Immunohistochemical localization of cathepsin D in ocular tissues.

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.

Ocular and systemic disposition of the dopamine agonist N-0437 in monkeys after ocular administration.

The ocular and systemic disposition of the new dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) was investigated in conscious monkeys after ocular administration of 0.56 mg N-0437. HCl (corresponding to 0.50 mg free base), containing 200 microCi [3H]N-0437, into the right eye. After killing the animal, the eyes were removed and several eye tissues were dissected and assessed for their content of radioactivity. In the treated eye, the iris contained by far the highest concentration of radioactivity, while in the untreated eye the lower conjunctiva, the iris the ciliary body and the choroid possessed the highest levels of radioactivity. In all dissected eye tissues, a substantially higher concentration of radioactivity was established for the treated eye compared to the untreated eye, except for the vitreous in which for both eyes about an equal concentration was measured. This result suggests the presence of a systemic transport, which was confirmed by the occurrence of bradycardia, starting immediately after ocular application of the drug and lasting for about 1.5 hr. As the total amount of radioactivity in both eyes 7 hr after ocular dosing is very low (0.3-0.5% of the dose), one can conclude that N-0437 is almost completely taken up into the general circulation. The radioactive measurements of bile and urine samples collected up to 7 hr after administration revealed that the elimination of N-0437 and its metabolites is very fast, with the urinary excretion (35% of the dose) slightly higher than to the biliary one (31% of the dose).

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

Characterization of ocular mucus extracts by crossed immunoelectrophoretic techniques.

Crossed immunoelectrophoresis (CIE) and crossed immunoelectrofocusing (CIEF) were used to characterize proteins and mucosubstance in the saline extract of human ocular mucus pooled from the normal eyes of donors. CIE resolved more components with greater specificity than previous techniques. Up to 25 components were identified. Lactoferrin, protein G, tear prealbumin, and ocular mucosubstance were found to be ocular-specific. CIE also allowed for the study of protein associations of: 1) albumin-alpha 1-antitrypsin; 2) albumin-tear prealbumin; and 3) IgA-secretory component and lactoferrin-mucosubstance. CIEF revealed that most of the proteins were in the pI range of 4.6-7.4. Up to 19 components were identified. Protein associations revealed by CIE were not evident by CIEF. These results provide a basis for future comparative analyses of tear and mucus from normal and diseased eyes, essential for a better understanding of tear and mucus function.

Genetic location and biochemical characterization of eye-colour mutants from natural populations of Drosophila melanogaster.

From six captures of Drosophila melanogaster carried out in three different habitats (cellar, vineyard, and pinewood) in two different seasons of the year (spring and autumn), 60 eye-colour mutations were isolated, which were reduced to 29 loci by means of allelism tests within and between populations. Forty-five of these mutations were analyzed genetically and biochemically; of these 33 turned out to be previously described mutants and mapped to a total of 17 loci. Twelve new mutants were discovered and they mapped to 12 new loci, distributed on chromosome X, II, and III. The eye-colour mutants show large effects on the red and brown pigments. The high variability of the eye-colour loci is discussed in relation to the mutation and selection hypotheses.

Chromatographic characterization of vasoactive intestinal polypeptide in guinea pig and rhesus monkey eyes.

Acid extracts of guinea pig and rhesus monkey anterior uvea, choroid and retina contain immunoreactive VIP. By reversed phase high performance liquid chromatography, the immunoreactive material from these ocular tissues elutes at a position similar to synthetic porcine VIP. Only in the guinea pig anterior uvea is a second smaller peak detected. By size exclusion high performance liquid chromatography, the peptide in the monkey anterior uvea, choroid and retina elutes in the identical position as the synthetic porcine VIP standard with an apparent molecular weight of 3450 daltons. We conclude that a single form of VIP, chromatographically similar to the porcine standard, is the predominant form of the peptide in the eye of guinea pig and rhesus monkey.

Distribution of transthyretin in the rat eye.

We reported previously synthesis of transthyretin (TTR), or prealbumin, a transport protein for thyroxine and retinol, in the eyes of rats and cows and showed that in the rat eye, TTR mRNA is localized exclusively in the retinal pigment epithelium (RPE). We now demonstrate by immunohistochemistry that TTR has a more widespread distribution in the rat eye than does its mRNA. Intense immunoreactivity for TTR was found in the RPE, ciliary epithelium, iris epithelium, corneal endothelium, optic nerve fiber layer of the retina, and lens capsule. Depending on the method of processing, immunoreactivity of varying intensity was found also in other ocular structures. In particular, the retinal ganglion cells were strongly immunoreactive on frozen sections but not on paraffin sections. Although vitreous humor was not included in the sections of adult rat eye, sections of a 25-mm rat embryo showed intense immunoreactivity in the vitreous humor. Since plasma TTR does not cross Bruch's membrane into the retina, our findings suggest that ocular TTR is synthesized, at least in part, in the RPE and is transported to specific locations within the eye. Although the physiologic role of ocular TTR is unknown, it is possible that it participates in retinol cycling within the eye. The widespread ocular distribution of TTR may account for the occurrence of various forms of ocular amyloidosis in the familial amyloidotic polyneuropathies, a group of dominantly inherited disorders caused by point mutations in the TTR gene.

