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Biomedical subjects

H Shichi

Publications and source records attributed to H Shichi.

At least 73 records · Page 4Linked to original sources
PubMed1989

Possible mechanism of immunosuppression by gramicidin S of S antigen-induced experimental autoimmune uveoretinitis.

Gramicidin S (GrS) suppressed the onset of experimental autoimmune uveoretinitis (EAU) induced with S-antigen in rats, and the proliferation of mitogen-stimulated lymphocytes in culture. As an immunosuppressive mechanism of the cyclic peptide antibiotic we first postulated that the drug exerts its effect as an ionophore. Although all ionophore compounds tested suppressed lymphocyte proliferation, no correlation was observed between changes in intracellular concentrations of Na+ and K+ and the degree of immunosuppression. For example, monensin inhibited lymphocyte proliferation without affecting intracellular Na+ and K+ levels. Thus it was likely that the immunosuppressive effects of the ionophore compounds including GrS were due to their ability to modify cell membrane properties rather than their ionophore activity. We then tested the hypothesis that GrS inhibits transport of metabolic intermediates or metabolites (thymidine and methionine) into lymphocytes. The idea was experimentally supported. Further, inhibition of metabolite transport by GrS was found to be reversible. To investigate whether inhibition of metabolite uptake can be a mechanism of immunosuppression of EAU, endogenously stimulated lymphocytes were isolated from S-antigen-immunized rats at different stages of EAU and the effect of GrS on metabolite uptake by the cells was determined. The activity of lymphocytes to transport metabolites was enhanced at pre-EAU stages and the enhanced metabolite uptake was markedly inhibited by GrS. We interpreted the result to support that inhibition of metabolite uptake by GrS is probably a mechanism of immunosuppression in vivo by this drug.

Animals↗
PubMed1989

Peroxisomal palmityl CoA oxidase activity in ocular tissues and cultured ciliary epithelial cells.

The activity of palmityl CoA oxidase, a peroxisomal enzyme, was determined in bovine ocular tissues. Significant levels of activity were found in the iris, ciliary body and pigmented epithelium-choroid but little or no activity was detected in the corneal epithelium, lens capsule-epithelium and retina. Treatment of bovine ciliary epithelial cells with clofibric acid for 72 hours in culture resulted in a several fold enhancement of palmityl CoA oxidase activity. These results suggest that peroxisomal enzymes can be induced in uveal tissues when peroxisome proliferation is stimulated by certain drugs in these tissues.

Animals↗
PubMed1989

Changes in immunoreactivity in S-antigen-induced pinealitis.

Changes in immunoreactivity in rat pineal gland were investigated in S-antigen (S-Ag)-induced pinealitis. Anti-S antibodies were used to study the accessibility of pathogenic epitopes in the organ, and phosphatidylinositol 4,5-bisphospate (PI[4,5]P2) antibodies were chosen to study changes in functional phospholipid in membranes. Control pineal gland (without inflammation) did not show much reactivity toward these antibodies. Immunoreactivity toward both antibodies was markedly increased throughout the organ at the peak of inflammation and decreased almost to control level in the postinflammatory stage. The rise and fall of immunoreactivity were attributed to changes in the accessibility of antigenic sites rather than changes in the amounts of S-Ag and P(4,5)P2. The transient enhancement in immunoreactivity, probably consequential to tissue damage, suggests that the pineal gland, unlike the retinal photoreceptors, is capable of quick repair of tissue.

Animals↗
PubMed1988

ADP-ribosylation of bovine S-antigen by cholera toxin.

The S-antigen (alias 48K protein or arrestin) of bovine rod photoreceptors contains two stretches of amino acid sequence homologous to the ADP-ribosylation sites of the alpha subunit of transducin (Ta). We have found that cholera toxin transfers the ADP-ribosyl group from NAD to purified bovine S-antigen as well as to S-antigen in rod outer segment membranes, while Bordetella pertussis toxin is unable to catalyze the transfer reaction efficiently. Under the same conditions, both toxins catalyzed ADP-ribosylation of Ta in rod outer segments. The ADP-ribosylation of S-antigen by cholera toxin indicates that S-antigen not only exhibits sequence homology with the ADP-ribosylation sites of Ta, but it must also resemble Ta in the tertiary structure of the domain which determines the susceptibility of S-antigen to the catalytic action of cholera toxin. These results suggest that S-antigen may function as a competitor of Ta in some stage of the cGMP cascade of visual transduction.

Adenosine Diphosphate Ribose↗
PubMed1988

Bovine non-pigmented and pigmented ciliary epithelial cells in culture: comparison of catalase, superoxide dismutase and glutathione peroxidase activities.

