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Masayasu Inoue

Publications and source records attributed to Masayasu Inoue.

53 records · Page 3Linked to original sources
PubMed2002

Oxidative stress-induced cell death of human oral neutrophils.

Polymorphonuclear leukocytes (PMN) play crucial roles in protecting hosts against invading microbes and in the pathogenesis of inflammatory tissue injury. Although PMN migrate into mucosal layers of digestive and respiratory tracts, only limited information is available of their fate and function in situ. We previously reported that, unlike circulating PMN (CPMN), PMN in the oral cavity spontaneously generate superoxide radical and nitric oxide (NO) in the absence of any stimuli. When cultured for 12 h under physiological conditions, oral PMN (OPMN) showed morphological changes that are characteristic of those of apoptosis. Upon agarose gel electrophoresis, nuclear DNA samples isolated from OPMN revealed ladder-like profiles characteristic of nucleosomal fragmentation. l-cysteine, reduced glutathione (GSH), and herbimycin A, a protein tyrosine kinase inhibitor, suppressed the activation of caspase-3 and apoptosis of OPMN. Neither thiourea, superoxide dismutase (SOD), nor catalase inhibited the activation of caspase-3 and apoptosis. Moreover, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibitor for caspase-3, inhibited the fragmentation of DNA. These results suggested that oxidative stress and/or tyrosine-kinase-dependent pathway(s) activated caspase-3 in OPMN, thereby inducing their apoptosis.

Antioxidants↗
PubMed2002

Down-regulation of spontaneous Epstein-Barr virus reactivation in the P3HR-1 cell line by L-arginine.

We tested the hypothesis that inhibition of Epstein-Barr virus (EBV) reactivation is controlled in part by nitric oxide (NO) generated from L-arginine (Arg). The spontaneous reactivation of EBV in the Burkitt's lymphoma (BL) cell line P3HR-1 was inhibited when the cells were cultured in L-Arg-supplemented medium. The expression of EBV early antigen (EA), immediate-early BZLF1 mRNA and the protein ZEBRA, and production of infectious virus were reduced by L-Arg supplementation in a dose-dependent manner. We demonstrated that inducible NO synthase (iNOS) mRNA was constitutively expressed in P3HR-1 cells, as quantitated by the reverse transcription-polymerase chain reaction. L-Arg supplementation enhanced iNOS and NOx expression in the cells. A specific NOS inhibitor, NG-monomethyl-L-Arg enhanced the expression of ZEBRA and early BMRF1 protein EA-D in the cells. L-Arg supplementation also inhibited the spontaneous EBV reactivation in another BL cell line EB1 and a B lymphoblastoid cell line OB. These results indicated that L-Arg induces iNOS and generates NO, which inhibits EBV reactivation in EBV-positive cells.

Arginine↗
PubMed2002

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats.

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.

Animals↗
PubMed2002

L-Carnitine inhibits cisplatin-induced injury of the kidney and small intestine.

Although cis-diamminedichloroplatinum (II) (cisplatin) is a potent anticancer drug, clinical use of this agent is highly limited predominantly because of its strong side effects on the kidney and gastrointestinal tracts. We found that cisplatin impaired respiratory function and DNA of mitochondria in renal proximal tubules and small intestinal mucosal cells, thereby inducing apoptosis of epithelial cells. Cisplatin-induced mitochondrial dysfunction and DNA (mtDNA) injury, lipid peroxidation, and apoptosis of epithelial cells in the kidney and small intestine were strongly inhibited by L-carnitine. However, carnitine had no appreciable effect on the tumoricidal action of cisplatin against cancer cells inoculated in the peritoneal cavity. These results indicate that L-carnitine may have therapeutic potential for inhibiting the side effects of cisplatin and other anticancer agents in the kidney and small intestine.

Animals↗
PubMed2002

Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate.

