Yuri Miura

In Vivo Electron Paramagnetic Resonance Studies on Oxidative Stress Caused by X-Irradiation in Whole Mice

The effect of x-irradiation on the reduction rates of nitroxyl radicals was examined in whole mice using in vivo EPR. One hour after irradiation, the reduction rates of nitroxyl increased up to 15 Gy irradiation, but decreased over this dose. The enhancement of the reduction rate of nitroxyl was suppressed by preadministration of a radioprotector, cysteamine, suggesting that the enhancement of nitroxyl reduction is related to the radiation damage. Thiobarbituric acid-reactive substances (TBARS) in liver homogenate were increased by x-irradiation, indicating that x-irradiation induced oxidative stress in mice. Endogenous antioxidant, alpha-tocopherol, and the activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase were not induced by x-irradiation under these experimental conditions. Eventually the nitroxyl reduction in whole mice should be enhanced by the oxidative stress due to x-irradiation. An in vivo EPR system probing the nitroxyl reduction should be applicable to the noninvasive study on the oxidative stress caused by radiation.

Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body.

A NOVEL LIPOPHILIC SPIN PROBE FOR THE MEASUREMENT OF RADIATION DAMAGE IN MOUSE BRAIN USING IN VIVO ELECTRON SPIN RESONANCE (ESR)

As a possible lipophilic spin probe of in vivo electron spin resonance (ESR), 3‐methoxy carbonyl‐2,2,5,5‐tetramethyl‐pyrrolidine‐1‐yloxy (MCPROXYL) was examined. The permeability of the blood‐brain barrier to this compound was evaluated with a brain uptake index and autoradiography, with result that this probe is well distributed in the brain. The in vivo ESR spectra were measured in the head and the abdomen of MCPROXYL‐injected living mice. The rate of signal decay of MCPROXYL in the head measured at one hour after X‐irradiation was about 75% of that of the controls. The decrease in the head seems to be related to the early response of the brain to X‐irradiation. This is the first report that the behavior of free radical such as MCPROXYL in the brain is influenced by X‐irradiation. MCPROXYL is thus useful as a novel spin probe for in vivo ESR to monitor the radiation damage in the brain.

Oxidation of spin-traps by chlorine dioxide (ClO2) radical in aqueous solutions: first ESR evidence of formation of new nitroxide radicals.

The reactivities of the chlorine dioxide (ClO2), which is a stable free radical towards some water-soluble spin-traps were investigated in aqueous solutions by an electron spin resonance (ESR) spectroscopy. The ClO2 radical was generated from the redox reaction of Ti3+ with potassium chlorate (KClO3) in aqueous solutions. When one of the spin-traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was included in the Ti3+-KClO3 reaction system, ESR spectrum due to the ClO2 radical completely disappeared and a new ESR spectrum [aN(1) = 0.72 mT, aH(2) = 0.41 mT], which is different from that of DMPO-ClO2 adduct, was observed. The ESR parameters of this new ESR signal was identical to those of 5,5-dimethylpyrrolidone-(2)-oxyl-(1) (DMPOX), suggesting the radical species giving the new ESR spectrum is assignable to DMPOX. The similar ESR spectrum consisting of a triplet [aN(1) = 0.69 mT] was observed when the derivative of DMPO, 3,3,5,5-tetramethyl-1-pyrroline N-oxide (M4PO) was included in the Ti3+-KClO3 reaction system. This radical species is attributed to the oxidation product of M4PO, 3,3,5,5-tetramethylpyrrolidone-(2)-oxyl-(1) (M4POX). When another nitrone spin-trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) was used as a spin-trap, the ESR signal intensity due to the ClO2 radical decreased and a new ESR signal consisting of a triplet [aN(1) = 0.76 mT] was observed. The similar ESR spectrum was observed when N-t-butyl-alpha- nitrone (PBN) was used as a spin-trap. This ESR parameter [a(N)(1) = 0.85 mT] was identical to the oxidation product of PBN, PBNX. Thus, the new ESR signal observed from POBN may be assigned to the oxidation product of POBN, POBNX. These results suggest that the ClO2, radical does not form the stable spin adducts with nitrone spin-traps, but oxidizes these spin-traps to give the corresponding nitroxyl radicals. On the other hand, nitroso spin-traps, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), and 2-methyl-2-nitrosopropane (MNP) did not trap the ClO2 radical. This result indicates that an unpaired electron of the ClO2 radical is localized on oxygen atom, because nitroso spin-traps cannot form the stable spin adduct with oxygen-centered radical.

