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H Shichi

Publications and source records attributed to H Shichi.

At least 19 recordsLinked to original sources
PubMed2001

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.

Animals↗
PubMed2001

Bovine eye 1-Cys peroxiredoxin: expression in E. coli and antioxidant properties.

Peroxiredoxins constitute a molecular family of novel antioxidant proteins and are distributed broadly in non-mammalian and mammalian tissues, including the eye. In this study, a recombinant bovine eye 1-Cys peroxiredoxin (BRPrx) was expressed in Escherichia coli (E. coli). The recombinant protein protected glutamine synthetase from oxidative damage caused by a metal ion-catalyzed oxidation system (ascorbate/Fe3+/O2) in the presence of dithiothreitol as an electron donor. The protector activity of BRPrx is attributed to its peroxidase activity exhibited in the presence of dithiothreitol. Both hydrogen peroxide and short chain hydroperoxides are substrates for the protein. Glutathione could not support antioxidant properties of the recombinant protein. The antioxidant activity of BRPrx in the glutamine synthetase protection assay was as high as the activity of catalase and about one order of magnitude lower than that of selenium glutathione peroxidase. These results support the premise that Prx is an important component of the antioxidant defense system in eye tissues.

Animals↗
PubMed2001

Peroxiredoxin in bovine ocular tissues: immunohistochemical localization and in situ hybridization.

Peroxiredoxins are widely distributed in nature and constitute a molecular family of antioxidant enzymes which decompose hydrogen peroxide and alkyl hydroperoxides. We have previously characterized a peroxiredoxin from bovine ciliary body and deduced its amino acid sequence from analysis of cDNA clones encoding the protein. In this work, we investigated the immunolocalization of this novel antioxidant enzyme and its mRNA expression in bovine eye tissues. High levels of immunoreactivity and mRNA for the enzyme were detected in corneal epithelium. Distinct immunoreactivity and mRNA expression for peroxiredoxin were also detected in uveal tissues, some of the retinal cell layers and ocular vasculature.

Animals↗
PubMed2001

Naphthoquinone-Induced cataract in mice: possible involvement of Ca2+ release and calpain activation.

N-acetyl-p-benzoquinone imine (NAPQI), a semiquinone metabolite of acetaminophen, produces cataract in mice. Naphthalene is biotransformed to the cataractogenic metabolite 1,2-naphthoquinone (NQ). Intracameral injection of NAPQI elicits a rapid increase in free intracellular Ca2+ in the lens epithelium and calpain activation before lens opacification begins. In order to test whether the cellular response is a common feature of quinone-induced cataracts, we injected in this work 1,2-naphthoquinone (NA) in the anterior chamber of mouse eye and followed cellular responses in the lens prior to opacity development. A marked rise in free intracellular Ca2+ in the lens epithelium and concurrent activation of calpain were observed within 1 hr after NQ injection preceding lens opacity development. These results support the suggestion that Ca2+ release and calpain activation are involved in the mechanism of quinone-induced cataractogenesis.

Animals↗
PubMed2000

Cataract formation by a semiquinone metabolite of acetaminophen in mice: possible involvement of Ca(2+)and calpain activation.

Acetaminophen, an analgesic/antipyretic, is metabolized by hepatic cytochrome P450 to N -acetyl- p -benzoquinone imine (NAPQI), which is transported by blood circulation to the eye and induces anterior cortical cataract in mice. In this study we injected NAPQI into the anterior chamber of mouse eye and investigated time-dependent cellular responses in the lens. After a lag period of about 2 hr following NAPQI injection, lens opacification as determined by measurement of light scattering by the lens became evident and progressively increased thereafter. There was no difference in the profile of opacity development between a P450-inducer responsive mouse strain and a non-responsive strain. During the lag period, a marked increase in free intracellular Ca(2+)in the lens epithelium was observed at 1 hr by confocal fluorescence microscopy with a Ca(2+)probe. Concurrent with the free Ca(2+)increase, there was a 300% rise in the activity of the non-lysosomal neutral protease calpain in the lens at 1 hr after NAPQI injection. Evidence indicated degradation of vimentin in the lens in which calpain activity was enhanced. Co-injection of calpain inhibitors (N-Ac-Leu-Leu-norleucinol and N-Ac-Leu-Leu-methioninal) with NAPQI protected animals completely from cataract development, although a rise in free intracellular Ca(2+)in the lens epithelium was still observed. Lenses from the protected mice did not exhibit enhanced calpain activity. These results suggest the following sequence of events as a possible mechanism of NAPQI-induced cataract. NAPQI introduced in the anterior chamber of the eye enters the lens epithelial cells and disturbs Ca(2+)homeostasis with a resultant rise in free intracellular Ca(2+)which in turn activates calpain in the epithelium. The neutral protease then degrades cellular proteins (e.g. cytoskeletal proteins) and initiates anterior cortical cataract formation.

