Makoto Akao

Acrolein induced DNA damage, mutagenicity and effect on DNA repair

Acrolein (Acr) is a ubiquitous environmental contaminant; it also can be generated endogenously by lipid peroxidation. Acr contains a carbonyl group and an olefinic double bond; it can react with many cellular molecules including amino acids, proteins and nucleic acids. In this review article we focus on updating information regarding: (i) Acr-induced DNA damage and methods of detection, (ii) repair of Acr-DNA damage, (iii) mutagenicity of Acr-DNA adducts, (iv) sequence specificity and methylation effect on Acr-DNA adduct formation and (v) the role of Acr in human cancer. We have found that Acr can inhibit DNA repair and induces mutagenic Acr-dG adducts and that the binding spectrum of Acr in the p53 gene in normal human bronchial epithelial cells is similar to the p53 mutational spectrum in lung cancer. Since Acr-DNA adduct has been identified in human lung tissue and Acr causes bladder cancer in human and rat models, we conclude that Acr is a major lung and bladder carcinogen, and its carcinogenicity arises via induction of DNA damage and inhibition of DNA repair.

Angiotensin II as candidate of cardiac cachexia

Purpose of reviewCongestive heart failure is increasing in prevalence and represents a major public health problem. The syndrome of advanced heart failure often includes muscle wasting, commonly termed cardiac cachexia, which is a predictor of poor outcome. Mechanisms of cardiac cachexia are poorly understood, but there is recent evidence that increased angiotensin II, interacting with the insulin-like growth factor-1 system, plays an important role. Recent findingsIn animals, angiotensin II produces weight loss through a pressor-independent mechanism, accompanied by decreased levels of circulating and skeletal muscle insulin-like growth factor-1 and increased mRNA levels of the ubiquitin ligases atrogin-1 and Muscle RING finger-1 in skeletal muscle. Reduced insulin-like growth factor-1 action in muscle leads to increased proteolysis, through the ubiquitin-proteasome pathway, and increased apoptosis. These changes are blocked by muscle-specific expression of insulin-like growth factor-1, likely to be via the Akt/mTOR/p70S6K signaling pathway. SummaryThe link between insulin-like growth factor-1, the ubiquitin-proteasome pathway, and angiotensin II effects has widespread clinical implications for the understanding of mechanisms of catabolic conditions. Therapeutic interventions targeting cross-talk mechanisms between angiotensin II and insulin-like growth factor-1 effects could provide new approaches for the treatment of muscle wasting.

Plasminogen activator-plasmin system potentiates the proliferation of hepatocytes in primary culture

BACKGROUND/AIMS Liver regeneration after partial hepatectomy is thought to be regulated by various molecules including the components of the plasminogen activator (PA)-plasmin system. We have examined the role of fibrinolytic factors, i.e., tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), and their substrate, plasminogen, in the proliferation of hepatocytes in primary culture. METHODS Hepatocyte and nonparenchymal liver cells were isolated from Wistar strain rat by a method perfusing the liver with collagenase. DNA synthesis was assessed by measuring the incorporation of [3H]-thymidine into cellular DNA fraction. tPA, uPA and type-1 plasminogen activator inhibitor (PAI-1) gene expressions were measured by Northern blotting. PA activity was measured by fibrin/agarose plate method. RESULTS Cellular density-dependent DNA synthesis was observed in the primary cultured hepatocytes; DNA synthesis was lower at high cell density (1.0 x 10(5) cells/cm(2)) than that at low cell density (0.2 x 10(5) cells/cm(2)). DNA synthesis in the hepatocytes cultured at a low cell density was increased by co-culture with nonparenchymal liver cells. Under these growth-stimulated culture conditions, tPA and uPA mRNAs were induced and up-regulated. On the contrary, the PAI-1 mRNA level was decreased under these conditions, and total PA activity was augmented accordingly. The synthetic plasmin inhibitor tranexamic acid, a competitive inhibitor for the plasmin molecule, and PASI-535, a plasmin active center-directed inhibitor, both suppressed hepatocyte proliferation in a dose-dependent fashion. Anti-plasmin antibody also suppressed hepatocyte proliferation. CONCLUSIONS The up-regulation of PA activity for ensuring plasmin activity should be an important mechanism in the proliferation of hepatocytes.

Plasminogen activator/plasmin system regulates formation of the hepatocyte spheroids

The isolated rat hepatocytes inoculated onto the surface of positively charged culture dishes are anchored initially and then begin to migrate and aggregate gradually to form multicellular spheroids detached from the dish. We studied the roles of fibrinolytic factors in the spheroid formation. The fibrinolytic factors, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), were increased in the course of spheroid formation. Then, we introduced fibrinolytic inhibitors into the spheroid cultures to determine functions of fibrinolytic factors. Plasmin inhibitor inhibited markedly the spheroid formation. Interestingly, the anti-plasmin antibody showed different effect depending on the timing of its administration. In summary, we demonstrated for the first time that induction of PAs and ensuing plasmin generation on the cell surface play important roles in hepatocyte spheroid formation, and that plasmin is involved in the different processes such as cell migration and cell detachment in the formation of hepatocyte spheroid.

