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Immunohistochemical localization of 2-Cys peroxiredoxins in human ciliary body
Tissue and Cell 39 (2007) 365–368
Short communication
Immunohistochemical localization of 2-Cys peroxiredoxins in human ciliary body Samin Hong a , Chan Yun Kim a , Joon H. Lee b , Gong Je Seong a,∗ a
b
Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Republic of Korea Myunggok Eye Research Institute at Kim’s Eye Hospital, Konyang University College of Medicine, Nonsan, Republic of Korea Received 26 January 2007; received in revised form 14 June 2007; accepted 19 June 2007 Available online 17 August 2007
Abstract 2-Cys peroxiredoxins (PRDX) are novel antioxidant enzymes that eliminate the hydrogen peroxide in cells to protect the cellular components from reactive oxygen species. To evaluate whether 2-Cys PRDX family plays a role in human ciliary body, the expression of PRDX I, II and III on normal human ciliary body was investigated. Three normal human ciliary body tissues obtained from three donor eyeballs were examined by an immunohistochemistry using light microscopy and fluorescent microscopy with antibodies directed against the PRDX I, II and III. In the normal human ciliary body, PRDX I, II and III were immunolocalized to the non-pigmented epithelial cells and ciliary muscle fibers. It suggests that 2-Cys PRDXs may have physiological functions to protect cells in human ciliary body. © 2007 Elsevier Ltd. All rights reserved. Keywords: Peroxiredoxin; Ciliary body
1. Introduction During normal metabolism as well as oxidative stress, reactive oxygen species (ROS) and free radicals are produced and have the potential of damaging cellular components. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. In ciliary body, there is evidence that several enzymes, like catalase and glutathione peroxidase, involve in the neutralization of hydrogen peroxide (H2 O2 ) and protect cells against oxidative stress induced by H2 O2 (Bhuyan and Bhuyan, 1977, 1978; Armstrong et al., 1981; Delamere and Williams, 1985; Shichi, 1990).
Abbreviations: FITC, fluorescein isothiocyanate; PRDX, peroxiredoxin; ROS, reactive oxygen species ∗ Corresponding author at: Institute of Vision Research, Department of Ophthalmology, Yongdong Severance Hospital, Yonsei University College of Medicine, 146-92 Dogok-dong, Kangnam-gu, Seoul 135-720, Republic of Korea. Tel.: +82 2 2019 3440; fax: +82 2 3463 1049. E-mail address: [email protected] (G.J. Seong). 0040-8166/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2007.06.003
Peroxiredoxin (PRDX) is a family of novel antioxidant enzymes that catalyze the H2 O2 in the presence of thioredoxin, thioredoxin reductase and NADPH (Flohe et al., 2003; Rhee et al., 2005; Kang et al., 2005; Immenschuh and Baumgart-Vogt, 2005). All PDRXs contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of PDRX (PRDX I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of cysteine residues that participate in catalysis. In particular, typical 2-Cys PRDXs (PRDX I to IV) appear to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2Cys PRDXs is clear for diseases that involve ROS. Even though the presence of PRDX in bovine uveal tissue has been reported (Peshenko et al., 2001; Singh and Shichi, 2001), studies about the human ciliary body and the isoforms of 2-Cys PRDXs are insufficient. The purpose of this study was to identify the presence of 2-Cys PRDXs and the distribution patterns of the
366
S. Hong et al. / Tissue and Cell 39 (2007) 365–368
three isoforms of 2-Cys PRDXs in normal human ciliary body.
protocol was approved by the Institutional Review Board. All specimens were handled in accordance with the Declaration of Helsinki.