Acetaminophen↗
PubMed2000

Acetaminophen produces cataract in DBA2 mice by Ah receptor-independent induction of CYP1A2.

The metabolic transformation of acetaminophen to N-acetyl-p-benzoquinone imine by cytochrome P450 enzymes (e.g., cytochrome P450 1A2) is a prerequisite for acetaminophen-induced cataract formation in mice. Aromatic hydrocarbons, such as beta-naphthoflavone, induce cytochrome P450 1A2 in C57BL6 mice via the mediation of the aromatic hydrocarbon receptor and render the animals susceptible to cataract formation by acetaminophen administration but not in DBA2 mice which do not respond to cytochrome P450 1A2 induction by these compounds. Polycyclic hydrocarbons, such as acenaphthylene, were recently found to induce cytochrome P450 1A2 gene expression in young DBA2 mice by aromatic hydrocarbon receptor-independent pathways. In this work, we investigated whether enhanced metabolism of acetaminophen to N-acetyl-p-benzoquinone by cytochrome P450 1A2 induction by acenaphthylene could produce cataract in young DBA2 mice. Fifteen-day-old DBA2 mice were pretreated with two intraperitoneal injections of acenaphthylene and, 24 hr later, with one injection of acetaminophen. In most mice, cataract developed 18-24 hr after acenaphthylene injection. Acenaphthylene treatment of young DBA2 mice resulted in a 2-fold increase in cytochrome P450 1A2-dependent methoxyresorufin O-demethylase activity in the liver. These results support the hypothesis that the aromatic hydrocarbon receptor-independent induction of cytochrome P450 1A2 enzyme leads to accumulation of sufficient N-acetyl-p-benzoquinone in the liver and cataract development in the eye.

Acenaphthenes↗
PubMed1999

Developmental changes in prostaglandin E(2) receptor subtypes in porcine ductus arteriosus. Possible contribution in altered responsiveness to prostaglandin E(2).

BACKGROUND: Prostaglandin E(2) (PGE(2)) is important in ductus arteriosus (DA) patency, but the types of functional PGE(2) receptors (EP) in the developing DA are not known. We postulated that age-dependent alterations in EP and/or their subtypes may possibly contribute to the reduced responsiveness of the newborn DA to PGE(2). METHODS AND RESULTS: We determined PGE(2) receptor subtypes by competition binding and immunoblot studies on the DA of fetal ( approximately 75% and 90% gestation) and newborn (<45 minutes old) pigs. We studied the effects of EP receptor stimulation on cAMP signaling in vitro and on term newborn (<3 hours old) DA patency in vivo. Fetal pig DA expressed EP(2), EP(3), and EP(4) receptors equivalently, but not EP(1). In neonatal DA, EP(1), EP(3), and EP(4) were undetectable, whereas EP(2) density was similar in fetus and newborn. Prostaglandin-induced changes in cAMP mirrored binding data. 16,16-Dimethyl PGE(2) and 11-deoxy PGE(1) (EP(2)/EP(3)/EP(4) agonist) produced more cAMP in fetus than newborn, but butaprost (selective EP(2) agonist) caused similar cAMP increases in both; EP(3) and EP(4) ligands (M&B28767 and AH23848B, respectively) affected cAMP production only in fetus. After birth, administration of butaprost alone was as effective as 11-deoxy PGE(1) and 16,16-dimethyl PGE(2) in dilating DA in vivo. CONCLUSIONS: The data reveal fewer PGE(2) receptors in the DA of the newborn than in that of the fetus; this may contribute to the decreased responsiveness of the DA to PGE(2) in newborn. Because EP(2) receptors seem to mediate the effects of PGE(2) on the newborn DA, one may propose that a selective EP(2) agonist may be preferred as a pharmacological agent to maintain DA patency in infants with certain congenital heart diseases.

