Tsuneko Fujii

Cloning of the peroxiredoxin gene family in rats and characterization of the fourth member.

Peroxiredoxin (PRx) exhibits thioredoxin‐dependent peroxidase activity and constitutes a family of proteins. Four members of genes from rat tissues were isolated by PCR using degenerated primers based on the sequences which encode a pair of highly conserved Cys‐containing domains, and were then cloned to full‐length cDNAs. These included two genes which have previously been isolated in rats, PRx I and PRx II, and two rat homologues of PRx III and PRx IV. We showed, for the first time, the simultaneous expression of all four genes in various rat tissues by Northern blotting. Since a discrepancy exists regarding cellular distribution, we further characterized PRx IV by expressing it in COS‐1 cells. This clearly demonstrates that PRx IV is a secretory form and functions within the extracellular space.

Alteration of Glutathione Reductase Expression in the Female Reproductive Organs During the Estrous Cycle

Abstract The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.

Differential Expression of Glutathione Reductase and Cytosolic Glutathione Peroxidase, GPX1, in Developing Rat Lungs and Kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.

Transient Elevation of Glutathione Peroxidase 1 Around the Time of Eyelid Opening in the Neonatal Rat

Glutathione peroxidase (GPX) reduces peroxides using reduced glutathione as the electron donor. Glutathione-dependent peroxidase activity in the soluble fraction of whole rat eye extracts (n = 3 or 4 at each stage) was the highest in the pre-natal stage (31.0 +/- 1.9 mU/mg protein) and gradually declined thereafter. The lowest value was 15.3 +/- 2.3 mU/mg protein at day 9. When the protein levels of the major selenium-containing glutathione peroxidase, GPX1, and the recently identified non-selenium-containing glutathione peroxidase, peroxiredoxin 6, were evaluated by immunoblotting using specific antibodies, they gradually declined after birth. An immunohistochemical analysis was carried out to identify the cells that express GPX1. Although the presence of GPX1 was evident only in restricted tissues, such as the corneal and lens epithelia in the adult, its levels were transiently augmented in ganglion cells, the layer of rods and cones, and pigment cells in the retina from 6 to 12 days after birth and then declined afterward. At the adult stage, the expression of GPX1 was negligible in these cells. Thus GPX1 appears to play a major role at this neonatal stage, corresponding to the period for eyelid opening. The decline in GPX1 levels after birth suggests that the detoxification of peroxides is important at this particular stage or that other, as yet unidentified peroxide-detoxifying enzymes are induced during this period.

Immunohistochemical study of glutathione reductase in rat ocular tissues at different developmental stages.

Glutathione, which is found in high levels in eye tissues, is involved in multiple functions, including serving as an antioxidant and as an electron donor for peroxidases. Although the activities of enzymes related to glutathione metabolism have been reported in the eye, the issue of which cells produce these proteins, where they are produced and at what levels is an important one. Glutathione reductase, an enzyme which recycles oxidized glutathione by transferring electrons from NADPH, was localized immunohistochemically in adult rat eye in this study. The reductase was distributed in the corneal and conjunctival epithelia, corneal keratocytes and endothelium, iridial and ciliary epithelia, neural retina, and retinal pigment epithelium. In addition, it was highly expressed in ganglion cells, which are responsible for transmitting photophysiological signals from the retina to the higher visual centres. To clarify the correlation of glutathione reductase expression and oxidative stress, the enzymatic activity and the level of protein expression at the pre- and postnatal stages was examined. Expression of the enzyme was detected first in the ganglion cell layer of a late prenatal stage, and appeared in the inner plexyform layer after birth. Along with an increasing differentiation between the inner nuclear and outer nuclear layers, glutathione reductase expression became detectable in the outer plexyform layer. Pigment epithelial cells were positively stained only after birth. Expression was also detected in the lens epithelium from the prenatal to early postnatal stages although its level was low in the adult lens. Collectively, these data, except for lens epithelia, suggest the pivotal role of glutathione reductase in recycling oxidized glutathione for the protection of the tissues against oxidative stress, which is caused by eye opening accompanied by the initiation of various ocular processes, such as accession of light and transduction of the photochemical signal.

Dysfunction of antioxidative enzymes in the trinitrobenzenesulfonic acid-induced colitis rat

Abstract Reactive oxygen and nitrogen species are thought to be involved in various inflammatory bowel diseases. We have previously shown a marked decrease in the levels of manganese superoxide dismutase (MnSOD) in the early stages of colitis in model rats after administration of unbuffered 2,4,6-trinitrobenzensulfonic acid (TNBS). Here we have extended the work in order to understand the relationship between colitis and the reactive nitrogen and oxygen species. Immunoblot analysis with an anti-nitrotyrosine antibody identified tyrosine nitration in TNBS-induced colitis tissues in rats. In addition, immunoprecipitation with an anti-MnSOD antibody followed by detection with the anti-nitrotyrosine antibody confirmed the nitration of the tyrosine residues of MnSOD. Then TNBS was administrated to rat aortic smooth muscle cells (RASMC) in culture to see if the same phenomenon occurred in vitro. RASMC exposed to TNBS neither produced NO nor induced tyrosine nitration in MnSOD. However, when activity of glutathione peroxidase (GPx), which is inactivated by various oxidants including NO, was measured, a significant decrease was observed in the early stage of experimental colitis as well as RASMC exposed to TNBS in culture. When direct effects of TNBS on these antioxidative enzymes were examined, GPx but neither MnSOD nor CuZnSOD was inactivated by TNBS and the effect was enhanced by the presence of ascorbate. Taken these data together, dysfunction of antioxidative enzymes are caused by both reactive nitrogen and oxygen species produced by activated inflammatory cells under pathological conditions and by TNBS and/or its metabolites under experimental conditions, and may be responsible for developing colitis through accumulation of such potentially harmful molecules.