2. Materials and methods
2.2. Immunohistochemical analysis
2.1. Tissue preparation
After serial paraffin sections were deparaffinized in xylenes and rehydrated with a graded concentration of ethanol, immunohistochemical staining was performed using the ABC Elite system (Vector Laboratories, Burlinghame, CA) according to the manufacturer’s instructions. Briefly, the sections were incubated with donkey serum for 30 min. After several washing steps, the sections were incubated with the primary antibodies specific for human PRDX I (1:500, goat, polyclonal, N-14, Santa Cruz Biotech, Santa Cruz,
Three normal human eyeballs (age 36–50 years) donated for keratoplasty were studied. After removal of corneal buttons for keratoplasty, remained tissue containing the ciliary body was fixed with 4% p-formaldehyde and subsequently embedded in paraffin. Each specimen was serially sectioned into 5-m sections and mounted on glass slides. Informed consent was obtained from each subject and the experimental
Fig. 1. Immunohistochemistry of normal human ciliary body probed with antibodies against 2-Cys peroxiredoxins (PRDX) I (A and B), II (C and D), and III (E and F). Sections were stained with 3,3 -diaminobenzidine (A, C and E) and FITC (B, D and F) resulting in a brown chromogen and a green fluorescence, respectively. Note that PRDX I to III are expressed in non-pigmented epithelial cells and ciliary muscle fibers. The magnification is 200×.
S. Hong et al. / Tissue and Cell 39 (2007) 365–368
CA), PRDX II (1:500, goat, polyclonal, N-13, Santa Cruz Biotech, Santa Cruz, CA), and PRDX III (1:500, goat, polyclonal, C-14, Santa Cruz Biotech, Santa Cruz, CA) for 12 h at 4 ◦ C. Binding of the primary antibodies was detected with peroxidase-conjugated rabbit anti-goat IgG (1:200, Zymed, Carlsbad, CA) after 1 h of incubation at room temperature. After washing, antibody-treated sections were developed with 3,3 -diaminobenzidine (Sigma, Deisenhofen, Germany) and subsequently with ELF substrate (Molecular Probes, Eugene, OR). Specimens were examined and with a light microscope. Also, the binding of the primary antibodies was detected with fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG (FITC-IgG; 1:200, Santa Cruz Biotech, Santa Cruz, CA) after 1 h of incubation at room temperature. The slides were examined by confocal immunofluorescence microscopy.
3. Results The results presented here localize PRDX I, II and III immunoreactivities within the normal human ciliary body. In the ciliary body, non-pigmented epithelial cells and ciliary muscle fibers presented staining for PRDX I to III (Fig. 1). When the tissue stained using 3,3 -diaminobenzidine, the melanin pigments interfere with brown chromogen in the pigmented ciliary epithelial cell layer. However, immunofluorescence staining technique using FITC could identify the immunoreactivity.
4. Discussion The major finding in this series is that three isoforms of 2-Cys PRDX (PRDX I to III) were present in the normal human ciliary body. PRDX I, II and III expressed in the non-pigmented epithelial cells and ciliary muscle fibers. The results support previous findings that PRDX is present in bovine ciliary body (Singh and Shichi, 2001). The surface of the non-pigmented ciliary epithelium adjoins the aqueous humor. Even though the human aqueous humor normally contains very low concentration of hydrogen peroxide (30 M), the various concentrations (60–600 M) of hydrogen peroxide have been reported in human eyes especially with cataract (Spector and Garner, 1981; Bhuyan et al., 1986; Ramachandran et al., 1991). Presence of hydrogen peroxide is generally regarded as being cytotoxic. It can cause cell membrane and DNA damage (Stanimirovic et al., 1995). The presence of hydrogen peroxide in aqueous humor raises the possibility that it could potentially constitute an oxidative challenge to the membrane of the non-pigmented ciliary epithelium. It has been suggested that the concentration of hydrogen peroxide in aqueous humor is regulated by some antioxidant enzymes like catalase and glutathione peroxidase
367
