Ah locus: genetic differences in susceptibility to cataracts induced by acetaminophen.

The Ahb/Ahb homozygous and the Ahb/Ahd heterozygous inbred mouse strains from the (C57BL/6)(DBA/2)F1 X DBA/2 backcross are genetically responsive to 3-methylcholanthrene. They both also develop, within 6 hours after a large intraperitoneal dose of acetaminophen, an irreversible opacity in the anterior portion of the lens. Such cataract formation does not occur in similarly treated nonresponsive inbred strains or nonresponsive Ahd/Ahd individuals from the same backcross. Differences in acetaminophen metabolism and toxicity are associated with the Ah locus in the mouse, and differences in heritability at the Ah locus exist in the human. Our ophthalmologic findings may be important clinically to certain patients receiving either a single large overdose of this drug or high doses over a long period.

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.

The N-terminal residue of bovine rhodopsin is acetylmethionine.

By affinity chromatography on a concanavalin A-Sepharose column and gel filtration on a Sephadex G-25 column, a glycopeptide of 16 amino acid residues has been separated from a tryptic digest of reduced and carboxymethylated bovine rhodopsin. The glycopeptide was treated with cyanogen bromide and products were subjected to high-voltage paper electrophoresis. N-Blocked homoserine separated was reacted first with anhydrous hydrazine and then with dansyl chloride. The product was identified by thin-layer chromatography to be 1-acetyl-2-dansyl hydrazine, thus showing that the N-terminal blocking group was an acetyl group. The remaining peptide after cyanogen bromide treatment was partially sequenced by the Edman dansylation method. The present results and previously reported findings on the binding site of the sugar moiety, taken together, indicate that the N-terminal heptapeptide has the following structure: acetylMet-Asn(carbohydrate)-Gly-Thr-Glx-Gly-Pro.

Developmental changes in prostaglandin E(2) receptor subtypes in porcine ductus arteriosus. Possible contribution in altered responsiveness to prostaglandin E(2).

BACKGROUND: Prostaglandin E(2) (PGE(2)) is important in ductus arteriosus (DA) patency, but the types of functional PGE(2) receptors (EP) in the developing DA are not known. We postulated that age-dependent alterations in EP and/or their subtypes may possibly contribute to the reduced responsiveness of the newborn DA to PGE(2). METHODS AND RESULTS: We determined PGE(2) receptor subtypes by competition binding and immunoblot studies on the DA of fetal ( approximately 75% and 90% gestation) and newborn (<45 minutes old) pigs. We studied the effects of EP receptor stimulation on cAMP signaling in vitro and on term newborn (<3 hours old) DA patency in vivo. Fetal pig DA expressed EP(2), EP(3), and EP(4) receptors equivalently, but not EP(1). In neonatal DA, EP(1), EP(3), and EP(4) were undetectable, whereas EP(2) density was similar in fetus and newborn. Prostaglandin-induced changes in cAMP mirrored binding data. 16,16-Dimethyl PGE(2) and 11-deoxy PGE(1) (EP(2)/EP(3)/EP(4) agonist) produced more cAMP in fetus than newborn, but butaprost (selective EP(2) agonist) caused similar cAMP increases in both; EP(3) and EP(4) ligands (M&B28767 and AH23848B, respectively) affected cAMP production only in fetus. After birth, administration of butaprost alone was as effective as 11-deoxy PGE(1) and 16,16-dimethyl PGE(2) in dilating DA in vivo. CONCLUSIONS: The data reveal fewer PGE(2) receptors in the DA of the newborn than in that of the fetus; this may contribute to the decreased responsiveness of the DA to PGE(2) in newborn. Because EP(2) receptors seem to mediate the effects of PGE(2) on the newborn DA, one may propose that a selective EP(2) agonist may be preferred as a pharmacological agent to maintain DA patency in infants with certain congenital heart diseases.

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.

Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies.

Two monoclonal antibodies (RSA1/83 and RSA2/83) were developed against a homogeneous preparation of bovine retinal S-antigen. The two hybridomas produced by mouse X mouse hybrid myeloma cells secrete immunoglobulin G. Indirect autoradiography on glutaraldehyde-fixed preparations of bovine explants was used to locate the antigenic site. Antibody RSA1/83 recognizes the antigen primarily in the apical region of the rod outer segment, while antibody RSA2/83 located the antigen both in the outer and inner segments of the rod photoreceptor cells. A distinct band of silver grains also appeared along the inner limiting membrane with both antibodies. Control explants showed no specific labeling pattern over the various retinal compartments.

Experimental autoimmune uveoretinitis induced by the gamma-subunit of cyclic guanosine monophosphate phosphodiesterase in rats.

