Junji Magae

A novel protein phosphatase inhibitor, tautomycin. Effect on smooth muscle.

The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type‐1 and type‐2A enzymes. For the catalytic subunits of the type‐1 and type‐2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca2+‐independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation.

Prodigiosin 25-C uncouples vacuolar type H+-ATPase, inhibits vacuolar acidification and affects glycoprotein processing

Prodigiosin 25‐C inhibited the accumulation of 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine and acridine orange in the acidic compartments of baby hamster kidney cells with little perturbation of cellular ATP levels. In rat liver lysosomes, prodigiosin 25‐C inhibited the proton pump activity with an IC50 of approximately 30 nM, but did not affect ATPase activity up to 1 μM. It also delayed the transport of vesicular stomatitis virus G protein and induced a drastic swelling of Golgi apparatus and mitochondria. These results indicate that prodigiosin 25‐C raises the pH of acidic compartments through inhibition of the proton pump activity of vacuolar type H+‐ATPase, thereby causing the functional and morphological changes to the Golgi apparatus.

Suppression Of Cytotoxic T Cell Induction In Vivo By Prodigiosin 25-c

Prodigiosin 25-C (PrG25-C) was discovered as an immunosuppressant in the course of our screening for immunomodulating substances. In this system, PrG25-C inhibited T lymphocytes proliferation and was less suppressive against B lymphocytes. PrG25-C was also a powerful inhibitor of cytotoxic T cell induction by mixed lymphocyte reaction and completely suppressed induction of H-2 specific cytotoxic cells at 12.7 nM. PrG25-C also inhibited in vivo induction of H-2 restricted cytotoxic T lymphocytes at a dose of 0.5 mg/kg but had little myelotoxicity because numbers of blood leukocytes and splenocytes of PrG25-C-treated mice were comparable to those of nonsensitized mice. No inhibitory effects of PrG25-C were observed on the production of anti-SRBC antibody. These results indicate that PrG25-C is a T-lymphocyte-specific immunosuppressant.

p53-Independent Induction of G1 Arrest and p21WAF1/CIP1 Expression by Ascofuranone, an Isoprenoid Antibiotic, through Downregulation of c-Myc

Ascofuranone has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we study the effects of ascofuranone on cell cycle progression in human cancer cells and find that ascofuranone induces G1 arrest without cytoxicity with upregulation of p53 and p21WAF1/CIP1 while downregulating c-Myc and G1 cyclins. Chromatin immunoprecipitation assay and RNA interference studies with cells deficient in p53 and p21 show that ascofuranone induces p21WAF1/CIP1 expression and subsequent G1 arrest through the release of p21WAF1/CIP1 promoter from c-Myc–mediated transcriptional repression, independent of p53. Ascofuranone-induced p21WAF1/CIP1 associates with CDK2 and prevents CDK2-cyclin E complex formation, leading to the inactivation of E2F transcriptional activity. These results suggest that ascofuranone upregulates p21WAF1/CIP1 through p53-independent suppression of c-Myc expression, leading to cytostatic G1 arrest. Thus, ascofuranone represents a unique natural antitumor compound that targets c-Myc independent of p53. Mol Cancer Ther; 9(7); 2102–13. ©2010 AACR.

Reduction in Mutation Frequency by Very Low-Dose Gamma Irradiation of Drosophila melanogaster Germ Cells

Abstract Ogura, K., Magae, J., Kawakami, Y. and Koana, T. Reduction in Mutation Frequency by Very Low-Dose Gamma Irradiation of Drosophila melanogaster Germ Cells. Radiat. Res. 171, 1–8 (2009). To determine whether the linear no-threshold (LNT) model for stochastic effects of ionizing radiation is applicable to very low-dose radiation at a low dose rate, we irradiated immature male germ cells of the fruit fly, Drosophila melanogaster, with several doses of 60Co γ rays at a dose rate of 22.4 mGy/h. Thereafter, we performed the sex-linked recessive lethal mutation assay by mating the irradiated males with nonirradiated females. The mutation frequency in the group irradiated with 500 μGy was found to be significantly lower than that in the control group (P < 0.01), whereas in the group subjected to 10 Gy irradiation, the mutation frequency was significantly higher than that in the control group (P < 0.03). A J-shaped dose–response relationship was evident. Molecular experiments using DNA microarray and quantitative reverse transcription PCR indicated that several genes known to be expressed in response to heat or chemical stress and grim, a positive regulator of apoptosis, were up-regulated immediately after irradiation with 500 μGy. The involvement of an apoptosis function in the non-linear dose–response relationship was suggested.

Ascochlorin suppresses oxLDL-induced MMP-9 expression by inhibiting the MEK/ERK signaling pathway in human THP-1 macrophages.

