Aruna K. Singh

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.

Analysis of adhesion, piliation, protease production and ocular infectivity of several P. aeruginosa strains

The role of bacterial piliation and protease production in Pseudomonas aeruginosa adhesion to the injured corneal epithelial surface and subsequent infectivity was examined using several bacterial strains, including three that were hyperpiliated. To initiate this study, bacteria were examined by transmission EM to confirm their piliation characteristics. The PAK strain, like pseudomonas ATCC 19660, possessed about 1-4 polar pili. The mutant PAK/PR11 lacked pili while PAK/PR1, DB2, a mutant of PAO1, and PA1244, a wild-type clinical isolate, were hyperpiliated. Ocular infectivity of these bacterial strains and mutants was examined macroscopically and histopathologically in mice and these data compared to the well-characterized ocular disease response of a murine model of infection with pseudomonas ATCC 19660. The PAK strain was infective, but less virulent than strain 19660 by both macroscopic grading and histopathological analysis of infected eyes. Infectivity of the PR11 mutant was similar to the PAK parent strain, while PR1, DB2 and 1244, all hyperpiliated, were not infective. To explore the hypothesis that hyperpiliated bacteria bound less well to cornea and thus failed to induce corneal disease, in vitro quantitative studies of bacterial adhesion were done using an ocular organ culture model. The PR1 hyperpiliated mutant bound significantly less well to cornea than the PAK parent strain, PR11 mutant or pseudomonas 19660, while DB2 and 1244 binding did not differ significantly from 19660 or PAK. Examination of protease production, another factor which may influence adhesion, revealed that only 19660 and DB2 produced detectable protease. This study provides evidence that non-piliated, non-protease producing strains such as PAK/PR11 possess alternate virulence mechanisms to facilitate binding to and infectivity of corneal tissue.

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.

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.

Induction of Immunotolerance in Rats by Intratesticular Administration of an Eicosapeptide of Bovine S-Antigen

Immunization of albino LEW rats with a retinal soluble antigen (S-antigen) induces experimental autoimmune uveoretinitis (EAU) which shows clinical features resembling those of human uveitis. Several uveitogenic epitopes have been identified in the antigen. This study reports that an intratesticular injection of low doses of a uveitogenic eicosapeptide (P343-362) of S-antigen prior to immunization with the same peptide prevented the onset of EAU by inducing systemic tolerance, designated orchidic tolerance. Splenic lymphocytes of both CD4+ and CD8+ subsets from tolerized rats transferred orchidic tolerance to syngeneic recipients and protected them from subsequent EAU induction. Orchidic tolerance elicited by low antigen dosage was mediated, in part, by active suppression due to suppressor or regulatory cells. At high antigen doses, however, regulatory activity was reduced possibly due to the induction of anergy in regulatory cells, and EAU severity increased. The CD4+ regulatory T cells from tolerized rats showed enhanced expression of IL-4 mRNA compared with CD4+ cells from control rats. Increased immunoreactivity for IL-4, IL-10 and TGF-beta was observed in the spleen and lymph nodes of tolerized animals. The results suggest that orchidic tolerance induced by low doses of P343-362 is mediated in part by CD4+ regulatory cells secreting Th2 cytokines.

Characteristics of immunotolerance induced by intratesticular injection of S-antigen.

Experimental autoimmune uveoretinitis (EAU) induced by immunization of male Lewis rats with bovine S-antigen (S-Ag) is prevented by injection of S-Ag into the testis prior to immunization. This protection of animals is due to the induction of systemic immunotolerance or immunosuppression, which the authors designate orchidic tolerance. In this paper they first present a brief review of several features of orchidic tolerance reported previously and features uncovered more recently, and then propose a possible sequence of events that is initiated by antigen challenge in the testis and results in the proliferation of specific subclasses of lymphocytes with immunosuppressive activity and prevention of the onset of EAU.

Pseudomonas aeruginosa: A Probe to Detail Change with Age in Corneal Receptor(s)

This in vitro study used Pseudomonas aeruginosa as a probe to examine the change with age of the host receptor(s) facilitating bacterial adherence to cornea in the immature eye and to pinpoint in time, when the host receptor(s) was no longer able to be recognized by the bacterium. Scanning EM was used to quantitate adherent organisms at the corneal surface of mice of select ages (5-37 postnatal days, P, and 28 months) at 15, 30 and 60 min following bacterial application. Organisms were most numerous at the surface of the 5-P eye at all time periods. In 9-21-P mice, no bacterial binding was observed until 30 min, and bacterial adherence modestly increased at 60 min after bacterial application. In the 37-P and in aged 28-month-old mice, no binding to cornea was observed until 60 min after bacterial application. Statistical analysis of these data revealed significant differences in the mean number of bacteria binding to cornea between age groups.

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.