Animals↗
PubMed1999

Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope.

The effects of prostaglandin E2 are thought to be mediated via G protein-coupled plasma membrane receptors, termed EP. However recent data implied that prostanoids may also act intracellularly. We investigated if the ubiquitous EP3 and the EP4 receptors are localized in nuclear membranes. Radioligand binding studies on isolated nuclear membrane fractions of neonatal porcine brain and adult rat liver revealed the presence of EP3 and EP4. A perinuclear localization of EP3alpha and EP4 receptors was visualized by indirect immunocytofluorescence and confocal microscopy in porcine cerebral microvascular endothelial cells and in transfected HEK 293 cells that stably overexpress these receptors. Immunoelectron microscopy clearly revealed EP3alpha and EP4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that nuclear EP receptors are functional as they affect transcription of genes such as inducible nitric-oxide synthase and intranuclear calcium transients; this appears to involve pertussis toxin-sensitive G proteins. These results define a possible molecular mechanism of action of nuclear EP3 receptors.

Animals↗
PubMed1999

Cytotoxic metabolite of acetaminophen, N-acetyl-p-benzoquinone imine, produces cataract in DBA2 mice.

Acetaminophen, or N-acetyl-p-aminophenol (APAP), is metabolized to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 enzymes in the liver. The biotransformation of APAP is enhanced in P450-inducible C57BL6 (B6) mice but not in non-inducible DBA2 (D2) mice. Our previous studies showed that high doses of APAP administered to B6 mice pretreated with beta-naphthoflavone (BNF), a P450 inducer, produced ocular tissue damage but not in D2 mice similarly treated. We then proposed that the ocular toxicity of APAP is due to accumulation of its metabolite, NAPQI. In the present work, we tested this hypothesis by injecting NAPQI (50 microg in 2 microl propyleneglycol/eye) intracamerally into B6 and D2 mice. NAPQI produced cataract within a few hours (mean = 4 hr) both in B6 and D2 mice. Lower concentrations of NAPQI did not produce lens opacification. Injection of the solvent propyleneglycol only did not cause cataract. Thus, when NAPQI was injected, P450 inducibility was not essential for cataract formation. In addition to vacuole formation in the lens epithelial cells, alterations were observed in the corneal endothelium and ciliary epithelium. The retinal cell layers remained intact. Extensive mitochondrial damage and changes in chromatin structure in the nucleus were evident in the affected lens epithelial cells. The present result dissociates APAP ocular toxicity from its metabolic potentiation by P450 enzymes and will allow us to investigate the mechanism of cataractogenesis in in vitro lens culture systems.

Acetaminophen↗
PubMed1998

Nuclear localization of prostaglandin E2 receptors.

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein-Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

3T3 Cells↗
PubMed1998

A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation.

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH2-terminal sequence of GPX detected GPX protein in all rat tissues examined.

Amino Acid Sequence↗
PubMed1998

Prevention of acetaminophen-induced cataract by a combination of diallyl disulfide and N-acetylcysteine.

Injection of acetaminophen (APAP) (350 mg/kg body weight) into C57BL/6 mice in which cytochrome P450 (CYP) 1A1/1A2 had been induced produced acute cataract and other ocular tissue damage. Treatment of APAP-injected mice with one of the major organosulfides in garlic oil, diallyl disulfide (DADS) (200 mg/kg body weight), prevented cataract development and prolonged survival time. N-acetyl L-cysteine (NAC) (500 mg/kg body weight), a prodrug that stimulates glutathione synthesis, also prolonged survival time but was effective only weakly to prevent cataract formation. A combination of DADS and NAC completely prevented cataractogenesis, and all of the treated animals survived APAP toxicity. Neither DADS nor NAC inhibited CYP 1A1/1A2 induction as determined by their effect on the induction of hepatic microsomal ethoxyresorufin O-dealkylase (ERD) activity. However, in the in vitro enzyme assay, DADS, but not NAC, was a potent inhibitor of ERD activity (IC50 = 3.5 mM). Treatment with DADS or NAC slowed but did not stop the decrease of hepatic glutathione (GSH) content. At 4 hours after APAP injection, hepatic GSH began to increase only when DADS and NAC were administered together. These results suggest that the protective effect of DADS is due to its inhibition of biotransformation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by CYP 1A1/1A2 enzymes and that NAC provides protection by increasing cellular cysteine level and GSH synthesis, thus facilitating detoxification of NAPQI by glutathione conjugation. Assay of plasma glutamate-pyruvate transaminase activity, an indicator of liver necrosis, showed that treatment with DADS and NAC together effectively protected the liver. Therefore, the decrease of GSH as much as 30% of normal concentration, by itself, is not responsible for liver damage. The primary cause of hepatic necrosis is rapid accumulation of NAPQI.

