Biomedical subjects

H Shichi

Publications and source records attributed to H Shichi.

At least 55 records · Page 3Linked to original sources

PubMed1992

Prostaglandin synthesis and accumulation by porcine ciliary epithelium.

The activity to synthesize prostaglandins (PGs*) from arachidonic acid [i.e. cyclooxygenase (CO) activity] was compared between nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells from porcine ciliary processes. PG synthesizing activities were about the same (0.58 and 0.52 pmol per mg protein per 15 min for NPE and PE, respectively), and inhibited by indomethacin and anti-CO antibody but not by nordihydroguaiaretic acid. By immunogold labeling, CO immunoreactivity was found both in NPE and PE. The present results and the previous observation on higher PG omega/omega-1 hydroxylation activities in NPE than in PE, taken together, suggest that controlled synthesis and inactivation of PGs may have a role in functions of NPE. When PGE2 was incubated with NPE or PE, NPE cells accumulated PGE2 more actively than did PE cells. PG uptake by the NPE cells may prevent aqueous and vitreous PGs from entering the anterior chamber and causing anterior uveitis.

Animals↗

PubMed1992

Ciliary body in experimental autoimmune uveitis: tissue repair and immunoreactivity of extracellular matrix substances.

Experimental autoimmune uveoretinitis was induced in female Lewis rats with bovine retinal soluble antigen (S-antigen). Tissue changes and immunoreactivities of transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), and extracellular matrix compounds in the anterior segment (ciliary body) were investigated by immunocytochemical methods. Control animals received adjuvant only. The immunized animals were sacrificed at day 0, 3, 7, 14, 20, and 30 postimmunization. Tissue changes that occurred at the peak of inflammation (day 14) included destruction of the inner basement membrane, epithelial cell loss, distortion of the ciliary stroma, and loss of epithelial basal infoldings. Ciliary body architecture was regenerated almost completely by day 30. Basement membrane laminin and collagen type IV levels did not change much during the inflammatory process. Fibronectin labeling level peaked at day 14 postimmunization. Collagen type V level was low at day 14 and elevated at day 20 and day 30. TGF-beta immunoreactivity peaked at day 14 and remained elevated thereafter. EGF labeling did not increase until day 20 and was maximal at day 30. Labeling of both growth factors was principally confinded to the stromal regions. The presence of TGF-beta and EGF in the ciliary stroma at well defined intervals suggests a coordinated effect upon the synthesis and reorganization of the extracellular matrix and possibly upon the inflammatory cell population in the anterior tissue.

Animals↗

PubMed1992

Suppression of experimental autoimmune uveoretinitis by intraorchidic administration of S-antigen.

Experimental autoimmune uveoretinitis (EAU) induced by immunization of albino Lewis rats with a retinal soluble antigen (S-antigen) has been studied extensively by many workers. An intraorchidic injection of S-antigen 4 days prior to immunization of the animal with the antigen emulsified in adjuvant was found to prevent the onset of the disease. Orchiectomy in 24 hr after the intraorchidic injection did not abolish the effect of treatment. The systemic suppression induced by the orchidic treatment persisted at least for 3 weeks after treatment. In rats that received orchidic treatment, delayed-type hypersensitivity was markedly inhibited but anti-S-antigen antibody levels in the serum were as high as those in immunized rats without orchidic pretreatment. These results indicate that antigen challenge to the testis is a novel method to systemic activation of the immunosuppressive mechanism.

Animals↗

PubMed1992

Immunosuppressive factors in porcine ciliary body.

Immunosuppressive factors locally present in the anterior tissues such as the ciliary body may contribute to the immunological privilege of the anterior chamber of the eye. Porcine ciliary body extracts contain at least three immunosuppressive factors (greater than 200 kDa, 25 kDa and 2.5 kDa) which can be separated by Sephacryl S300 gel filtration. On the basis of its molecular weight and reversal of inhibitory activity by anti-transforming growth factor beta (TGF beta) antibody, 25 kDa factor is identified with TGF beta. The high molecular factor is low in protein content and its activity is not affected by anti-TGF beta antibody. The 2.5 kDa factor is most likely to be identical with a low molecular factor present in the vitreous body and aqueous humor. This low molecular factor is extracted from nonpigmented ciliary epithelial cells but seems to be absent in the extracts of pigmented epithelial cells. The regional distribution suggests that the factor may be synthesized and secreted with the aqueous humor by the nonpigmented cells. All three factors suppress lymphoblastogenesis without affecting the expression of CD4 and CD8. Identical or similar factors may be present in other immunologically privileged tissues and be responsible for maintaining the immunologically suppressed environment.