Induction of Hepatic Tissue-Type Plasminogen Activator and Type 1 Plasminogen Activator–Inhibitor Gene Expressions and Appearance of Their Translation Products in the Bile Following Acute Liver Injury in Rats

BACKGROUND/AIMS The plasminogen activator (PA)-plasmin system is primarily involved in fibrinolysis, but is also in the patho/physiological events in which breakdown of extracellular matrices is evoked topically. In this present study, we examined the expression of fibrinolytic factors, tissue-type PA (t-PA) and Type 1 PA inhibitor (PAI-1), in acute liver injury. METHODS Acute liver injury was produced in rats by the intraperitoneal administration of carbon tetrachloride (CCl(4)). Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured to verify the hepatocellular damage. t-PA and PAI-1 gene expressions were measured by Northern blotting, and the cell type(s) expressing these genes was identified by in situ hybridization. t-PA and PAI-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS A single intraperitoneal administration of CCl(4) caused severe acute parenchymatous liver injury. Both t-PA and PAI-1 gene expressions were induced by the acute liver injury, and plasma t-PA and PAI-1 concentrations were also increased. In situ hybridization studies demonstrated that the hepatocytes were the cells expressing t-PA and PAI-1 genes during the acute liver injury. t-PA was also augmented in the bile, whereas PAI-1 was decreased there. CONCLUSIONS t-PA and PAI-1 gene expressions are induced in the hepatocytes of rats with acute liver injury. These fibrinolytic factors induced by liver injury may play important roles in liver regeneration.

Evaluation of Reduced Allergenicity of Deamidated Gliadin in a Mouse Model of Wheat-Gliadin Allergy Using an Antibody Prepared by a Peptide Containing Three Epitopes

Gliadin is the principal allergen of wheat-dependent exercise-induced anaphylaxis (WDEIA). The primary structure of IgE-binding epitopes in wheat gliadin includes tandem sequencing sites of glutamine residues. Therefore, deamidation would be an effective approach to reduce the allergenicity of wheat proteins. In our previous study, we deamidated wheat gliadin without causing peptide-bond hydrolysis or polymerization by use of carboxylated cation-exchange resins, and we found that the deamidated gliadin scarcely reacted with the sera of patients radioallergosorbent test (RAST)-positive to wheat. In this study, we examined the allergenicity of deamidated gliadin in a mouse model of wheat-gliadin allergy. Oral administration of deamidated gliadin to gliadin-sensitized mice suppressed enhancement in intestinal permeability, serum allergen level, serum allergen-specific IgE level, mast-cell-surface expression of FcεRI, and serum and intestinal histamine levels. Our results indicate that gliadin deamidated with no peptide-bond hydrolysis by cation-exchange resins has low allergenicity even under in vivo conditions.

Binding of plasminogen to hepatocytes isolated from injured mice liver and nonparenchymal cell-dependent proliferation of hepatocytes

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in α2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/α2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of α2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen–plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.

Cellular density regulation of plasminogen gene expression in mouse hepatocytes.

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.

Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of Blood Glucose and Plasma Insulin Levels after Oral Glucose Loading

The suppressive effect of rice albumin (RA) of 16 kDa on elevation of blood glucose level after oral loading of starch or glucose and its possible mechanism were examined. RA suppressed the increase in blood glucose levels in both the oral starch tolerance test and the oral glucose tolerance test. The blood glucose concentrations 15 min after the oral administration of starch were 144 ± 6 mg/dL for control group and 127 ± 4 mg/dL for RA 200 mg/kg BW group, while those after the oral administration of glucose were 157 ± 7 mg/dL for control group and 137 ± 4 mg/dL for RA 200 mg/kg BW group. However, in the intraperitoneal glucose tolerance test, no significant differences in blood glucose level were observed between RA and the control groups, indicating that RA suppresses the glucose absorption from the small intestine. However, RA did not inhibit the activity of mammalian α-amylase. RA was hydrolyzed to an indigestible high-molecular-weight peptide (HMP) of 14 kDa and low-molecular-weight peptides by pepsin and pancreatin. Furthermore, RA suppressed the glucose diffusion rate through a semipermeable membrane like dietary fibers in vitro. Therefore, the indigestible HMP may adsorb glucose and suppress its absorption from the small intestine.

A Simple Synthesis of Alliin and allo-Alliin: X-ray Diffraction Analysis and Determination of Their Absolute Configurations.

A simple method for the isolation of the bioactive compound alliin from garlic, as well as a method for the synthesis of diastereomerically pure alliin and allo-alliin on a preparative laboratory scale, was developed. The absolute configuration of the sulfur atom in alliin and allo-alliin was assigned on the basis of enzyme reactivity, optical rotatory dispersion, and circular dichroism analyses. A comparison of the results from these analyses, in combination with an X-ray diffraction study on a protected allo-alliin derivative, revealed S and R configurations of the sulfur atoms in alliin and allo-alliin, respectively. In addition, the same (1)H NMR spectrum was observed for synthetic and natural alliin. The absolute configuration of natural alliin was assigned for the first time on the basis of the NMR spectrum and X-ray coordinates.