Acetaminophen↗
PubMed1997

Regulatory cells generated by testicular tolerization to retinal S-antigen: possible involvement of IL-4, IL-10, and TGF-beta in the suppression of experimental autoimmune uveoretinitis.

Intratesticular injection of a retinal protein (S-antigen) into Lewis rats induces systemic immunotolerance (designated orchidic tolerance) and renders animals refractory to experimental autoimmune uveoretinitis (EAU) produced by S-antigen immunization. We demonstrated in this work that the immunotolerance could be transferred to syngeneic naive rats by both CD4+ and CD8+ regulatory cells. Attempts were then made to characterize the cytokines involved in the immunosuppressive activity of these regulatory cells. Using the in vitro lymphoproliferation assay, the inhibitory effect of CD4+ cells on effector cells was found to be reversed by antibodies against IL-4 and IL-10 but not by anti-TGF-beta antibody. IL-4 (IC50 = 1.6 ng/10(6) cells) and IL-10 (IC50 = 0.6 ng/10(6) cells) added to the assay medium were potent inhibitors of effector cell proliferation. Increased immunoreactivity and mRNA expression for IL-4 and IL-10 was observed for CD4+ regulatory cells. The inhibitory effect of CD8+ regulatory cells was reversed by anti-TGF-beta antibody but not by antibodies against IL-4 and IL-10. Compared with control CD8+ cells, CD8+ cells from tolerized rats demonstrated higher immunoreactivity for TGF-beta but did not show an enhanced expression of mRNA for TGF-beta. TGF-beta 1 and TGF-beta 2 added to effector cells showed dichotomous effects; both isoforms stimulated cell proliferation at 2.5 ng/10(6) cells and inhibited at lower or higher concentrations. These results led us to conclude that IL-4 and IL-10 are important cytokines for the immunosuppressive effect of CD4+ regulatory cells generated in orchidic tolerance. TGF-beta is an important immunosuppressive cytokine for CD8+ regulatory cells but further studies will determine whether other cytokines are also involved.

Animals↗
PubMed1997

Microplate assay of ascorbic acid in aqueous humor with bicinchoninic acid.

An inexpensive microplate assay method was developed to determine ascorbic acid in human aqueous humor samples. The method is based on the selective oxidation of ascorbic acid at alkaline pH and determination of ascorbic acid and other reducing substances in samples with bicinchoninic acid/CuSO4 before and after the alkaline treatment. The two-point measurement eliminates the effect of interfering substances, such as glucose, uric acid and glutathione, which are stable at alkaline pH. The advantages of the method are that it requires small sample volumes and allows handling of a large number of samples simultaneously.

Aqueous Humor↗
PubMed1997

Acetaminophen cytotoxicity in mouse eye: mitochondria in anterior tissues are the primary target.