Phthalate esters have been used extensively as plasticizers of synthetic polymers. Recent studies have revealed that these esters induce atrophy of the testis, although its pathogenesis remains unknown. The present study describes the possible involvement of oxidative stress in the pathogenesis of atrophy of the rat testis induced by di(2-ethylhexyl)phthalate (DEHP). Biochemical and immunohistochemical analysis revealed that oral administration of DEHP increased the generation of reactive oxygen species, with concomitant decrease in the concentration of glutathione and ascorbic acid in the testis, and selectively induced apoptosis of spermatocytes, thereby causing atrophy of this organ. Oxidative stress was selectively induced in germ cells, but not in Sertoli cells, treated with mono(2-ethylhexyl)phthalate (MEHP), a hydrolysed metabolite of DEHP. Furthermore, MEHP selectively induced the release of cytochrome c from mitochondria of the testis. These results indicate that oxidative stress elicited by MEHP principally injured mitochondrial function and induced the release of cytochrome c, thereby inducing apoptosis of spermatocytes and causing atrophy of the testis.

Animals↗
PubMed2002

Smoking a single cigarette rapidly reduces combined concentrations of nitrate and nitrite and concentrations of antioxidants in plasma.

BACKGROUND: Cigarette smoking is a well-known risk factor for the development of cardiovascular disease, yet the mechanism of action involved is not completely understood. Because cigarette smoke contains superoxide and other reactive oxygen species, it has been hypothesized that some of the adverse effects of smoking may result from oxidative damage to endothelial cells, which results in nitric oxide (NO) shortage. However, little information is available regarding the acute effects of smoking on plasma concentrations of NO and antioxidants. We measured the changes in the combined plasma concentrations of nitrate and nitrite as an index of NO concentration, as well as changes in concentrations of major serum antioxidants (ascorbic acid, cysteine, methionine, and uric acid) in smokers after smoking a single cigarette. METHODS AND RESULTS: A randomized crossover study of the effects of smoking a single cigarette was performed in 20 smokers. Smoking a sham cigarette induced no significant changes in all assayed parameters. However, smoking a single cigarette significantly decreased nitrate and nitrite plasma concentrations by 3.5 +/-1.2 and 3.4 +/- 1.1 micromol/L, compared with plasma concentrations at presmoking and sham smoking, respectively. The concentrations of ascorbic acid and other antioxidants were also significantly lower after smoking a single cigarette. These parameters returned to preexperimental levels 60 minutes after smoking cessation. CONCLUSION: The present findings indicate that smoking a single cigarette temporarily decreases nitrate, nitrite, and serum antioxidant concentrations in the plasma. These transient changes may partially contribute to coronary vasoconstriction, which is routinely observed after smoking.

Adult↗
PubMed2002

Association of Cu,Zn-type superoxide dismutase with mitochondria and peroxisomes.

The subcellular localization of Cu,Zn-type superoxide dismutase (Cu,Zn-SOD) was investigated in rat tissues and cultured human fibroblasts. Subcellular fractionation, Nycodenz gradient centrifugation, and immunoblot analysis using specific antibodies showed that Cu,Zn-SOD was localized in cytosol, mitochondria, and peroxisomes of rat liver and brain. Treatment of highly purified mitochondria from rat liver with either Chaps or Triton X-100 released the bound Cu,Zn-SOD into supernatant fraction. Depolarization of mitochondria by inorganic phosphate and Ca(2+) released both Cu,Zn-SOD and cytochrome c from mitochondria. Digitonin also released Cu,Zn-SOD but not cytochrome c from mitochondria. Confocal immunofluorescence microscopy revealed that anti-Cu,Zn-SOD antibody in cultured human fibroblasts was found to colocalize with antibodies to Mn-SOD and PMP-70, markers of mitochondria and peroxisomes, respectively. Incubation of human Cu,Zn-SOD with purified mitochondria resulted in their association. These results indicate that Cu,Zn-SOD associates with mitochondria and peroxisomes in various cell types such as those in brain, liver, and skin.

Animals↗
PubMed2002

Molecular characterization of a novel protein disulfide isomerase in carrot.

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.

