Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen.

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.

Axonal transport of tritium-labeled putrescinein the embryonic visual system of zebrafish.

The transport of [(3)H]putrescine is demonstrated by autoradiography in the retino-tectal tract of Brachydanio rerio embryos. Transport of [(3)H]putrescine appears to be more rapid than that of tritium-labeled protein and is not inhibited by a colchicine effect on axonal neurotubules as is protein transport. The radioactivity transported to the brain is found, on electrophoresis, in the putrescine fraction.

Elemental analysis of biological specimens in air with a proton microprobe.

The unique capabilities of the proton microprobe in an atmospheric environment as a biological tool are illustrated in studies of arsenic and mercury distributions in siingle strands of hair from poisoning victims and of the distributions of several abundant elements in frozen hydrated eye and kidney specimens from rats.

Structure of a light-adapting hormone from the shrimp, Pandalus borealis.

The structure of a light-adapting hormone of the shrimp, Pandalus borealis, has been determined. The hormone, which had been isolated from Pandalus eyestalks and which adapts the shrimp to brighter light conditions by causing the pigment in the distal retinal pigment cells of the eye to move into a more proximal position, is the peptide: Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH2. The structure was obtained by sequence analysis by the dansyl-Edman method of the intact hormone and of isolated tryptic and thermolytic peptides.

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.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis.

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport.

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Neuropeptide Y and the ocular innervation of rat, guinea pig, cat and monkey.

By the immunohistofluorescence technique, peripheral nerves of the rat, guinea pig, cat and monkey eye contain a neuropeptide Y-like immunoreactive peptide. A broad distribution of immunoreactive nerve fibers is present in all four animals, innervating tissues of the aqueous humor outflow apparatus, the limbal blood vessels, and uveal blood vessels. A dense plexus of neuropeptide Y-like immunoreactive nerve fibers is present to the ciliary processes. A rich innervation exists to the iris dilator muscle, but that to the iris sphincter is modest. Throughout all regions of the uvea, neuropeptide Y-like immunoreactive nerves are associated closely with melanocytes. When acid extracts of anterior uvea and choroid from rat and guinea pig are analyzed by radioimmunoassay and reverse phase high performance liquid chromatography, the immunoreactive ocular peptide occurs in a single molecular form indistinguishable from porcine neuropeptide Y. The present findings indicate that neuropeptide Y is present in ocular nerves of rat, guinea pig, cat, and monkey. Their distribution, with a few small exceptions, closely parallels that of ocular adrenergic nerves as revealed by histofluorometric techniques. While no ocular effects of neuropeptide Y have been reported to date, its other known biological effects imply potential functions in the eye.

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies.

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.

Localization of catecholamines in the eyes and other tissues of Aplysia.

A green fluorescence indicative of catecholamines (CA) was localized in the secondary cells (nonreceptor neurons), neuropile and optic nerve of the eye and other tissues in Aplysia by using the formaldehyde-induced fluorescence method for the demonstration of biogenic amines. The specificity of the induced fluorescence was confirmed by its absence in tissue not exposed to formaldehyde vapor, relatively rapid decay upon exposure to UV light and its chemical reduction by sodium borohydride. The fluorescence was greatly reduced in eyes treated with reserpine (depletes serotonin and catecholamines). Furhter confirmation that the green fluorescence in the eye was due to a CA and not to serotonin was obtained by showing that it was decreased or eliminated by alpha-methyl-para-tyrosine (an inhibitor of catecholamine synthesis), increased by incubation in dopamine and exhibited a peak emission (470 nm) characteristic of CA fluroescence. CA fluorescence was also observed in the neuropiles of the cerebral, pedal, pleural and parieto-visceral ganglia and in cells in the pedal ganglion, statocyst, mantle, anterior tentacles and siphon. The finding of CA in secondary neurons of the eye was unusual since CA-containing cells have not been observed in other gastropod eyes. The distribution of CA in Aplysia, in tissue other than the eye, is similar to that of other gastropod molluscs.

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits.

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.

Autoradiographic localization of beta-adrenergic receptors in rabbit eye.