Non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells were dissociated from bovine ciliary processes by brief digestion with pronase and grown in a culture medium containing high fetal bovine serum for at least 25 generations. Both types of cells grown to confluence showed the presence of intermediate junctions with associated tonofilaments. PE cells were distinguished from NPE cells by pigmentation during the early passages. Gamma-glutamyl transpeptidase activity was associated almost exclusively with NPE cells and proved to be a useful enzymatic marker to distinguish NPE from PE. A comparison was made between NPE and PE cells as to the levels of enzymes involved in the detoxification of active oxygen species. Catalase, Se-dependent glutathione peroxidase and superoxide dismutase activities were significantly higher in NPE than in PE cells. The results suggest that NPE cells play the major role in detoxification of active oxygen species during aqueous humor formation.

Animals↗
PubMed1988

Identification of proteins in retinas and IPM from eyes with retinitis pigmentosa.

Opsin, the alpha-subunit of transducin, S-antigen, interphotoreceptor retinoid-binding protein (IRBP) and cathepsin D were assessed in autopsy eyes from patients with retinitis pigmentosa (RP) and normal autopsy eyes. Immunochemical methods were used to determine the presence of these proteins on Western blots of retinal homogenates from five RP donors and on blots of interphotoreceptor matrix (IPM) preparations from six other RP eyes. The amounts of immunoreactive opsin, S-antigen, alpha-transducin, and IRBP appeared below normal in retinas from RP eyes. All six IPM samples from patients with advanced RP had reduced amounts of S-antigen and no detectable IRBP or transducin. Cathepsin D (an RPE protein) was present in IPM or RP eyes in amounts comparable to that in IPMs from normal eyes. Small amounts of cathepsin D were also detected in retinas from both normal and RP eyes. These studies show that proteins specific to the photoreceptor-pigment epithelium complex in normal eyes can be detected in autopsy eyes from patients with RP and suggest that the observed reductions in photoreceptor-specific proteins occur as a consequence of photoreceptor loss.

Adult↗
PubMed1988

Immunosuppressive and anti-proliferative effects of a macrotetrolide antibiotic, tetranactin.

The macrotetrolide tetranactin suppressed the appearance of experimental autoimmune uveoretinitis induced in Lewis rats with a soluble retinal antigen (S-antigen). The drug at 1 ng/ml inhibited the mitogen activation of unfractionated lymphocytes; incorporation of radiolabelled precursors such as thymidine, uridine and leucine into the cells was markedly reduced. The synthesis and release of IL-2 by mitogen-activated lymphocytes was significantly suppressed in the presence of tetranactin. Incorporation of 45Ca was also inhibited, while intracellular Na+ levels were increased. In view of the ionophore property of tetranactin, it was suggested that the drug might demonstrate its immunosuppressive effect by affecting intracellular cation concentrations.

Animals↗
PubMed1987

Enzymes of mercapturate pathway in cultured bovine ciliary epithelial cells.

Relative enzyme activities of the mercapturate pathway were determined in bovine ciliary pigmented (PE) and nonpigmented (NPE) epithelial cells in culture. Glutathione S-transferase activity was greater in PE cells than in NPE cells. Gamma-glutamyl transpeptidase activity appeared to be exclusively associated with NPE cells; the enzyme activity was virtually absent in PE cells. The level of cystine aminopeptidase activity was about the same in NPE and PE cells. N-acetyl transferase was predominantly found in NPE cells. If a similar enzyme distribution should exist in vivo in the bicellular layer of ciliary epithelium, the present result suggests that both NPE and PE cells are involved in the detoxification of xenobiotics via the mercapturate pathway, glutathione conjugation occurring primarily in the PE cells and the reactions catalyzed by the membrane-bound enzymes gamma-glutamyl transpeptidase and N-acetyl transferase taking place mainly in the NPE cells.

Acetylcysteine↗
PubMed1987

Immunochemical evidence for the light-regulated modulation of phosphatidylinositol-4,5-bisphosphate in rat photoreceptor cells.

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate (PIP2) in the rat rod photoreceptor outer segments (OS) was investigated with rabbit antiPIP2 antibodies. The OS of the light-adapted rat eye showed little or no staining, whereas the OS of the dark-adapted eye were intensely stained for PIP2. The immunoreactivity of photoreceptor PIP2 in the eye exposed to a brief flash of light was markedly reduced. However, subsequent dark-adaptation of the flash-bleached eye resulted in a rapid recovery of PIP2 immunoreactivity; dark-adaptation for 5 min was sufficient for recovery to the fully dark-adapted level. In dark-adapted eyes exposed to graded light intensities, the PIP2 immunostaining varied with light levels and was correlated with unbleached rhodopsin concentrations. These results suggest that PIP2 in the rat photoreceptor cells is rapidly hydrolyzed upon light exposure and rapidly synthesized in the dark and that the decrease of PIP2 level is triggered by photic bleaching of rhodopsin.