PURPOSE: To investigate the capacity of the recombinant gamma-subunit (P gamma) of cyclic guanosine monophosphate phosphodiesterase to induce experimental autoimmune uveoretinitis in Lewis rats. METHODS: Bovine P gamma was expressed in Escherichia coli cells and purified by fast protein liquid chromatography. Lewis rats were immunized by a single footpad injection of P gamma emulsified in complete Freund's adjuvant. Clinical and histopathologic changes in the eye and pineal gland were examined. Lymphocytes were prepared from the lymph nodes of rats with uveitis and transferred by intraperitoneal injection to naive recipient rats. RESULTS: Immunization of rats with P gamma induced panuveitis and pinealitis with clinical and histopathologic changes similar to those induced by S-antigen. Lymphocytes from the lymph nodes of diseased rats transferred uveitis to naive recipients. CONCLUSIONS: P gamma, a retina-specific protein of molecular weight less than 10,000 kDa, is capable of inducing uveoretinitis in Lewis rats. The disease can be transferred adoptively to naive rats by injection of lymphocytes from donor rats with experimental autoimmune uveoretinitis. Inflammation of the pineal gland of immunized rats suggests that P gamma is not only localized to the retina but also to the pineal gland.

Preparation of a uveitogenic peptide by chymotryptic digestion of bovine S-antigen.

Limited proteolysis of bovine S-antigen with alpha-chymotrypsin resulted in the accumulation of three peptides of MW 24,000, 16,000, and 12,000 daltons, respectively. By ELISA (enzyme-linked immunosorbent assay), MW 24,000 peptide was found to react with anti-S antibodies, but the other two peptides did not react with the antibodies under the assay conditions. The reactive peptide was separated from the smaller peptides by gel filtration on Sephadex G-75 and Sephadex G-50. When the MW 24,000 peptide was injected into Lewis rats, severe to mild uveitis was produced in all injected animals. The results indicate that the pathogenic determinant is on the MW 24,000 peptide.

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.

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.

Genetic differences in drug metabolism associated with ocular toxicity.

The tissue localization and subcellular distribution of drug-metabolizing enzymes in the eye are described. With the use of inbred strains of mice, the [Ah] complex is shown to be an important experimental system for probing genetic differences in drug metabolism and related drug toxicities. Although the genetic system described in detail here involves mice, there is ample evidence that the same system operates in man. Genetic differences in acetaminophen- and naphthalene-induced cataract formation and and other ocular degeneration are shown to be related to the [Ah] complex. Because this toxicity appears similar to senile cataracts, we propose that certain types of drug-induced cataracts might exist among clinical populations of senile cataracts but that any cause-and-effect relationship would be very difficult to determine because of underlying interindividual differences in genetic predisposition. It is therefore suggested that genetic differences in drug metabolism be an important consideration in the clinical assessment of ocular toxicity caused by drugs and other environmental pollutants.

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.

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.

Phosrestin I, an arrestin homolog that undergoes light-induced phosphorylation in dipteran photoreceptors.

Two classes of phosphorylated homologs of vertebrate arrestins, designated phosrestins I (PRI) and phosrestin II (PRII), are expressed in the photoreceptors of a fruit fly, Drosophila melanogaster. This study presents evidence that the housefly, Musca domestica, also has a protein similar to Drosophila PRI. Our conclusion is based on the following evidence. (1) We identified a Musca photoreceptor protein exhibiting a molecular mass (51 kDa) and an isoelectric point (pI = 8.6) similar to those of Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. Drosophila PRI. This Musca protein, designated Musca PRI, changes its pI upon illumination in vivo. (2) Rabbit antibodies raised against Musca PRI, against bovine arrestin, and against a synthetic peptide based on the Drosophila PRI sequence stained the Drosophila and Musca PRIs specifically on 1 and 2-dimensional Western immunoblots. (3) Both Drosophila and Musca PRIs incorporated 32P-radioactivity from gamma-32P-ATP in cell-free homogenates of retinas. Partial peptide digestions of Drosophila and Musca PRIs revealed similarity between these proteins. We observed that Drosophila PRI exists in the random preparation, but it also exists in other subcellular fractions. Immunocytochemistry at the EM level revealed a distribution of both Drosophila and Musca PRI epitopes in membranous vesicular structures in the cytosol as well as in the rhabdomeric microvillar membranes where the visual pigment, rhodopsin, exists. Such distribution of PRI epitopes suggests that PRI and its light-dependent phosphorylation may function in a space remote from the rhabdomere as well as the immediate milieu of photoreception.

Regulation of rod GTP binding protein by guanine nucleotides.

The rod GTP-binding protein in the bovine disk membrane seems to exist as oligomers in the dark. G alpha and G beta do not interact strongly, and G gamma may be required for G alpha . GDP to dimerize.

Carbohydrate structures of bovine kidney gamma-glutamyltranspeptidase.

The carbohydrate moieties of gamma-glutamyltranspeptidase (gamma-GTP) purified from bovine kidney were chemically released as oligosaccharides. The oligosaccharide mixture was fractionated into a neutral fraction and three groups of acidic fractions containing one, two, and three sialic acid residues. Both the neutral fractions and the neutral oligosaccharide mixture obtained from the pooled sample of all acidic fractions by sialidase treatment were separated into seven components by Bio-Gel P-4 column chromatography. Structural studies of these components by sequential exoglycosidase digestion in combination with methylation analysis indicated that the gamma-GTP contains at least twelve neutral sugar chains which are either the high mannose type, or the bi- and triantennary complex type, and thirteen acidic sugar chains of the bi-, tri-, and tetraantennary complex type. The characteristics feature of the sugar chains of the gamma-GTP is that most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the beta-mannosyl residue of their trimannosyl core, and their outer chain moieties are enriched in non-reducing terminal beta-N-acetylglucosamine residues.