Amino Acid Sequence↗
PubMed2002

Antioxidant protection of propofol and its recycling in erythrocyte membranes.

alpha-Tocopherol is a potent antioxidant that effectively protects biological membranes against oxidative injury through coordination with ascorbic acid. Because propofol has a phenolic structure similar to that of alpha-tocopherol, this intravenous anesthetic may also have similar antioxidant activity. To test this hypothesis, the effect of propofol on oxidative injury of human erythrocytes was examined. Propofol inhibited oxidative hemolysis and cis-parinaric acid oxidation in erythrocyte membranes (ED(50) = 6 microM). Although ascorbic acid alone has no appreciable effect, the protective effect of propofol was enhanced by ascorbic acid. An electron spin resonance (ESR) study showed that propofol-derived radicals (g = 2.005) were continuously generated during the oxidation of erythrocyte membranes by an ascorbic acid-inhibitable mechanism. These and other results suggest that propofol interacts with ascorbic acid, thereby exhibiting potent antioxidant activity in and around membranes as does alpha-tocopherol. Kinetic analysis revealed that propofol increased the membrane fluidity of erythrocytes, thereby increasing their resistance to physical and hemodynamic stress. Further, a greater preservation of red blood cell counts was seen after surgery with propofol compared with conventional sevoflurane anesthesia. Thus, propofol may protect erythrocytes against both oxidative and physical stress, indicating its potential as an efficient and safe antioxidant.

Antioxidants↗
PubMed2002

Functional analysis of recombinant pancreatic secretory trypsin inhibitor protein with amino-acid substitution.

BACKGROUND: We hypothesized that mutation of the pancreatic secretory trypsin inhibitor ( PSTI) gene may promote a predisposition to pancreatitis, possibly by reducing the inhibition of trypsin activity. Based on this hypothesis, we performed a biochemical analysis of recombinant PSTI protein. METHODS: The trypsin inhibitory activity of the recombinant protein was analyzed. The activity of PSTI protein with a point mutation of the most common type: (34)Asn (AAT)-to-Ser (AGT)(101A>G N34S: N34S) in exon 3, was compared with that of the wild type. RESULTS: The function of N34S PSTI remained unchanged under both the usual alkaline and acidic conditions compared with the wild-type PSTI. The calcium concentration did not affect the activity of recombinant PSTI. The trypsin susceptibility of the N34S protein was not increased either. CONCLUSIONS: Mechanisms other than the conformational change of PSTI associated with amino-acid substitution, such as abnormal splicing, may underlie the predisposition to pancreatitis in patients with the N34S mutation.

Amino Acid Substitution↗
PubMed2002

Inhibition of vancomycin-induced nephrotoxicity by targeting superoxide dismutase to renal proximal tubule cells in the rat.

Vancomycin, a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). Because vancomycin induces renal dysfunction, the dose and the duration of its administration are limited. The mechanism of vancomycin-induced renal dysfunction is not known. We recently synthesized a hexamethylenediamine-conjugated cationic superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. The present work reports the protective effects of AH-SOD against vancomycin-induced renal dysfunction. Male Wistar rats (200-210 g) were intraperitoneally administered with either 200 or 400 mg/kg of vancomycin twice a day for 7 days. Either 5 mg/kg/day AH-SOD or saline was subcutaneously injected 5 min before every vancomycin injection. Biochemical analysis revealed that plasma levels of blood urea nitrogen and creatinine increased significantly in vancomycin-treated group by an AH-SOD-inhibitable mechanism. Histological examination revealed that vancomycin also elicited a marked destruction of glomeruli and necrosis of proximal tubule by an AH-SOD inhibitable mechanism. These results suggest that oxidative stress underlies the pathogenesis of vancomycin-induced nephrotoxicity and that targeting SOD and/or related antioxidants to renal proximal tubule cells might permit the administration of higher doses of vancomycin sufficient for eradication of MRSA without causing renal injury.

Animals↗
PubMed2002

[Reactive oxygen].

Mammalian tissues have large amounts of available ATP which are generated by oxidative phosphorylation in mitochondria. To maintain the body, large amounts of oxygen are required to regenerate the ATP molecules. A small fraction of the inspired oxygen is converted to superoxide radicals and related metabolites even under physiological conditions. Most reactive oxygen species react rapidly with a variety of molecules, thereby interfering with cellular functions and various diseases.

Hemoglobins↗
PubMed2002

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium.

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.

Animals↗
PubMedDate not supplied

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells.

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

Acetophenones↗