Adaptive response and the influence of ageing: effects of low-dose irradiation on cell growth of cultured glial cells

Purpose : To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of ageing on the response. Materials and methods : Glial cells were cultured from young and older rats (1 and 24 months). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signalling pathway factors in RAR was investigated using their inhibitors, activators, and mutated and knockout glial cells. Results : RAR was observed in cells cultured from young rats but was not in cells from older animals. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (P13K) suppressed RAR. The activators of PKC instead of low-dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxiatelangiectasia mutated (Atm) knockout mice showed no RAR. Conclusion : The results indicated that PKC, ATM, DNAPK and/or P13K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with ageing.

Survival responses to oxidative stress and aging.

Oxidative stress is recognized as an important environmental factor in aging; however, because reactive oxygen species (ROS) and related free radicals are normally produced both intra‐ and extracellularly, air‐living organisms cannot avoid the risk of oxidative stress. Consequently, these organisms have evolved various anti‐oxidant systems to prevent ROS, scavenge free radicals, repair damaged components and adaptive responses. This review will focus on the repair and adaptive response to oxidative stress, and summarize the changes of these systems as a result aging and their relationship to premature aging. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S1–S9.

Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4

PurposeThe objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes.MethodsO-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA).ResultsO-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules.ConclusionThese results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy.

Integrin β4 and vinculin contained in exosomes are potential markers for progression of prostate cancer associated with taxane-resistance

Treatment with taxanes for castration-resistant prostate cancer often leads to the development of resistance. It has been recently demonstrated that exosomes present in the body fluids contain proteins and RNAs in the cells from which they are derived and could serve as a diagnostic marker for various diseases. In the present study, we aimed to identify proteins contained in exosomes that could be markers for progression and taxane-resistance of prostate cancer. Exosomes were isolated by differential centrifugation from the culture medium of taxane-resistant human prostate cancer PC-3 cells (PC-3R) and their parental PC-3 cells. Isolated exosomes were subjected to iTRAQ-based quantitative proteomic analysis. Exosomes were also isolated from the culture medium by using anti-CD9 antibody-conjugated magnetic beads. Protein expression was knocked down by siRNA transfection followed by analysis of the silencing effects. Proteomic analysis showed that integrin β4 (ITGB4) and vinculin (VCL) were upregulated in exosomes derived from PC-3R cells compared to PC-3 cells. The elevation of ITGB4 and VCL was confirmed in exosomes captured by anti-CD9 antibody from the culture medium of PC-3R cells. Silencing of ITGB4 and VCL expression did not affect proliferation and taxane-resistance of PC-3R cells, but ITGB4 knockdown attenuated both cell migration and invasion and VCL knockdown reduced invasion. Our results suggest that ITGB4 and VCL in exosomes could be useful markers for progression of prostate cancer associated with taxane-resistance, providing the basis for development of an exosome-based diagnostic system.

O-GlcNAc modification affects the ATM-mediated DNA damage response

BACKGROUND O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein. METHODS The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM. RESULTS Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress. GENERAL SIGNIFICANCE ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.

Oxidation of linoleic acid by copper(II) complexes: effects of ligand

Abstract The oxidation of linoleic acid (LH) by cupric (Cu(II)) complexes was investigated by absorption spectroscopy and high performance liquid chromatography (HPLC). The following Cu(II) complexes were used: Cu(II)(BC) 2 (BC: bathocuproinedisulfonic acid), Cu(II)(CyHH) 2 (CyHH: cyclo( L -histidyl- L -histidyl)), Cu(II)(OP) 2 (OP: o -phenanthroline), Cu(II)(HGG) (HGG: L -histidylglycylglycine), and Cu(II)(en) 2 (en: ethylenediamine). The absorbance at 234 nm observed during Cu(II)-catalyzed oxidation of LH increased with incubation time, reached a maximum level, and subsequently decreased. HPLC chromatograms of oxidation products indicated the appearance of peaks corresponding to four isomers of linoleic acid hydroperoxide (LOOH) and subsequent complete decomposition of LOOH. The time to reach the maximal value of absorbance at 234 nm within 24 h was in the following order: Cu(II)(BC) 2 >Cu(II)(CyHH) 2 =Cu(II)(OP) 2 >Cu(II)(en) 2 >Cu(II)(HGG). These observations suggested that Cu(II)(BC) 2 can produce LOOH from linoleic acid more rapidly than Cu(II)(HGG) and decompose the LOOH generated more easily. The absorbance at 234 nm observed in the reaction of Cu(II)(HGG) or Cu(II)(en) 2 with LH in the presence of H 2 O 2 increased with incubation time more rapidly than in the absence of H 2 O 2 . Hydroxyl radical generation from the reaction of Cu(II)(HGG) or Cu(II)(en) 2 with H 2 O 2 may initiate the oxidation of LH. In contrast, the addition of H 2 O 2 inhibited the oxidation of LH by Cu(II)(BC) 2 .