Animals↗

PubMed1991

Induction of alkoxyresorufin O-dealkylases and UDP-glucuronosyl transferase by phenobarbital and 3-methylcholanthrene in primary cultures of porcine ciliary epithelial cells.

Our previous studies have shown that drug-metabolizing activities in the eye are highest in the ciliary body, a tissue responsible for aqueous humor production. In this work, we have separated nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells from porcine ciliary body and determined basal and induced activities of 7-ethoxyresorufin (ER) O-dealkylase, 7-pentoxyresorufin (PR) O-dealkylase, and UDP-glucuronosyl transferase (UDP-GT) using primary cultures of separated cells. ER and PR activities were associated primarily with NPE cells and were very low in PE cells. Treatment of NPE cells with phenobarbital (PB) for 48 hr resulted in about a 4-fold increase in PR O-dealkylase activity but only a 1.3-fold rise in ER O-dealkylase activity. Conversely, 3-methylcholanthrene (MC) treatment augmented the ER O-dealkylase activity of NPE cells 6 times over the basal activity in 48 hr but had little effect on PR O-dealkylase activity. Both NPE and PE cells had low basal UDP-GT activities. UDP-GT activity increased about 5-fold in PB-treated PE cells and about 4-fold in PB-treated NPE cells in 48 hr. The results of MC treatment were similar to those of PB treatment; enhancement of UDP-GT was more pronounced in PE cells than in NPE cells. Induction by PB and MC of ER O-dealkylase, PR O-dealkylase and UDP-GT activities in ciliary NPE and PE cells was inhibited almost completely by 3.5 microM cyclohexamide and 40 nM actinomycin D. The heterogeneous distribution of these enzymes suggests that a harmonious interplay between NPE and PE cells is important for metabolic detoxification of blood plasma prior to aqueous humor formation.

Animals↗

PubMed1991

Breakdown of the blood-retinal barrier in S-antigen-induced uveoretinitis in rats.

Experimental autoimmune uveoretinitis was induced in Lewis rats by footpad injection of purified, bovine retinal soluble antigen (S-antigen) with complete Freund's adjuvant. Control animals received adjuvant only. At the peak of ocular inflammation the animals received an intravenous injection of the tracer horseradish peroxidase, and the eyes were removed and processed for peroxidase cytochemistry. A breakdown of the blood-retinal barrier occurred at the level of the retinal venules. Peroxidase reaction product was demonstrable in the interendothelial tight junctions and in the perivascular spaces. Heavy infiltration of inflammatory cells was also found in and around the venular wall. The endothelial cells of venules and capillaries were hypertrophied and showed swollen endoplasmic reticulum and Golgi apparatus as well as increased numbers of ribosomes and cytoplasmic fibrils; pericytes and smooth-muscle cells showed a similar synthetic phenotype and the basal lamina of these cells was thickened.

Animals↗

PubMed1991

Immunocytochemical localization of gamma-glutamyl transpeptidase in porcine ciliary epithelium.

Our previous work on cultured bovine ciliary epithelial cells indicated that gamma-glutamyl transpeptidase (GGTP) activity is primarily associated with the non-pigmented epithelial cells and is virtually absent in the pigmented epithelial cells [Ng and Shichi, (1987) J. Ocular Pharmacol. 3, 341-348]. We have now investigated the localization of GGTP in porcine ciliary processes by immunocytochemical methods using goat anti-rat GGTP serum detected by rabbit anti-goat IgG secondary antibodies conjugated to fluorescein isothiocyanate or horseradish peroxidase, or biotin-conjugated secondary antibodies and streptavidin (Auroprobe). The non-pigmented cells were labeled uniformly and intensely by fluorescent antibodies, while only the plasma membrane (on the stroma side) was labeled in the pigmented cells. Immunoperoxidase reactions demonstrated that only the plasma membrane was labeled also in non-pigmented cells. The apparent uniform labeling of the cytoplasm of non-pigmented cells by fluorescent antibodies was attributed to the extensively infolded plasma membrane. The peroxidase reaction product was not found in the mitochondria, nucleus or endoplasmic reticulum of non-pigmented or pigmented cells. Immunogold labeling indicated that antigenic sites are associated with the plasma membrane infoldings (vitreous side) of non-pigmented cells, and the plasma membrane (stroma side) of pigmented cells. For enzymatic localization of GGTP activity in ciliary epithelial cells, non-pigmented and pigmented epithelial cells were carefully separated from fresh porcine ciliary body and homogenized to isolate microsomal fractions containing vesicles of plasma membrane and endoplasmic reticulum. The specific activity of microsomes from non-pigmented cells was 23 times higher than that of microsomes from pigmented cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Regional distribution of lipoxygenase activities in porcine ciliary epithelium.

Nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells were separated from porcine ciliary processes without protease treatment. The relative capabilities of NPE cells and PE cells to synthesize 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 5,12-diHETE from 1-[14C] arachidonic acid were determined. HETE compounds were separated by HPLC. The capability of NPE cells to synthesize 5-lipoxygenase and 12-lipoxygenase products was, respectively, five-fold and 2.5 fold higher than that of PE cells. The formation of 5-HETE, 12-HETE and 5,12-diHETE was inhibited virtually completely by 50 microM nordihydroguaiaretic acid but not by 5 microM indomethacin. The major product formed both by NPE cells and PE cells was 12-HETE.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1991

Immunocytochemical study of phenobarbital- and 3-methylcholanthrene-inducible cytochrome P450 isozymes in primary cultures of porcine ciliary epithelium.

We found in the previous study that the induction of 7-pentoxyresorufin O-dealkylase and 7-ethoxyresorufin O-dealkylase activities by phenobarbital and 3-methylcholanthrene, respectively, is more pronounced in porcine ciliary nonpigmented epithelial cells than in pigmented epithelial cells. In order to determine whether cytochrome P450 isozymes that mediate the O-dealkylase activities are also induced in nonpigmented cells under the conditions, primary cultures of porcine ciliary processes were treated with phenobarbital and 3-methylcholanthrene and the expression and localization of cytochrome P450 isozymes induced by these compounds were investigated by immunocytochemical methods using antibodies against the individual P450 isozymes. Intense labeling of nonpigmented epithelial cells was observed when ciliary processes treated with phenobarbital were reacted with anti-P450 (phenobarbital) antibody and when the processes treated with 3-methylcholanthrene were incubated with anti-P450 (methylcholanthrene) antibody. The labeling patterns supported the conclusion that the O-dealkylase activities and P450 isozymes specific for these activities are co-induced in and localized to the endoplasmic reticulum. This study is the first report presenting direct evidence of cytochrome P450 induction in primary cultures of ocular tissues and demonstrates the usefulness of porcine ciliary epithelial cells for studying the induction of ocular drug metabolizing enzymes.

Animals↗

PubMed1991

Increased immunoreactivity of collagen types I, III and V, fibronectin and TGF-beta in retinal vessels of rats with experimental autoimmune uveoretinitis.

The immunoreactivity of types I, III and V collagen, fibronectin and transforming growth factor-beta (TGF-beta) was studied by an immunogold labeling method in retinal vessels of rats with experimental autoimmune uveoretinitis (EAU) induced by retinal S-antigen. The basal lamina of retinal capillaries in normal rats showed low immunoreactivities for the extracellular matrix components and TGF-beta. However, at the peak of inflammation (day 13-16 postimmunization), labeling of all three types of collagen and fibronectin increased considerably in the basal lamina of retinal vessels. TGF-beta immunoreactivity was detected mainly in the vascular endothelium and the pericytes. The results suggest that TGF-beta synthesized by the endothelial cells and pericytes may regulate the synthesis and composition of extracellular matrix components in the vascular basal lamina during the course of EAU development.

Animals↗

PubMed1991

Immunosuppressive factors in porcine vitreous body.