Expression of novel basement membrane components in the developing human kidney and eye.

The distribution of two novel human, basement-membrane (BM) collagens has been characterized by immunohistochemical analysis of developing and mature tissue using monoclonal antibodies specific for the non-collagenous (NC1) domain of each molecule. A distribution more restricted than that of type IV collagen was observed. In the kidney, the 28K parent molecules appear relatively late, at the early capillary-loop stage of glomerular development, whereas type IV collagen is present in all BM, including those of the ureteric bud, S-form, primitive glomerulus, and vessels. Antibody to the Alport familial nephritis antigen (a 26K peptide), which is missing from epidermal BM and glomerular BM in Alport syndrome, reacted with the ureteral bud BM and all stages of glomerular BM development from the early capillary-loop stage onward, but not with BM of more primitive glomeruli (vesicles and S forms). In the human fetal eye, the collagen molecules from which the 28K NC1 peptides are derived appear later in development than type IV collagen. They are present in trace amounts in Bruch's membrane but are not detected until after birth in the retinal internal limiting membrane and cuticular and non-pigmented epithelial BM of the ciliary process. In contrast, the BM of the lens capsule and Descemet's membrane were reactive with anti-28K antibodies early in development. In all instances, the 28K peptides are detected in BM that also contain the Alport antigen, although the later is present in some BM not containing the 28K peptides. The distribution of Alport antigen and type IV collagen in developing eye is similar to that observed in the mature eye. The 28K parent molecules appear to be expressed in concert with the maturation of the BM, coincident with fusion of glomerular endothelial and epithelial BM, whereas the lens capsule BM and Descemet's membrane contain these restricted components much earlier in gestation.

Purification and partial characterization of a novel cellular retinol-binding protein, type three, from the piscine eyes.

A novel cellular retinol-binding protein, termed type three (CRBP III), was isolated from eyes of the bigeye of tuna. CRBP III showed a molecular weight of 15,400, an isoelectric point of 4.80, alpha 1-mobility in electrophoresis, and a lambda max of 350 nm. All-trans-retinol, the endogenous ligand, could be competitively displaced by retinoic acid but not by retinal. CRBP III was differentiated from purified piscine and rat cellular retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins (CRABP) by its amino-acid composition, electrophoretic mobility, fluorescence spectra and ligand-binding specificity.

1H magnetic resonance imaging study of bovine ocular tissue.

The present study used magnetic resonance imaging (MRI) to study bovine eyes using proton magnetic resonance at a 7-tesla field. The MRI images provide detailed structural information on various sections of the ocular tissues. Detailed images of the lens show several well-defined areas of water content. Relative rankings of various structures of the eye are obtained based on T1, T2 and spin density.

Immunohistochemical localization of peroxidative enzymes in ocular tissue.

Localization of the peroxidative enzymes catalase and glutathione peroxidase in rabbit ocular tissue was investigated by immunohistochemical methods. We raised antisera to both enzymes in rabbits using commercially available purified enzymes. Immunoreactive catalase and glutathione peroxidase were found in the corneal epithelium and endothelium, the choroid, the inner segment of photoreceptors, and the retinal pigmented epithelium.

Beta adrenergic binding sites in the human eye: an autoradiographic study.

Beta adrenergic binding sites were localized and characterized in the human eye by means of "in vitro" autoradiography, using [125I] (-) iodocyanopindolol (125ICYP) as radioligand. Binding sites were visualized by apposition of isotope sensitive film to slide mounted eye sections. Receptor sites were present in the extraocular muscles, in the conjunctiva, in the epithelium and endothelium of the cornea, in the trabeculum and in the ciliary muscle. They were also present in the lens epithelium and in the retina. The pigmented ocular structures were heavily labelled but the binding was nonspecific. Characterization of these binding sites was achieved by testing the ability of selective adrenergic compounds to displace 125ICYP binding. These studies suggested that the majority of adrenergic binding sites in nonpigmented structures of human eye were of a beta2 type.

Renin in the bovine eye.

Recently, high levels of prorenin were found in human vitreous and subretinal fluid. In this study we attempted to identify and quantitate renin and prorenin in the bovine eye. Both these substances are present in the bovine eye in concentrations that cannot be explained by plasma contamination. Concentrations of total renin, i.e. renin plus prorenin, are highest in the posterior uveal tract [15.4 ng angiotensin (Ang) l/g per h]; in the anterior uveal tract the total renin concentration was 10.1, in plasma 6.3, in vitreous fluid 5.7 and in the retina 5.1 ng Ang l/g per h. Vitreous fluid contains mainly prorenin (99%), whereas the retina, and the anterior and posterior uveal tract contain less prorenin (respectively, 78, 47 and 32%). The absence of renin in vitreous fluid is consistent with the general finding that extrarenal renin synthesis is often associated with the release of mainly or exclusively prorenin into the extracellular fluid. Synthesis of renin and prorenin may take place in the eye.