(Bhuyan and Bhuyan, 1977, 1978; Armstrong et al., 1981; Delamere and Williams, 1985; Shichi, 1990). 2-Cys PRDXs are a new class of peroxidases (Flohe et al., 2003; Rhee et al., 2005; Kang et al., 2005; Immenschuh and Baumgart-Vogt, 2005). They are coupled with thioredoxin and thioredoxin reductase, forming an electron-conveying system. Thioredoxin reductase transfers electrons from NADPH to thioredoxin, which in turn reduces a disulfide linkage formed during the peroxidase reaction in 2-Cys PRDX. Thus far, five 2-Cys PRDX isoforms have been recognized in mammals. On the basis of the peptide regions that harbor a conserved cysteine residue, they can be classified into typical (PRDX I-IV) and atypical 2-Cys PRDX (PRDX V). Even though the catalytic efficiency of 2-Cys PRDXs is less than that of catalase and glutathione peroxidase, 2-Cys PRDXs have important and interesting roles in hydrogen peroxidemediated signaling (Kang et al., 2005). Therefore, 2-Cys PRDXs suggest new potential therapeutic approaches in variety human diseases related to ROS. Distribution of 2-Cys PRDXs in human ciliary body is still not well-known, even though the presence of PRDX in bovine uveal tissue has been reported (Peshenko et al., 2001; Singh and Shichi, 2001). So, this study was performed to identify the presence of 2-Cys PRDXs and the distribution patterns of the three isoforms of 2-Cys PRDXs in normal human ciliary body. In conclusion, PRDX I, II and III of 2-Cys PRDXs were immunolocalized to the non-pigmented ciliary epithelium and ciliary muscle fibers. It suggests that 2-Cys PRDXs may have physiological functions to protect cells in human ciliary body.
Acknowledgements This work was supported by the Students’ Association of the Graduate School of Yonsei University funded by the Graduate School of Yonsei University, Seoul, Republic of Korea.
References Armstrong, D., Santangelo, G., Connole, E., 1981. The distribution of peroxide regulating enzymes in the canine eye. Curr. Eye Res. 1, 225–242. Bhuyan, K.C., Bhuyan, D.K., 1977. Regulation of hydrogen peroxide in eye humors. Effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye. Biochim. Biophys. Acta 497, 641–651. Bhuyan, K.C., Bhuyan, D.K., 1978. Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage. Biochim. Biophys. Acta 542, 28–38. Bhuyan, K.C., Bhuyan, D.K., Podos, S.M., 1986. Lipid peroxidation in cataract of the human. Life Sci. 38, 1463–1471. Delamere, N.A., Williams, R.N., 1985. Detoxification of hydrogen peroxide by the rabbit iris-ciliary body. Exp. Eye Res. 40, 805–811. Flohe, L., Budde, H., Hofmann, B., 2003. Peroxiredoxins in antioxidant defense and redox regulation. Biofactors 19, 3–10. Immenschuh, S., Baumgart-Vogt, E., 2005. Peroxiredoxins, oxidative stress, and cell proliferation. Antioxid. Redox Signal. 7, 768–777.
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S. Hong et al. / Tissue and Cell 39 (2007) 365–368
Kang, S.W., Rhee, S.G., Chang, T.S., Jeong, W., Choi, M.H., 2005. 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol. Med. 11, 571–578. Peshenko, I.V., Singh, A.K., Shichi, H., 2001. Bovine eye 1-Cys peroxiredoxin: expression in E. coli and antioxidant properties. J. Ocul. Pharmacol. Ther. 17, 93–99. Ramachandran, S., Morris, S.M., Devamanoharan, P., Henein, M., Varma, S.D., 1991. Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine. Exp. Eye Res. 53, 503–506. Rhee, S.G., Chae, H.Z., Kim, K., 2005. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med. 38, 1543–1552.
Shichi, H., 1990. Glutathione-dependent detoxification of peroxide in bovine ciliary body. Exp. Eye Res. 50, 813–818. Singh, A.K., Shichi, H., 2001. Peroxiredoxin in bovine ocular tissues: immunohistochemical localization and in situ hybridization. J. Ocul. Pharmacol. Ther. 17, 279–286. Spector, A., Garner, W.H., 1981. Hydrogen peroxide and human cataract. Exp. Eye. Res. 33, 673–681. Stanimirovic, D.B., Wong, J., Ball, R., Durkin, J.P., 1995. Free radicalinduced endothelial membrane dysfunction at the site of blood-brain barrier: relationship between lipid peroxidation, Na, K-ATPase activity and 51 Cr release. Neurochem. Res. 20, 1417–1427.