Adaptation, Ocular↗
PubMed1987

Immunosuppressive effects of polynactins (tetranactin, trinactin and dinactin) on experimental autoimmune uveoretinitis in rats.

Macrotetrolide antibiotic polynactins [dinactin, trinactin and tetranactin (1:4:5)] are hydrophobic cyclic esters produced by Streptomyces aureus. Polynactins (PN) and their major component tetranactin (TN) delayed or suppressed the onset of S-antigen-induced experimental autoimmune uveoretinitis (EAU) in Lewis rats. Termination of treatment with PN or TN before day 14 of immunization resulted in a delayed onset of EAU in many animals. Thus, the immunosuppressive effect of PN and TN was not lasting. PN and TN suppressed anti-S-antigen antibody formation. Skin hypersensitivity tests indicated suppression by PN of the delayed-type rather than Arthus type hypersensitivity to S-antigen. PN, TN and trinactin all inhibited 3H-thymidine incorporation into concanavalin A-treated lymphocytes at the early stage of cell activation. For each drug, 50% inhibition was obtained at about 0.1 ng/ml. Under the incubation condition that the cells were exposed to TN for 21 hours, cell viability remained unchanged up to 100 ng/ml of TN. It is evident that PN and TN suppress T-lymphocyte proliferation without cell injury. These results suggest that PN and TN inhibit the onset of EAU primarily through the suppression of cell-mediated immunity but also by affecting humoral immunity.

Animals↗
PubMed1986

Purification and properties of anionic glutathione S-transferase from bovine ciliary body.

An anionic glutathione S-transferase was purified from bovine ciliary body by DEAE-agarose chromatography and affinity chromatography on GSH-agarose and Orange A. The enzyme accounts for about 25% of total soluble glutathione S-transferase activity of the tissue. The purified enzyme has a molecular mass of about 50,000 Da and is composed of two identical subunits of about 25,000 Da. The enzyme has a pI of 5.8. The enzyme conjugates GSH with 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 1,2-epoxy-3-(p-nitrophenyl)propane, 1,2-dinitrobenzene and 3,4-dinitrobenzoic acid. The Km values for 1-chloro-2,4-dinitrobenzene and GSH are 0.40 mM and 0.57 mM respectively. Haematin is a non-competitive inhibitor (Ki = 4.5 microM) when tested with various concentrations of 1-chloro-2,4-dinitrobenzene. The enzyme shows no glutathione peroxidase activity with either H2O2 or cumene peroxide as substrate. On the basis of substrate specificities, pI values, amino acid composition and peptide maps, it is concluded that the ciliary-body enzyme is probably identical with the anionic form of glutathione S-transferase from bovine lens and liver.

Amino Acids↗
PubMed1986

Purification and properties of glutathione reductases from bovine ciliary body.

Two glutathione reductases, designated GR-I and GR-II, were purified from bovine ciliary body to apparent homogeneity by ion-exchange chromatography on DEAE-agarose and gel filtration on Sephacryl S-200. The two enzymes were isolated whether protease inhibitors were included or not in the medium during tissue homogenization. GR-I was found to have a molecular weight of about 140,000 by gel filtration and be composed of two identical subunits. On the other hand, GR-II was shown to exist as aggregates with molecular weights larger than 670,000. The subunit molecular weight of GR-II was estimated to be about 45,000 by SDS-polyacrylamide gel electrophoresis. The high molecular aggregate dissociated into smaller species by treatment with mercaptoethanol. The isoelectric points of GR-I and GR-II were determined to be 5.8 and 6.4, respectively. The K'm values for NADPH and oxidized glutathione (GSSG) were 22- and 84 microM for GR-I and 11- and 98 microM for GR-II. Coenzyme A-glutathione disulfide served as a substrate, though it was much less reactive than oxidized glutathione, for both enzymes. S-alkyl glutathiones were inactive as substrates. Nitrofurantoin was a non-competitive inhibitor of the enzymes. 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) inhibited GR-I and GR-II irreversibly. The two enzymes are similar, if not identical, in their amino-acid composition. The two enzymes are also very similar on the basis of the relative position of tryptic peptides separated in two-dimensional peptide maps. From the tissue content and turnover numbers of these enzymes it is concluded that bovine ciliary body has a sufficient capability for rapid reduction of oxidized glutathione produced in the tissue during glutathione-dependent peroxide detoxification.