[Age-related changes in fibronectin of the eye outflow drainage system]. / Vozrastnaia dinamika fibronektina drenazhnogo apparata glaza.

The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in ageing. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxta-canalicular zone was demonstrated along with ageing. Comparison of fibronectin accumulation in glaucoma and ageing support the idea that ageing is a risk factor of glaucoma.

[The dynamic postmortem changes of the nucleoproteins in the tissues of the liver and eye]. / Dinamika posmertnykh izmenenii nukleoproteidov v tkaniakh pecheni i glaza.

Postmortal changes in nucleoproteins were examined in the hepatocytes and anterior corneal and crystalline lenticular epithelial cells from 106 cadavers within 48 h after death by microfluorometry. Cryostat tissue sections were stained by acridine yellow by the method of R. Rigler (1966). The author found a natural decrease in fluorescence intensity of the examined tissue cellular nuclei at 530 nm within 4-24 h. and an increase in their fluorescence intensity at 640 nm within 4-48 h. after death. Data obtained may be used to establish the time of death.

Age determination in the adult screwworm (Diptera: Calliphoridae) by pteridine levels.

Fluorescence spectrophotometry was used to assess the possible use of pteridines in the compound eyes to estimate the age of adult screwworms, Cochliomyia hominivorax (Coquerel). Factors affecting the quantities of pteridines include temperature and head size. No difference in pteridine levels was found among flies fed protein or carbohydrate. A regression model for estimating the age of female screwworms was constructed. The model uses head capsule size and relative pteridine quantities and assumes a constant body temperature of 30 degrees C. This regression formula has an r2 of 0.74. Our study extends the use of pteridine accumulation for age determination from obligate sanguinivorous Diptera to an autogenous species that feeds facultatively on nectar and wound exudates. The technique appears to provide a valid means to determine age of these flies.

Effect of environmental temperature and photoperiod on the melatonin levels in the pineal, lateral eye, and plasma of the frog, Rana perezi: importance of ocular melatonin.

Day-night melatonin concentrations were studied in the pineal body, lateral eye, and plasma of the frog Rana perezi in animals maintained in February and July under long (18L:6D) or short (6L:18D) photoperiod and high (25 +/- 1 degree) or low (6 +/- 1 degree) temperature in order to evaluate the influence of these environmental factors. When frogs were kept under short photoperiod and low temperature in February, no melatonin rhythm was observed in the pineal, ocular tissue, and plasma. High temperature at this period of the year induced a day-night rhythm of melatonin levels in the lateral eye and plasma. In July, under long photoperiod and high temperature, animals showed pronounced rhythms of melatonin in the pineal, eye, and plasma. A decrease of environmental temperature in this season abolished the melatonin rhythm. When animals were maintained in August under high (25 +/- 1 degree) temperature and long (18L:6D) or short (6L:18D) photoperiod, the duration of high night time ocular melatonin levels was correlated to the length of the dark phase. In all experiments the high ocular melatonin concentrations and the close parallelism observed between ocular and circulating melatonin profiles suggest that in this species melatonin could be released from the eyes in the general circulation.

Presence and actions of vasopressin-like peptides in the rabbit anterior uvea.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

Radioimmunoassay for thyrotropin-releasing hormone precursor peptide, Lys-Arg-Gln-His-Pro-Gly-Arg-Arg.

A radioimmunoassay for thyrotropin-releasing hormone (TRH) precursor peptide Lys-Arg-Gln-His-Pro-Gly-Arg-Arg (pro-TRH), has been developed. Anti-pro-TRH antibody was raised in rabbits immunized with a conjugate of synthetic pro-TRH analog, Cys-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Cys (pCC10) to bovine serum albumin. This antibody did not cross-react with TRH, TRH-OH, His-Pro-diketopiperazine, neuropeptides, pituitary hormones and peptides which are included in prepro-TRH. Radioiodination of pCC 10 was performed by chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Pro-TRH was extracted from tissues, using 1.0 N acetic acid. The assay was performed with a double antibody system. The values are expressed as an equivalent of pCC 10. The dilution curves of acetic acid-extracts of rat hypothalamus and stomach in radioimmunoassay system were parallel to the standard curve. The recovery of tissue pro-TRH was 80%, the intra-assay and interassay variation was 5.2% and 8.9%, respectively. The elution profiles of acetic acid-extracts of the rat hypothalamus and stomach on Sephadex G-50 showed a single peak corresponding that of pCC 10. Immunoreactive pro-TRH was found in the rat brain, spinal cord, eye, stomach, intestine, pancreas and adrenal gland. These data suggest that this assay system is a suitable to measure pro-TRH in the tissues, and that pro-TRH is widely distributed in the rats.