Short communication
Immunohistochemical localization of 2-Cys peroxiredoxins in human ciliary body Samin Hong a , Chan Yun Kim a , Joon H. Lee b , Gong Je Seong a,∗ a
b
Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Republic of Korea Myunggok Eye Research Institute at Kim’s Eye Hospital, Konyang University College of Medicine, Nonsan, Republic of Korea Received 26 January 2007; received in revised form 14 June 2007; accepted 19 June 2007 Available online 17 August 2007
Abstract 2-Cys peroxiredoxins (PRDX) are novel antioxidant enzymes that eliminate the hydrogen peroxide in cells to protect the cellular components from reactive oxygen species. To evaluate whether 2-Cys PRDX family plays a role in human ciliary body, the expression of PRDX I, II and III on normal human ciliary body was investigated. Three normal human ciliary body tissues obtained from three donor eyeballs were examined by an immunohistochemistry using light microscopy and fluorescent microscopy with antibodies directed against the PRDX I, II and III. In the normal human ciliary body, PRDX I, II and III were immunolocalized to the non-pigmented epithelial cells and ciliary muscle fibers. It suggests that 2-Cys PRDXs may have physiological functions to protect cells in human ciliary body. © 2007 Elsevier Ltd. All rights reserved. Keywords: Peroxiredoxin; Ciliary body
1. Introduction During normal metabolism as well as oxidative stress, reactive oxygen species (ROS) and free radicals are produced and have the potential of damaging cellular components. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. In ciliary body, there is evidence that several enzymes, like catalase and glutathione peroxidase, involve in the neutralization of hydrogen peroxide (H2 O2 ) and protect cells against oxidative stress induced by H2 O2 (Bhuyan and Bhuyan, 1977, 1978; Armstrong et al., 1981; Delamere and Williams, 1985; Shichi, 1990).
Abbreviations: FITC, fluorescein isothiocyanate; PRDX, peroxiredoxin; ROS, reactive oxygen species ∗ Corresponding author at: Institute of Vision Research, Department of Ophthalmology, Yongdong Severance Hospital, Yonsei University College of Medicine, 146-92 Dogok-dong, Kangnam-gu, Seoul 135-720, Republic of Korea. Tel.: +82 2 2019 3440; fax: +82 2 3463 1049. E-mail address: [email protected] (G.J. Seong). 0040-8166/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2007.06.003
Peroxiredoxin (PRDX) is a family of novel antioxidant enzymes that catalyze the H2 O2 in the presence of thioredoxin, thioredoxin reductase and NADPH (Flohe et al., 2003; Rhee et al., 2005; Kang et al., 2005; Immenschuh and Baumgart-Vogt, 2005). All PDRXs contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of PDRX (PRDX I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of cysteine residues that participate in catalysis. In particular, typical 2-Cys PRDXs (PRDX I to IV) appear to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2Cys PRDXs is clear for diseases that involve ROS. Even though the presence of PRDX in bovine uveal tissue has been reported (Peshenko et al., 2001; Singh and Shichi, 2001), studies about the human ciliary body and the isoforms of 2-Cys PRDXs are insufficient. The purpose of this study was to identify the presence of 2-Cys PRDXs and the distribution patterns of the
366
S. Hong et al. / Tissue and Cell 39 (2007) 365–368
three isoforms of 2-Cys PRDXs in normal human ciliary body.
protocol was approved by the Institutional Review Board. All specimens were handled in accordance with the Declaration of Helsinki.
