currently employed has been decreasing due to drug resistance and side effects of the commonly used drugs, the dual inhibition of α- and/or s-CAs of H. pylori could be applied as an alternative therapy in patients with H. pylori infection or for the prevention of gastroduodenal diseases provoked by this widespread pathogen.

Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver

To identify Ito cells in normal and pathological adult human livers, immunohistochemical studies were performed by the avidin-biotin-peroxidase complex method using monoclonal antibodies for α-smooth muscle actin (ASMA), desmin, and vimentin. Fifty one needle biopsies, 7 surgically resected specimens, and 5 autopsy specimens were studied. In the normal adult liver vascular smooth muscle cells and pericytes, together with perisinusoidal cells with thin cytoplasmic processes were positive for ASMA. These latter cells formed a loose and discontinuous layer along the sinusoidal walls. Immunoelectron microscopy showed that the ASMA-positive perisinusoidal cells were Ito cells containing fat droplets. The other sinusoidal lining cells were negative for ASMA. In chronic liver disease, ASMA-positive Ito cells showed an increase in number, size, and the intensity of immunostaining in areas of piecemeal necrosis), and formed a continuous cellular network. These cells were dendritic in shape with irregularly elongated cytoplasmic processes and contained an increased amount of microfilaments, in association with loss of the characteristic fat droplets. Thus, their ultrastructural features corresponded to those of myofibroblastic cells. Ito cells showed no staining for desmin in both normal and pathological livers. These results indicate that immunohistochemistry using an anti-ASMA antibody is a sensitive and reliable method for the identification of both normal and transformed Ito cells in adult human livers.

Human Mitochondrial Carbonic Anhydrase VB cDNA CLONING, mRNA EXPRESSION, SUBCELLULAR LOCALIZATION, AND MAPPING TO CHROMOSOME X

A cDNA clone for a novel carbonic anhydrase (CA) isozyme was isolated from human pancreas and salivary glands. The cDNA sequence of 1182 base pairs encoded a 317-amino acid protein with a predicted mass of 36.4 kDa. The highest similarity of this cDNA and the deduced amino acid sequence is to human CA V (mitochondrial CA), hereafter referred to as CA VA. Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmic granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that the cDNA clone presented herein encodes a novel human mitochondrial CA isozyme, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signal sequence (33 amino acid residues). Western blot analysis showed a 36-kDa protein precursor and a 32-kDa mature protein for CA VB. Similar to CA VA, CA VB is a “low activity” enzyme with a sensitivity to acetazolamide. The CA VB gene is located on Xp22.1. Northern blot analysis in normal human tissues demonstrated expression of a 1.3-kilobase transcript in heart and skeletal muscle, and reverse transcription-polymerase chain reaction analysis showed expression of CA VB in pancreas, kidney, salivary glands, and spinal cord but not in liver. CA VA mRNA expression was observed only in liver. These findings indicate these are two genetically distinct isoforms of human CA V, designated CA VA and CA VB, which have different patterns of tissue-specific distribution, suggest different physiological roles for the two mitochondrial isozymes.

DNA typing of HLA class II genes; DRB1*0803 increases the susceptibility of Japanese to primary biliary cirrhosis

The association between human leukocyte antigens and primary biliary cirrhosis is controversial, but major histocompatibility complex class II antigen DR8 was recently reported to be associated with increased susceptibility for primary biliary cirrhosis in some Caucasians and Japanese. Accordingly, we performed DNA typing of HLA class II genes in Japanese patients with primary biliary cirrhosis. The genotypes of HLA DRB1, DRB3-5, DQA and DQB were determined by polymerase chain reaction and subsequent hybridization with sequence specific oligonucleotides in 31 primary biliary cirrhosis patients and 215 racially matched local controls. DR8 was found in 24 of the 31 primary biliary cirrhosis patients and was highly concentrated in DRB1*0803. The gene frequency of DRB1*0803 was significantly increased in the patients (35.5% vs 7.4%, relative risk = 6.84, p < 0.0001). DQA1*0103 and DQB1*0601 were also increased in the primary biliary cirrhosis patients, in relation to linkage disequilibrium with DRB1*0803 on the same haplotype. In contrast, DQA1*0102 showed a significantly lower frequency in the primary biliary cirrhosis patients (p < 0.05). These data suggest that DRB1*0803 is one of the HLA class II genes related to an increased risk of primary biliary cirrhosis in Japanese individuals.

The efficacy of ursodeoxycholic acid and bezafibrate combination therapy for primary biliary cirrhosis: A prospective, multicenter study

Aim:  Treatment with ursodeoxycholic acid (UDCA) improves the survival of stage I and II primary biliary cirrhosis (PBC) patients. However, new therapeutic options are needed for patients who are refractory to UDCA and for those whose disease is at an advanced stage. Bezafibrate could be useful in PBC treatment, since it increases phospholipid output into the bile and reduces the cytotoxicity of hydrophobic bile acids, which are increased with cholestasis.

Development of hepatic sinusoidal structure with special reference to the Ito cells

To elucidate sinusoidal cell structure and function under normal conditions and their behavior in diseased settings, an understanding of their developmental aspects is needed.

Etiology and prognosis of fulminant hepatitis and late-onset hepatic failure in Japan : Summary of the annual nationwide survey between 2004 and 2009

To summarize the annual nationwide survey on fulminant hepatitis (FH) and late‐onset hepatic failure (LOHF) between 2004 and 2009 in Japan.

Serum antibodies to carbonic anhydrase IV in patients with autoimmune pancreatitis

Background and aims: Serum antibodies to carbonic anhydrase (CA) II have been reported in patients with autoimmune pancreatitis (AIP) and Sjögren’s syndrome (SjS). However, their significance in the pathogenesis of these diseases is controversial. The aim of this study was to identify serum antibodies to CA isozymes, which are expressed in ductal cells of the pancreas. Methods: Recombinant proteins of human CAs IV, IX, and XII were obtained using a bacterial expression system, and five CA IV peptides with theoretically high antigenicity were synthesised. Western blotting and enzyme linked immunosorbent assay (ELISA) were used to detect serum antibodies to the CA isozymes. Results: The first screening analysis by western blot showed serum antibodies to CA IV among three CA isozymes in patients with idiopathic chronic pancreatitis, including AIP patients. Further analysis by ELISA showed a significantly increased prevalence of serum antibodies to the truncated CA IV protein and the CA IV synthetic peptide (LGS LTT PTC DEK VVW TVF REP I) in patients with definite AIP (4/15 and 6/20, respectively; p<0.01), probable AIP (6/14 and 3/14; p<0.02), and SjS (9/20 and 8/40; p<0.001) compared with normal controls (0/26). There was no significant difference in the antibody prevalence rates between normal controls and patients with alcoholic chronic pancreatitis (2/15 in each) or pancreatic cancer (2/14 and 1/14, respectively). The presence of serum antibodies to the CA IV peptide showed significant correlations with serum gamma-globulin and IgG levels in AIP patients. Conclusions: These findings suggest that CA IV may be a target antigen that is commonly expressed in epithelial cells of specific tissues involved in AIP and its related diseases.