High molecular (designated Sv, greater than 3K) and low molecular (designated Sv, less than 3K) fractions were prepared by filtration of porcine vitreous extracts through a membrane filter with a molecular weight cut-off of 3,000 daltons. Both fractions inhibited mitogen-stimulated proliferation of lymphocytes from rat spleen, as determined by [3H]-thymidine incorporation. The inhibitory activity of Sv, greater than 3K was markedly enhanced by acid treatment and abolished almost completely by incubation with anti-transforming growth factor beta (TGF-beta) antibody. The major immunosuppressive factor in Sv, greater than 3K was therefore concluded to be TGF-beta. On the other hand, the inhibitory activity of Sv, less than 3K was not affected by acid treatment or by incubation with anti-TGF-beta antibody but was decreased by proteolytic treatment. From these results the immunosuppressive factor in Sv, less than 3K was attributed to a low molecular substance which is probably a peptide structurally unrelated to TGF-beta. The inhibitory effects of both Sv, greater than 3K and Sv, less than 3K were reversible and therefore not cytotoxic. Both factors seemed to suppress lymphoproliferation without affecting the expression of Interleukin-2 receptor, CD4 and CD8 on the cell surface of cultured lymphocytes.

Animals↗

PubMed1990

Non-selenium glutathione peroxidase without glutathione S-transferase activity from bovine ciliary body.

A glutathione peroxidase was purified from bovine ciliary body by ammonium sulfate fractionation. Sephacryl S-300 gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography and hydroxyapatite chromatography. The purified enzyme has an apparent mw of 112 kDa by gel filtration and 29 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme therefore is composed of four identical subunits. The ciliary enzyme is active with H2O2 (25), cumene hydroperoxide (170), t-butyl hydroperoxide (22), triphenylcarbinyl hydroperoxide (12), linoleic hydroperoxide (34) and 5-phenylpentenyl hydroperoxide (22): the numbers after substrates are K'm in microM. Glutathione is essential for the reaction; L-cysteine, dithiothreitol and 2-mercaptoethanol are inactive. Mercaptosuccinate (10 microM) inhibits the enzyme competitively (Ki = 7 microM) when cumene hydroperoxide is substrate, and uncompetitively (Ki = 10 microM) when H2O2 is substrate. No selenium was found in the enzyme by the fluorometric assay with 2.3-diaminonaphthalene. The enzyme demonstrates no glutathione S-transferase activity when tested with 1-chloro-2,4-dinitrobenzene, and several other compounds. A partial sequence of the enzyme shows some similarities both to Se-glutathione peroxidases and a glutathione S-transferase isozyme.

Amino Acid Sequence↗

PubMed1990

Glutathione-dependent detoxification of peroxide in bovine ciliary body.

Perfusion of the bovine eye with a buffer solution containing t-butyl hydroperoxide and the glutathione reductase inhibitor nitrofurantoin caused significant decreases in reduced glutathione level in ciliary body and iris. The result was interpreted to suggest that the organic hydroperoxide was decomposed by the glutathione peroxidase-reductase system. The glutathione reductase reaction requires NADPH. Since the level of NADPH is maintained by the hexose monophosphate shunt in many tissues, we investigated whether this is also the case with bovine uveal tissues. CO2 formation from [1-14C]glucose but not from [6-14C]glucose was markedly stimulated by t-butyl hydroperoxide and was inhibited by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, thus supporting the importance of the hexose monophosphate shunt for hydroperoxide decomposition through the glutathione peroxidase-reductase system. The peroxidase-reductase activity was found both in non-pigmented and pigmented ciliary epithelial cells in culture. Purification studies isolated two forms of glutathione reductase [GR I (140 kDa) with subunit Mr of 70 kDa and GR II (greater than 670 kDa) with subunit Mr of 45 kDa] and a novel glutathione peroxidase (112 kDa with subunit Mr of 29 kDa). The peroxidase is active both with H2O2 and organic hydroperoxides, does not contain selenium and shows no glutathione S-transferase activity.

Amino Acid Sequence↗

PubMed1990

Distribution and induction of alkoxyresorufin O-dealkylases and gamma-glutamyl transpeptidase in porcine ciliary epithelium.

Nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells were separated from porcine ciliary body by a simple surgical procedure. Using primary cultures of separated cells, basal and induced activities of 7-ethoxy resorufin (ER) O-dealkylase, 7-pentoxy resorufin (PR) O-dealkylase, and gamma-glutamyl transpeptidase (GGTP) were determined. These enzymatic activities were primarily associated with NPE cells; PE cells showed little or no activity. Treatment of NPE cells with phenobarbital for 48 hours resulted in about 5-fold increase in PR O-dealkylase activity but not ER-Odealkylase activity. Conversely, 3-methylcholanthrene treatment increased ER O-dealkylase activity 5-6 times over the basal level but had little or nor effect on PR O-dealkylase activity. The enhancement of enzymatic activities by inducers was completely inhibited by cyclohexamide. NPE cells exhibited higher GGTP activity than PE cells. Both phenobarbital and 3-methylcholanthrene augmented the GGTP activity of NPE cells but not of PE cells. The physiological significance of the regional differences in enzyme distribution was discussed.