2. Materials and methods
2.2. Immunohistochemical analysis
2.1. Tissue preparation
After serial paraffin sections were deparaffinized in xylenes and rehydrated with a graded concentration of ethanol, immunohistochemical staining was performed using the ABC Elite system (Vector Laboratories, Burlinghame, CA) according to the manufacturer’s instructions. Briefly, the sections were incubated with donkey serum for 30 min. After several washing steps, the sections were incubated with the primary antibodies specific for human PRDX I (1:500, goat, polyclonal, N-14, Santa Cruz Biotech, Santa Cruz,
Three normal human eyeballs (age 36–50 years) donated for keratoplasty were studied. After removal of corneal buttons for keratoplasty, remained tissue containing the ciliary body was fixed with 4% p-formaldehyde and subsequently embedded in paraffin. Each specimen was serially sectioned into 5-m sections and mounted on glass slides. Informed consent was obtained from each subject and the experimental
Fig. 1. Immunohistochemistry of normal human ciliary body probed with antibodies against 2-Cys peroxiredoxins (PRDX) I (A and B), II (C and D), and III (E and F). Sections were stained with 3,3 -diaminobenzidine (A, C and E) and FITC (B, D and F) resulting in a brown chromogen and a green fluorescence, respectively. Note that PRDX I to III are expressed in non-pigmented epithelial cells and ciliary muscle fibers. The magnification is 200×.
S. Hong et al. / Tissue and Cell 39 (2007) 365–368
CA), PRDX II (1:500, goat, polyclonal, N-13, Santa Cruz Biotech, Santa Cruz, CA), and PRDX III (1:500, goat, polyclonal, C-14, Santa Cruz Biotech, Santa Cruz, CA) for 12 h at 4 ◦ C. Binding of the primary antibodies was detected with peroxidase-conjugated rabbit anti-goat IgG (1:200, Zymed, Carlsbad, CA) after 1 h of incubation at room temperature. After washing, antibody-treated sections were developed with 3,3 -diaminobenzidine (Sigma, Deisenhofen, Germany) and subsequently with ELF substrate (Molecular Probes, Eugene, OR). Specimens were examined and with a light microscope. Also, the binding of the primary antibodies was detected with fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG (FITC-IgG; 1:200, Santa Cruz Biotech, Santa Cruz, CA) after 1 h of incubation at room temperature. The slides were examined by confocal immunofluorescence microscopy.
3. Results The results presented here localize PRDX I, II and III immunoreactivities within the normal human ciliary body. In the ciliary body, non-pigmented epithelial cells and ciliary muscle fibers presented staining for PRDX I to III (Fig. 1). When the tissue stained using 3,3 -diaminobenzidine, the melanin pigments interfere with brown chromogen in the pigmented ciliary epithelial cell layer. However, immunofluorescence staining technique using FITC could identify the immunoreactivity.
4. Discussion The major finding in this series is that three isoforms of 2-Cys PRDX (PRDX I to III) were present in the normal human ciliary body. PRDX I, II and III expressed in the non-pigmented epithelial cells and ciliary muscle fibers. The results support previous findings that PRDX is present in bovine ciliary body (Singh and Shichi, 2001). The surface of the non-pigmented ciliary epithelium adjoins the aqueous humor. Even though the human aqueous humor normally contains very low concentration of hydrogen peroxide (30 M), the various concentrations (60–600 M) of hydrogen peroxide have been reported in human eyes especially with cataract (Spector and Garner, 1981; Bhuyan et al., 1986; Ramachandran et al., 1991). Presence of hydrogen peroxide is generally regarded as being cytotoxic. It can cause cell membrane and DNA damage (Stanimirovic et al., 1995). The presence of hydrogen peroxide in aqueous humor raises the possibility that it could potentially constitute an oxidative challenge to the membrane of the non-pigmented ciliary epithelium. It has been suggested that the concentration of hydrogen peroxide in aqueous humor is regulated by some antioxidant enzymes like catalase and glutathione peroxidase
367
(Bhuyan and Bhuyan, 1977, 1978; Armstrong et al., 1981; Delamere and Williams, 1985; Shichi, 1990). 2-Cys PRDXs are a new class of peroxidases (Flohe et al., 2003; Rhee et al., 2005; Kang et al., 2005; Immenschuh and Baumgart-Vogt, 2005). They are coupled with thioredoxin and thioredoxin reductase, forming an electron-conveying system. Thioredoxin reductase transfers electrons from NADPH to thioredoxin, which in turn reduces a disulfide linkage formed during the peroxidase reaction in 2-Cys PRDX. Thus far, five 2-Cys PRDX isoforms have been recognized in mammals. On the basis of the peptide regions that harbor a conserved cysteine residue, they can be classified into typical (PRDX I-IV) and atypical 2-Cys PRDX (PRDX V). Even though the catalytic efficiency of 2-Cys PRDXs is less than that of catalase and glutathione peroxidase, 2-Cys PRDXs have important and interesting roles in hydrogen peroxidemediated signaling (Kang et al., 2005). Therefore, 2-Cys PRDXs suggest new potential therapeutic approaches in variety human diseases related to ROS. Distribution of 2-Cys PRDXs in human ciliary body is still not well-known, even though the presence of PRDX in bovine uveal tissue has been reported (Peshenko et al., 2001; Singh and Shichi, 2001). So, this study was performed to identify the presence of 2-Cys PRDXs and the distribution patterns of the three isoforms of 2-Cys PRDXs in normal human ciliary body. In conclusion, PRDX I, II and III of 2-Cys PRDXs were immunolocalized to the non-pigmented ciliary epithelium and ciliary muscle fibers. It suggests that 2-Cys PRDXs may have physiological functions to protect cells in human ciliary body.