Using an in vitro autoradiography, beta-adrenergic receptors were localized in the rabbit eye. Autoradiograms were generated by apposition of isotope-sensitive film to slide-mounted eye sections, labelled with [125I](-)Iodocyanopindolol. A high density of silver grains was obtained in conjunctival, corneal and ciliary process epithelium. Binding sites were also present in corneal endothelium, iris epithelium, lens epithelium, choroid and extraocular muscles. In some areas, retina was also labelled. Studies with beta-adrenergic compounds showed that the majority of beta-adrenergic receptors, detectable by autoradiography, were of the beta2 type in the rabbit eye.

Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye.

The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non-human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate electrophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin-4-sulfate and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS.

Ocular renin-angiotensin: immunohistochemical evidence for the presence of prorenin in eye tissue.

Angiotensin II (A2) is a vasoconstrictor generated by the renin-angiotensin system. A2 appears to act also as an angiogenic factor. Recent evidence suggests that renin is synthesized at many tissue sites and may generate A2 locally. Local A2 may have important functions in the normal and diseased eye. We examined eight human eyes by immunostaining with an antibody to prorenin, the biosynthetic precursor of renin. In all eyes, prorenin staining was extensive in the pars plicata of the ciliary body suggesting that the ciliary body synthesizes renin and this renin may be part of an ocular A2 generating system.

Ocular penetration of 5-fluorocytosine.

Ocular penetration of 5-fluorocytosine (5FC) was studied in uninfected rabbits after subconjunctival and oral administration. With oral administration, 5FC achieved therapeutic levels in both the vitreous and aqueous humors. The use of a pharmacokinetic model permitted objective comparison of kinetic events within the eye chambers and the serum. The rates of entry and elimination in the vitreous were found to be slower than those in the aqueous, but the mean concentration over 24 hr was the same. The therapeutic levels achieved in the aqueous after subconjunctival administration were of shorter duration, and no detectable levels occurred in the vitreous. Oral administration is clearly therapeutically superior to subconjunctival administration in this model.

Rhodopsin determinations in C57BL/6J-pallid strain mice.

The influence of light environment on rhodopsin concentration per eye was determined in littermate pigmented and nonpigmented C57BL/6J-pallid gene mice reared under cyclic light or continuous dark environments. Attempts to exacerbate a congenital manganese deficiency in pallid strain mice included dietary deprivation and supplementation with manganese and exposure to intense light followed by the determination of rhodopsin recovery rates in darkness. Homozygous pallid mice (pa/pa) reared in cyclic light had rhodopsin levels which were significantly lower than heterozygous (+/pa) or homozygous (+/+) black control mice. Dark-rearing resulted in a significant increase in rhodopsin per eye in pallid strain mice and equivalent levels in adult mice, but young pallid strain mice did not achieve the same rhodopsin concentration as young +/+ mice. Although dietary manganese deprivation or supplementation did not significantly alter rhodopsin levels among pallid mice, the deficient diet resulted in lower rhodopsin per eye in the young +/+ control animals. The recovery of rhodopsin in darkness following intense light exposure was equal and complete within 24 hr for most genotypes. However, recovery by pallid mice after 24 hr was significantly lower than by pigmented or albino genotypes.

Vitamin A in human eyes: amount, distribution, and composition.

The amount, distribution, and composition of vitamin A stored in the eyes of 29 postmortem donors was determined by a combination of techniques, including high-pressure liquid chromatography. The vitamin A concentration in the pigment epithelium-choroid (RPE-Ch) was the highest observed for human non-liver tissue and amounted to 7.9 +/- 4.3 nmol/eye (n = 28), or 10.4 +/- 7.1 microgram/gm (n = 27). There was no evidence for significant losses during the interval between death and enucleation or during subsequent storage at 4 degrees C. The vitamin A extracted from the retina was 15.3% of that in the corresponding RPE-Ch. By measuring rhodopsin regeneration in retinal homogenates incubated with 11-cis retinal, we estimated that the amount of vitamin A in the RPE-Ch of fully dark-adapted eyes would represent 2.5 mole equivalents of the retinal rhodopsin, a value similar to that found in the frog. A preponderance of the vitamin A in the eye was esterified (98.3% in the RPE-Ch, 79.3% in the retina) and consisted principally of stearate and palmitate in the ratio of 1:4.8. A small amount of oleate was also detected. The ratio of 11-cis isomer over the all-trans averaged 1.52 +/- 0.48 (n - 11). Variable, usually small proportions of 13-cis retinyl esters were also present. Intact RPE-Ch or isolated RPE cells esterified exogenous all-trans-3H2-retinol to the same fatty acids in roughly the same proportions as in the endogenous stores. The all-trans configuration was mainly retained during uptake and esterification, although some isomerization to 13-cis also occurred. No 11-cis isomer was formed under these conditions.