The oligosaccharide moiety of rhodopsin-its structure and cellular location.

The sugar chains of bovine rhodopsin released from opsin by hydrazinolysis were reduced with NaB((3)H)(4) and fractionated by paper chromatography. Three oligosaccharides were obtained. The structure of the major ((3)H)-oligosaccharide (ca. 60% of total) was elucidated by sequential exoglycosidase digestion, methylation analysis and endo-?-N-acetyl glucosaminidase D digestion. The structure of the sugar moiety of rhodopsin was thus identified as: Since the terminal GlcNac serves as galactose acceptor, the location of the sugar moiety of rhodopsin in the disc membrane was studied by incorporation of ((3)H)-galactose (from UDP-((3)H)-galactose) into the disk membrane. After inversion of disks by freeze-thawing, rhodopsin in the membrane incorporated one mole of ((3)H)-galactose per mole purified pigment. Intact disks incorporated lower amounts of ((3)H)-galactose. It was therefore concluded that the sugar moiety of rhodopsin is located on the internal surface of disk membrane. The results of lectin binding studies are consistent with the conclusion. Inverted disks, but not intact disks, bind concanavalin A (specific for ?-mannosyl residue) and wheat germ lectin (specific for N-acetylglucosamine). Since intact sealed rod outer segments bind concanavalin A, the sugar moiety of rhodopsin in the plasma membrane is probably exposed on the external surface of the rod.

Bimodal regulation of adenylate cyclase by prostaglandin E2 receptors in porcine ciliary epithelium.

Prostaglandin E2 (PGE2) binding revealed that porcine ciliary epithelial membranes possess two subclasses of binding sites or receptors. Ciliary nonpigmented epithelial (NPE) membranes have two binding sites (Kd,1 = 35 x 10(-9) M, Bmax,1 = 485 x 10(-12) mol/mg protein; Kd,2 = 0.723 x 10(-6) M, Bmax,2 = 1473 x 10(-12) mol/mg protein), while pigmented epithelial (PE) membranes have one binding site (Kd = 82 x 10(-9) M, Bmax = 377 x 10(-12) mol/mg protein). The three sites of NPE/PE membranes were found to show the highest affinity for PGE2 by competitive binding assay; affinity for PGF2 alpha and PGD2 was several orders of magnitude less. PGE2 binding to the higher affinity site of NPE membranes induced inhibition of adenylate cyclase activity. Activation of the lower affinity site resulted in activation of the cyclase activity. PGE2 binding to PE membranes caused inhibition of adenylate cyclase activity. There is evidence that cyclic adenosine monophosphate (cAMP) affects transport of ions and water in the ciliary epithelium, hence modulates aqueous humor inflow. The present results, therefore suggest that aqueous humor production by the ciliary NPE cells may be influenced by changes in the concentration of PGE2 which binds to the receptor subtypes having opposing effects on adenylate cyclase and regulates intracellular cAMP level.

GTP binding protein: properties and lack of activation by phosphorylated rhodopsin.

Taking advantage of the capability of GTP binding protein to bind GTP, we purified the catalytic subunit (G alpha) of bovine rod GTP binding protein by nucleotide-affinity chromatography on Blue Sepharose CL6B. Purified G alpha was essentially free of bound guanine nucleotide and activated by photoactivated rod membranes. Circular dichroism spectra suggested that a significant portion of the protein would be in alpha-helical conformation. No appreciable differences were detected in the circular dichroism spectra when G alpha . GDP and G alpha . GppNp were compared. The extent of G protein activation by rod membranes was reduced moderately by phosphorylation of rhodopsin during photolysis. However, if the pigment had been phosphorylated and regenerated, the ability of rhodopsin to activate G protein was markedly suppressed.

Resemblance between rhodopsin kinase and S-antigen induced uveitis.

The retinal S-antigen (S-Ag) has been shown to induce uveitis effectively in subhuman primates, and lymphocytes from patients with certain uveitic conditions show cell-mediated responses to this antigen. Rhodopsin kinase (RK), an enzyme probably unique to the mammalian eye, is reported here to resemble the retinal S-Ag in its capacity to induce uveitis in experimental animals. A histological comparison of rat eyes taken 2 and 3 weeks after immunisation with either RK or S-Ag reveals essentially identical pathological alterations. Ocular inflammation is seen in both the anterior and posterior portion of the globe. Areas of focal degeneration of the photo-receptor layer, from which both the S-Ag and RK are extracted, could be seen in both RK and S-Ag immunised animals. Cells from draining lymph nodes of both groups responded by increased thymidine incorporation when cultured in the presence of either RK or S-Ag. In addition antibodies directed against the S-Ag were detected in both groups. These findings, in addition to the biochemical similarities of these preparations, reported elsewhere, would strongly suggest that RK and S-Ag are one and the same. The identification of potentially uveitogenic ocular antigens could help to reclassify uveitic entities that at present have clinically similar courses.