Amino Acids↗
PubMed1986

Superoxide in ocular inflammation: human and experimental uveitis.

A possible involvement of superoxide in the pathogenesis of uveal inflammation in man and experimental animals was investigated. Superoxide production by the leukocytes of Behcet patients was significantly higher in the attack phase than in the remission phase. Leukocyte superoxide generation was also enhanced in guinea pigs with S-antigen-induced experimental autoimmune uveoretinitis (EAU). If the animals were treated with superoxide dismutase (SOD) at the onset of EAU, aqueous humor cell count was significantly lower than that of control (i.e., without SOD treatment). Infiltration of the inflammatory cells in the anterior retina was markedly reduced in SOD-treated animals. A similar protective effect of SOD against tissue damage was also observed in a bovine serum albumin-induced passive Arthus type uveitis in rabbits. These results suggest that superoxide may play a role in causing tissue damage in animal models of ocular inflammation and possibly in Behcet disease.

Animals↗
PubMed1986

Stimulation of the hexose monophosphate shunt in bovine ciliary body under oxidative stress.

The effect of t-butyl hydroperoxide (TBHP) on the oxidation of C1-radiolabeled glucose and C6-radiolabeled glucose by bovine ciliary body was investigated in the presence and absence of the glutathione reductase inhibitors nitrofurantoin (NF) and 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]. 14CO2 production from 1-14C-glucose was markedly stimulated by TBHP and inhibited by BCNU. Using tissue extracts, BCNU was shown to inhibit both glutathione reductase and glucose-6-phosphate dehydrogenase. These results support the hypothesis that bovine ciliary body has high NADPH regenerating capacity by the hexose monophosphate shunt.

Animals↗
PubMed1986

Immunocytochemical localization of phosphatidylinositol-4,5-bisphosphate in dark- and light-adapted rat retinas.

Light-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) to 1,2-diacylglycerol and D-myo-inositol 1,4,5-triphosphate (IP3) has been reported in the visual photoreceptor cells. We have investigated the localization of the TPI antigenic sites in dark- and light-adapted rat retinas using rabbit anti TPI antibodies (Ab). Sprague-Dawley rats were dark-, or light-adapted, or were exposed to a light flash. The eyes were fixed immediately and the tissue sections stained with the rabbit anti TPI Ab. The peroxidase-antiperoxidase method was used to find the localization of the TPI antigenic site. Image analysis of the sections was performed to obtain optical density profiles of the stain. Dark-adapted retinas showed intense staining of the rod outer segment (OS) layer but much less staining of the rod inner segment layer. Compared with the OS of dark-adapted retinas, those of the flash-bleached retinas were stained much less. The OS of fully bleached retinas showed little or no staining. The anti TPI Ab-protein A-gold conjugate intensely stained disks from dark-adapted retina but those from bleached retina much less. Our results suggest that rapid hydrolysis of TPI in rat rod outer segments occurs in vivo in response to light.

Animals↗
PubMed1985

Glutathione depletion and oxidative stress: study with perfused bovine eye.

A bovine eye perfusion system was developed for detoxification studies. The viability of the system was examined by two criteria: the permeability of the aqueous-blood barrier to protein, and the turnover of aqueous humor formation. Although the usefulness of the system for physiological studies is limited, the bovine eye perfusion system proved to be useful for biochemical studies of detoxification. When the system was perfused with t-butyl hydroperoxide plus the glutathione reductase inhibitor nitrofurantoin, glutathione levels of the ciliary body and iris were markedly reduced. The result was interpreted to suggest that glutathione and its redox cycling may play an important role in cellular defense against oxidative stress.

Animals↗
PubMed1985

Localization and properties of an immunoreactive protein in bovine ciliary body similar to retinal S antigen.

Immunocytochemical studies showed that monoclonal antibodies to bovine retinal S-antigen recognize an immunoreactive protein present in the bovine ciliary body epithelium. This protein was purified by DEAE-agarose chromatography and found to have a M.W. of 28,000 daltons by SDS polyacrylamide gel electrophoresis. Crude and purified preparations of this protein cross-react with polyclonal antibodies to retinal S-antigen. The relative reactivity of S-antigen monoclonal antibodies to ciliary body protein was determined by ELISA. Lewis rats immunized with crude and purified ciliary body protein failed to develop experimental uveitis. The results indicate that the ciliary body protein contains an antigenic peptide domain which is similar to that of S-antigen but lacks the immunopathogenic domain.

Animals↗