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Purpose: To determine the safety, sensitivity, and specificity of a novel flash photorecovery timing instrument with response verification in differentiating normal from abnormal maculae, and in detecting worsening macular disease., Methods: Right and left eye photorecovery times were determined at baseline and after 5 min using a xenon arc, flash filtered for infrared, ultraviolet, and visible short wavelengths, delivered through an aperture in a hand-held tube. A push-button actuated timer and flash and stopped timer when lighted numbers became visible post-flash. A numeric keypad verified responses. Normal subjects (two eyes tested, n = 144; one eye tested, n = 108) ranged in age from 15 to 84. Photorecovery times were measured in one eye of subjects with small drusen and 20/20 acuity (53-55 correct ETDRS letters; n = 57); in both eyes of subjects with dry age-related macular degeneration (AMD; n = 118); wet AMD with (n = 19) or without (n = 17) macular fluid; and eyes of diabetics with background retinopathy with (n = 19) or without (n = 17) macular retinal thickening. Once-weekly photorecovery measurements for 6 months in each eye of 10 dry AMD subjects and 10 dry diabetic maculopathy subjects provided longitudinal data., Results: Normal subjects' mean right eye recovery time was 9.6 sec (+/- 1.9 SD); left 10.8 sec (+/- 1.0 SD). Photorecovery lengthened after age 55, nearly doubling that of young subjects by age 80. Macular edema, serous macular detachment, or worsened dry AMD were accompanied by prolonged photorecovery (p < .01). When abnormal new vessels or retinal thickening appeared in three serially followed patients, photorecovery at least doubled (p < .01). In all three, photorecovery prolongation occurred without clinical symptoms. None of the 499 tested subjects reported adverse events due to the flash testing., Conclusions: These findings support the usefulness of a reproducible light flash macular vision recovery measurement as an indicator of macular pathology and worsening disease.

Purpose: To test the hypothesis that daily use of zinc-monocysteine (ZMC) supplement will be well tolerated and result in improved macular function in persons with dry age-related macular degeneration (AMD)., Methods: Eligible, consenting subjects were randomized to either ZMC 25 mg or placebo twice daily for 6 months. Both ZMC and placebo groups enrolled 40 participants, with best corrected visual acuity 20/25 to 20/70, macular drusen, and pigment changes. Masked personnel determined baseline, 3- and 6-month best-corrected visual acuity, contrast sensitivity, and light flash recovery time. Differences between ZMC and placebo were analyzed by a one-sided unpaired t-test of the paired differences between baseline and 3- and 6-month timepoints for right and left eyes separately., Results: By 6 months the ZMC group showed improved visual acuity (p < 0.0001) and contrast sensitivity (p < 0.0001). Macular light flash recovery time shortened in the ZMC group at 3 months by 2.1 sec (left eye, p = 0.0001) to 3.6 sec (right eye, p < 0.0001), and at 6 months by 7.2 sec (left eye, p < 0.0001) to 7.4 sec (right eye, p < 0.0001). This variable had no improvement in the placebo group. ZMC had a gastrointestinal irritation rate of under 2%., Conclusion: ZMC 25 mg twice daily was well tolerated and was associated with improved macular function in comparison to a placebo in persons with dry AMD.

The net mass transfer resistance for gas molecules permeating through zeolite membranes includes contributions from intracrystalline diffusion and contributions from interfacial effects. These interfacial effects can arise either from gas-zeolite interfaces or from interfaces that exist within zeolite crystals due to grain boundaries. We present the first atomically detailed simulations that examine interfacial mass transfer resistance due to internal grain boundaries in zeolites that are relevant for membrane applications. Our calculations examine twinned silicalite crystals in crystallographic configurations that have been identified in previous experiments. We used the dual control volume grand canonical molecular dynamics method to simulate the permeance of CH(4) and CF(4) through thin twinned silicalite crystals. The magnitudes of the grain boundary resistances are quite substantial, at least for the thin crystals that are accessible in our simulations.

Carbon nanotubes have significant promise as gas separation membranes. Gas permeation through nanopores involves mass transfer resistances from molecules entering and leaving pores (so-called surface resistances) and diffusion within the pores. We use molecular simulations to give the first estimates of surface resistances for gas transport through nanotubes. For CH4 transport through (20,0) carbon nanotubes at 300 K, surface resistances are small for nanotubes 5-10 mum in length but can be significant for shorter nanotubes.

Purpose: The aim of this study was to document the effect of a novel zinc amino acid combination on the concentrations of important antioxidants in cultured human retinal pigment epithelial (hRPE) cells., Methods: Primary confluent hRPE cells were treated with 30 microM of zinc acetate, zinc chloride, zinc cysteine, and zinc sulfate. The antioxidants catalase, glutathione, glutathione peroxidase, and metallothionein were measured. MTT assays were performed to determine the relative protection of the zinc compounds from the cytotoxic effects of H202 and t-butyl hydroperoxide., Results: Catalase and glutathione peroxidase activities were increased by the zinc formulations compared with the untreated control. Glutathione and metallothionein content were also increased. The greatest increases occurred with zinc conjugated to the amino acid cysteine. The MTT assays showed that zinc monocysteine protected cultured RPE cells from the toxicity of H2O2 and t-butyl hydroperoxide., Conclusions: Our results demonstrate that zinc treatment of RPE cells increases antioxidants and protects cultured RPE cells from the cytotoxic effects of H2O2 and t-butyl hydroperoxide. The results show that zinc conjugated to cysteine offers greater benefits than either zinc salts or cysteine alone.