Animals↗

PubMed1990

Immunosuppression by gramicidin S of experimental autoimmune uveoretinitis, pinealitis and autoimmune encephalomyelitis.

Using an in vitro lymphocyte proliferation assay we screened several cyclic peptide antibiotics (bacitracin, oleandomycin, capreomycin, colistin, virginiamycin, and gramicidin S) for their immunosuppressive activity. Gramicidin S (GrS) was found to inhibit [3H]-thymidine incorporation into concanavalin A-stimulated and E coli lipopolysaccharide-stimulated lymphocytes. In vivo studies, experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in female Lewis rats by immunization with bovine S-antigen and experimental autoimmune encephalomyelitis (EAE) were induced by immunization of rats with rat brain homogenates. GrS suppressed the onset of these inflammatory diseases at nontoxic concentrations. Evidence was obtained that GrS inhibits [3H]-thymidine incorporation into lymphocytes by preventing transport of the compound across the membrane. Since GrS binds to various cell membranes, GrS would suppress the proliferation of not only lymphocytes but also of other immune cells by modifying cell membrane properties. The present study indicates that a search for compounds which cause proper cell membrane modification should be a worthwhile strategy for development of immunosuppressive drugs.

Amino Acid Sequence↗

PubMed1990

The sugar moiety of bovine S-antigen.

To obtain insight into the glycoprotein nature of S-antigen (S-Ag), we have investigated the affinity of bovine S-Ag for plant lectins, location of the sugar moiety within the molecule, and incorporation of radiolabelled mannose and glucosamine into S-Ag. It was found (i) that only about 10% of purified S-Ag was bound to concanavalin A (Con A) and wheat germ agglutinin (WGA) columns, (ii) that both concanavalin A and wheat germ agglutinin bound chymotryptic peptides derived from the C-terminal half of S-Ag, and (iii) that radiolabelled D-mannose and D-glucosamine were incorporated into S-Ag and the incorporation was inhibited by tunicamycin, an N-glycosylation inhibitor. 14C-Mannose-labelled S-Ag was identified by affinity chromatography on an anti-S-Ag antibody column. These results support the supposition that only a small population of S-Ag is glycosylated (probably in N-glycosylated form), and the sugar moiety is located in the C-terminal half of the molecule.

Animals↗

PubMed1990

Immunosuppressive effect of gramicidin S on experimental ocular neuritis and allergic encephalomyelitis.

Investigations were carried out on the immunosuppressive effect of gramicidin S (GrS), a cyclic peptide antibiotic produced by Bacillus brevis, on the onset of experimental ocular neuritis and allergic encephalomyelitis in Lewis rats immunized with rat brain homogenates. The criteria for evaluation of the drug effect were changes in body weight, activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), clinical manifestations such as paralysis of lower extremities and histopathological changes. Clinical symptoms and body weight reduction were effectively prevented by GrS treatment of immunized animals. The activity of the myelin marker enzyme CNPase was markedly decreased in the lumbar spinal cord of encephalitogen-immunized animals on day 16 (ie 16 days after immunization) and the decrease of enzymatic activity was partially prevented by GrS administration. On the other hand, the CNPase activity of the retrobulbar optic nerve of inoculated animals remained essentially the same as that of healthy control animals, although inflammatory changes were prominent in the optic nerve. Histopathological changes observed in the optic nerve and spinal cord of diseased animals were virtually absent in GrS-treated animals. A possible mechanism of the immunosuppressive activity of GrS is discussed.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1989

The amino acid sequence of S-antigen: N-terminus and uveitogenic peptides.

Bovine S-antigen purified in the presence of proteinase inhibitors contained a protein (30%) having an acetylated N-terminus (Ac-Met-Lys-Ala-Asn-Lys-Pro-Ala...) and a protein (70%) having an N-terminus unblocked but four residues less (Lys-Pro-Ala-Pro-Asn-His-Val-Ile-Phe...). Sequencing studies showed that uveitogenic peptides produced by chymotryptic digestion of S-antigen derived from the C-terminal half of the protein.

Amino Acid Sequence↗