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP‐1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low‐density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92‐kDa matrix metalloproteinase‐9 (MMP‐9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL‐induced MMP‐9 expression and activity in human THP‐1 macrophages. ASC reduced oxLDL‐induced MMP‐9 expression and activity in a time‐dependent and dose‐dependent manner. Also, an analysis of MMP‐9 activity using pharmacologic inhibitors showed that ASC inhibits MMP‐9 activity via the extracellular signal‐regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents. J. Cell. Biochem. 102: 506–514, 2007.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway

A natural compound C23H32O4Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti‐cancer, anti‐hypolipidemic, and anti‐hypertension agent. In this study, anti‐inflammatory activity has been investigated in lipopolysaccharide (LPS)‐induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1–50 μM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in a dose‐dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)‐1β and IL‐6 but not tumor necrosis factor (TNF)‐α in LPS‐stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor‐κB (NF‐κB). Furthermore, ASC down‐regulated phospho‐extracellular signal‐regulated kinase 1/2 (p‐ERK1/2) and p‐p38. These results demonstrate that ASC exhibits anti‐inflammatory effects in RAW 264.7 macrophage cells. J. Cell. Biochem. 117: 978–987, 2016.

Suppressive Effects of Continuous Low-Dose-Rate γ Irradiation on Diabetic Nephropathy in Type II Diabetes Mellitus Model Mice

It has been proposed that the development of diabetic nephropathy is caused in large part by oxidative stress. We previously showed that continuous exposure of mice to low-dose-rate &ggr; radiation enhances antioxidant activity. Here, we studied the ameliorative effect of continuous whole-body irradiation with low-dose-rate &ggr; rays on diabetic nephropathy. Ten-week-old female db/db mice, an experimental model for type II diabetes, were irradiated with low-dose-rate &ggr; rays from 10 weeks of age throughout their lives. Nephropathy was studied by histological observation and biochemical analysis of serum and urine. Antioxidant activities in kidneys were determined biochemically. Continuous low-dose-rate &ggr; radiation significantly increases life span in db/db mice. Three of 24 irradiated mice were free of glucosuria after 80 weeks of irradiation. Histological studies of kidney suggest that low-dose irradiation increases the number of normal capillaries in glomeruli. Antioxidant activities of superoxide dismutase, catalase and glutathione are significantly increased in kidneys of irradiated db/db mice. Continuous low-dose-rate &ggr; irradiation ameliorates diabetic nephropathy and increases life span in db/db mice through the activation of renal antioxidants. These findings have noteworthy implications for radiation risk estimation of non-cancer diseases as well as for the clinical application of low-dose-rate &ggr; radiation for diabetes treatment.

Amelioration of Type II Diabetes in db/db Mice by Continuous Low-Dose-Rate γ Irradiation

Abstract Tsuruga, M., Taki, K., Ishii, G., Sasaki, Y., Furukawa, C., Sugihara, T., Nomura, T., Ochiai, A., and Magae, J. Amelioration of Type II Diabetes in db/db Mice by Continuous Low-Dose-Rate γ Irradiation. Radiat. Res. 167, 592–599 (2007). Low-dose-rate radiation modulates various biological responses including carcinogenesis, immunological responses and diabetes. We found that continuous irradiation with low-dose-rate γ rays ameliorated type II diabetes in db/db mice, diabetic mice that lack leptin receptors. Whole-body exposure of db/db mice to low dose-rate γ radiation improved glucose clearance without affecting the response to insulin. Histological studies suggested that degeneration of pancreatic islets was significantly suppressed by the radiation. Insulin secretion in response to glucose loading was increased significantly in the irradiated mice. These results suggest that low-dose-rate γ radiation ameliorates type II diabetes by maintaining insulin secretion, which gradually decreases during the progression of diabetes due to degeneration of pancreatic islets. We also inferred that protection from oxidative damage is involved in the anti-diabetic effect of low-dose-rate γ rays because expression and activity of pancreatic superoxide dismutase were significantly elevated by low-dose-rate γ radiation.

Prolongation of Life Span in the Accelerated Aging klotho Mouse Model, by Low-Dose-Rate Continuous γ Irradiation

While lifespan studies provide basic information for estimating the risk of ionizing radiation, findings on the effect of low-dose/low-dose-rate irradiation on the lifespan of mammals are controversial. Here we evaluate the effect of continuous exposure to low-dose-rate γ radiation on the lifespan of mice with accelerated aging caused by mutation of the klotho gene. While control mice died within 80 days after birth, more than 10% of mice exposed continuously to 0.35 or 0.7 or mGy/h γ radiation from 40 days after birth survived for more than 80 days. Two of 50 mice survived for more than 100 days. Low-dose-rate irradiation significantly increased plasma calcium concentration in mutant mice, and concomitantly increased hepatic catalase activity. Although hepatic activity of superoxide dismutase in mutant mice decreased significantly compared to wild-type mice, continuous γ irradiation decreased the activity in mutant mice significantly. These results suggest that low-dose-rate ionizing radiation can prolong the lifespan of mice in certain settings.