Concentration of 3H-8-methoxypsoralen and its metabolites in the rat lens and eye after a single oral administration.

Concentrations of 3H-8-methoxypsoralen (MOP), its lipid- and water-soluble metabolites, tritiated water, and an insoluble compound have been measured in rat serum, lens, and eye tissue by liquid scintillation, thin-layer chromatography, and other techniques. The radioactivity has been measured in albino and pigmented rats up to 1 week after medication, and 3H-8-MOP and metabolite concentrations have been measured in pigmented rats from 10 min to 24 hr after oral administration by stomach tube of a solution of 3H-8-MOP (1 mg/kg body weight). The animals were kept in the dark. The maximum 3H-8-MOP concentrations were seen 10 min after medication in all the organs examined. The concentrations (in microgram/kg) at 10 min, 2 hr, and 24 hr, respectively, after medication were: serum, 698, 55, and 0.8; lens, 11.3, 5.2, and 1.4; other eye tissue, 393, 23, and 2.5. Water soluble metabolites might accumulate in the lens, but the absolute concentration is small. There was no accumulation of metabolites in the other organs. The considerable amount of radioactivity still present in tissues 1 week after a single medication was a result of the formation of tritiated water from the degraded 3H-8-MOP.

Distinct substance P and calcitonin gene-related peptide immunoreactive nerves in the guinea pig eye.

Using a double labeling indirect immunofluorescent technique, we studied the guinea pig trigeminal ganglion and eye for co-localization of substance P and calcitonin gene-related peptide. In the trigeminal ganglion, the number of neurons immunoreactive for calcitonin gene-related peptide significantly outnumber those immunoreactive for substance P, but virtually all substance P positive neurons are immunoreactive for calcitonin gene-related peptide. In the eye, a complex pattern of co-localization is present; both peptides co-localize in most immunoreactive nerve fibers. Nerve fibers immunoreactive only for calcitonin gene-related peptide tend to be concentrated in the cornea and posterior ciliary body. Nerve fibers immunoreactive only for substance P are present in relation to both iris muscles. Sensory denervation by intracranial transection of the ophthalmic and maxillary nerves fails to eliminate these substance P positive but CGRP negative iris nerve fibers. These findings indicate an alternative origin for substance P immunoreactive nerves supplying the iris muscles in this species.

Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.

The permeability of the blood-retinal and blood-aqueous barriers to fluorescein (F) and the rate of aqueous flow can be estimated by measurements of F in the vitreous, aqueous, and plasma after systemic administration. F is commonly measured by fluorescence, but fluorescein glucuronide (FG), a metabolite of F, also fluoresces. To assess the influence of FG on the quantitation of F by fluorescence, we studied the pharmacokinetics of F and FG for 38 hr in the plasma of five normal subjects given 14 mg/kg of sodium fluorescein intravenously. The plasma and the plasma ultrafiltrate were measured by fluorescence and by high performance liquid chromatography. In our fluorophotometer, FG was 0.124 times as fluorescent as F. F was rapidly converted to FG, and within 10 min the concentration of unbound FG exceeded that of unbound F. The terminal half-lives of F and FG in the plasma ultrafiltrate were 23.5 and 264 min, respectively, so that FG contributed almost all of the plasma fluorescence after 4-5 hr. Because FG was less bound in the plasma than F, the ratio of the fluorescence of the plasma ultrafiltrate to that of the plasma increased with time. The greatest proportion of the total F available to penetrate into the ocular compartments occurred shortly after injection. We concluded that FG is an important contributor to the fluorescence of the plasma ultrafiltrate after intravenous injection and that accurate quantitation of physiologic parameters calculated from the plasma F requires taking this factor into account.