Acetaminophen (APAP) injected into C57BL/6 mice (cytochrome P450 inducer-responsive strain) that had been pretreated with b-naphthoflavone (BNF) produced ocular tissue damage, including cataract. Our previous histocytochemical studies showed that tissue damage spread in association with the flow of the aqueous humor and appeared first in the ciliary epithelium, followed by the iris and corneal endothelium and, finally, the lens. The neural retina, retinal pigmented epithelium and choroid remained unaffected. A close examination of the affected tissues indicated that mitochondria are the primary target of APAP cytotoxicity. In order to investigate whether the respiratory capacity of mitochondria is more sensitive to APAP cytotoxicity than mitochondrial morphology, we determined in this work the oxygen uptake by eye tissues dissected from BNF-pretreated and APAP-injected C57BL/6 mice. Oxygen uptake by the ciliary body/iris decreased about 60% at 90 min and 85% at 120 min after APAP administration. The oxygen uptake was inhibited about 50% by 10 microM rotenone. Since the earliest sign of mitochondrial damage was noted at 120 min, the result indicates that mitochondrial energy dysfunction precedes morphological alterations. It was also observed that oxygen uptake by the retina remained unaffected at least for 120 min after APAP administration; therefore, it is evident that the retina and, possibly, other posterior tissues as well are resistant to APAP cytotoxicity, not only in their morphology but, also, in their capacity of mitochondrial energy metabolism.

Acetaminophen↗
PubMed1997

Immunotolerance induced by intratesticular antigen priming: expression of TGF-beta, Fas and Fas ligand.

The authors have previously reported that an injection of S-antigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (designated orchidic tolerance) and protects the animals from experimental autoimmune uveoretinitis (EAU) and that the signal for orchidic tolerance induction emigrates from the testis within a few hours after antigen priming of the testis. In order to understand the mechanism by which the signal or signal carrier is generated, they determined in this study changes in immunoreactivity for transforming growth factor-beta (TGF-beta), IFN-gamma, IL-2, Fas and Fas ligand in the testis following an injection of S-Ag. Immunoreactivity for TGF-beta increased with time, reaching a maximum in six hours and declining thereafter. The time required for the maximum expression of TGF-beta coincided well with the time-dependent profile of orchidic tolerance signal generation within the testis. Little or no immunoreactivity was observed for IFN-gamma and IL-2 in normal (control) and S-Ag-injected testes. Immunoreactivity for Fas and Fas ligand was detected both in control and experimental testes and did not change appreciably with time following Ag-priming of the testis. Fas immunoreactivity was found in spermatids and virtually absent in the interstitial tissue, while Fas ligand immunoreactivity was primarily associated with the interstitial cells such as Leydig cells. Fas ligand immunoreactivity was very weak, if any, in the germ cells and Sertoli cells. These results suggest that TGF-beta and Fas ligand expressed in MHC-positive interstitial cells may play an important role in the generation of orchidic tolerance induction signal. A preliminary study showed that splenocytes preincubated with testis extracts and S-Ag, when transferred to naive rats, induced systemic tolerance in recipient animals. Inclusion of anti-TGF-beta or a carboxyl terminal peptide of Fas in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. These results indicate that generation of the orchidic tolerance signal does not require the anatomical structure of the testis but is mediated by molecular entities such as TGF-beta and Fas ligand.

Amino Acid Sequence↗
PubMed1996

Porcine S-antigen: cDNA sequence and expression in retina, ciliary epithelium and iris.

cDNA clones encoding S-antigen (arrestin) were isolated from the expression library constructed from porcine retina and sequenced. The 1490 base pair fragment contained a 1215 base pair open reading frame. From the nucleotide sequence, an amino acid sequence consisting of 405 residues was deduced and a molecular mass of 45,102 daltons was calculated. In order to determine whether the S-antigen mRNA transcript was expressed in anterior eye tissues, mRNA from ciliary non-pigmented epithelial cells and pigmented epithelial cells and iris was analysed by the reverse transcription polymerase chain reaction (PCR) using primers taken from sequences flanking the coding and non-coding regions of retinal S-antigen. Sequence analysis of the expected 611 base pairs in the 5' region and 672 base pairs in the 3' region of DNA fragments indicated that an identical mRNA for S-antigen was expressed in the anterior tissues examined. To investigate the in situ expression of S-antigen mRNA, 35S-labeled sense and antisense RNA probes were synthesized from the cDNA to label frozen sections of retina, ciliary body and iris and the treated sections were examined by autoradiography. The antisense probe labeled the layer between retinal pigmented epithelium and the outer nuclear layer of the retina, ciliary epithelium, and iris epithelium. From the results of sequencing PCR products and in situ hybridization, we concluded that, in porcine eye, the mRNA for S-antigen is expressed not only in the retina but also in the anterior tissues such as the ciliary epithelium and iris epithelium.

Animals↗