Acknowledgements This work was supported by the Students’ Association of the Graduate School of Yonsei University funded by the Graduate School of Yonsei University, Seoul, Republic of Korea.
References Armstrong, D., Santangelo, G., Connole, E., 1981. The distribution of peroxide regulating enzymes in the canine eye. Curr. Eye Res. 1, 225–242. Bhuyan, K.C., Bhuyan, D.K., 1977. Regulation of hydrogen peroxide in eye humors. Effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye. Biochim. Biophys. Acta 497, 641–651. Bhuyan, K.C., Bhuyan, D.K., 1978. Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage. Biochim. Biophys. Acta 542, 28–38. Bhuyan, K.C., Bhuyan, D.K., Podos, S.M., 1986. Lipid peroxidation in cataract of the human. Life Sci. 38, 1463–1471. Delamere, N.A., Williams, R.N., 1985. Detoxification of hydrogen peroxide by the rabbit iris-ciliary body. Exp. Eye Res. 40, 805–811. Flohe, L., Budde, H., Hofmann, B., 2003. Peroxiredoxins in antioxidant defense and redox regulation. Biofactors 19, 3–10. Immenschuh, S., Baumgart-Vogt, E., 2005. Peroxiredoxins, oxidative stress, and cell proliferation. Antioxid. Redox Signal. 7, 768–777.
368
S. Hong et al. / Tissue and Cell 39 (2007) 365–368
Kang, S.W., Rhee, S.G., Chang, T.S., Jeong, W., Choi, M.H., 2005. 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol. Med. 11, 571–578. Peshenko, I.V., Singh, A.K., Shichi, H., 2001. Bovine eye 1-Cys peroxiredoxin: expression in E. coli and antioxidant properties. J. Ocul. Pharmacol. Ther. 17, 93–99. Ramachandran, S., Morris, S.M., Devamanoharan, P., Henein, M., Varma, S.D., 1991. Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine. Exp. Eye Res. 53, 503–506. Rhee, S.G., Chae, H.Z., Kim, K., 2005. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med. 38, 1543–1552.
Shichi, H., 1990. Glutathione-dependent detoxification of peroxide in bovine ciliary body. Exp. Eye Res. 50, 813–818. Singh, A.K., Shichi, H., 2001. Peroxiredoxin in bovine ocular tissues: immunohistochemical localization and in situ hybridization. J. Ocul. Pharmacol. Ther. 17, 279–286. Spector, A., Garner, W.H., 1981. Hydrogen peroxide and human cataract. Exp. Eye. Res. 33, 673–681. Stanimirovic, D.B., Wong, J., Ball, R., Durkin, J.P., 1995. Free radicalinduced endothelial membrane dysfunction at the site of blood-brain barrier: relationship between lipid peroxidation, Na, K-ATPase activity and 51 Cr release. Neurochem. Res. 20, 1417–1427.