The diffusive transport of molecules through nanoporous membranes is determined by both intracrystalline diffusion and mass transport resistances associated with entering and leaving the membrane material. We compare two methods for assessing the relative importance of these resistances based on atomically detailed descriptions of the membrane material. For extremely thin membranes, net transport can be assessed using dual control volume grand canonical molecular dynamics (DCV-GCMD). We show that previous implementations of this technique may have been influenced by nonisothermal effects in interfacial regions and suggest a simple remedy to this situation. We also introduce an approximate method that uses information only from equilibrium MD simulations, which avoids the significant computational expense associated with DCV-GCMD. This approximate method can be used to rapidly assess the importance of interface-related resistances to mass transport over broad ranges of membrane operating conditions. This method will be useful in allowing a rapid determination of whether these interface resistances are significant in practical experimental situations. These two methods are compared by considering the transport of CH(4) and CF(4) through defect-free silicalite membranes.

Purpose: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex., Methods: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression., Results: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina., Conclusions: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.

Purpose: Due to a possible link in humans between atherosclerosis, a high-fat diet, and the development of age-related retinal degenerations, we investigated retinal changes with age and diet in high-fat-fed C57BL/6 mice, a mouse model of human atherosclerosis., Methods: We fed C57BL/6J mice either a normal chow diet or an atherogenic diet containing 15% total fat for either 15, 30 or 45 weeks. We sacrificed the animals and examined the eyes using fluorescence microscopy, light microscopy and transmission electron microscopy., Results: At 15 weeks, in high-fat-fed mice, there was an increase in the number of autofluorescent granules in the retinal pigment epithelium (RPE) compared to chow-fed mice. By light microscopy, we noted no remarkable differences in the inner retinal layers between mice fed either diet for 15 or 30 weeks. In contrast, we noted major changes in transmission electron micrographs from the 30 week high-fat group. In the RPE, these included: (1) an increase in the number and size of autophagocitic and empty cytoplasmic vacuoles; (2) accumulations of lipid-like droplets in the cytoplasm, and (3) RPE atrophy. Changes in Bruch's membrane included: (1) thickening; (2) fragmentation of the elastic lamina, and (3) the accumulation of electron dense particulate and vesicular structures within the inner and outer collagenous zones. These changes were not seen in mice fed normal diets even at 45 weeks., Conclusions: We suggest that this model may prove useful for investigations into mechanisms on the effects of diet on the RPE and Bruch's membrane.

Purpose: To determine the effect of moderate zinc deficiency on antioxidant defenses and measures of oxidative stress in the retina and retinal pigment epithelium (RPE) of Brown Norway Rats., Methods: Twenty-four rats were housed individually and divided into three groups of 8 rats each. Group 1 was fed ad libitum a semipurified control diet formulated to contain 50 parts per million [ppm] total zinc; group 2 was fed ad libitum an identical diet but containing 5 ppm total zinc; and group 3 was pair-fed the control diet but restricted in amount to that consumed by group 2. Food intake was measured daily and the rats weighed weekly. After 6 weeks, the rats were killed and the following measurements were made: serum zinc, serum alkaline phosphatase, retinal zinc, RPE-choroid zinc, RPE-choroid catalase, liver metallothionein (MT), retinal MT, RPE-choroid MT, retinal catalase, and retinal thiobarbituric reactive substances (TBARS)., Results: The following showed statistically significant differences between groups 2 and 3, respectively: serum Zn (1216 micro/l versus 1555 microg/l, P < or = 0.01), serum alkaline phosphatase (3.75 U/mg versus 5.10 U/mg, P < or = 0.05), liver MT (4.3 microg/mg protein versus 16.7 microg/mg, P < or = 0.0001), RPE-choroid MT (1.3 microg/mg protein versus 2.2 microg/mg, P < or = 0.02), retinal MT (0.85 microg/mg protein versus 2.8 microg/mg, P < or = 0.05), and retinal TBARS (6.2 nM/mg protein versus 2.2 nM/mg, P < or = 0.05)., Conclusions: The results show that retinal MT and RPE MT concentrations are very sensitive to intake of dietary zinc. The increase in retinal TBARS in group 2 indicates that moderate zinc deficiency increases oxidative stress to the retina. The results also suggest that MT is protective against lipid peroxidation of retinal membranes.

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 microM total zinc, or in medium containing 0.55 microM total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of H2O2 or paraquat on RPE cells. Conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured in RPE cells treated with 0, 0.5 mM H2O2, 10 microM FeSO4 + 0.5 mM H2O2 or 10 microM FeSO4 + xanthine/xanthine oxidase for 24 h or paraquat for 7 d. Oxidized proteins were determined by the formation of carbonyl residues. The antioxidants metallothionein, catalase, superoxide dismutase, and glutathione peroxidase were also measured. The MTT assays showed that zinc protected cultured RPE from the toxicity of H2O2 and paraquat. RPE cells in 0.55 microM zinc medium contained higher levels of TBARS, conjugated dienes and protein carbonyls due to the oxidative stresses, compared to cells in 14 microM zinc. Catalase and MT content were reduced in cells cultured in 0.55 microM zinc medium and were reduced additionally when treated with above stresses. Superoxide dismutase activity increased in 0.55 microM zinc medium in response to these stresses. Our results show RPE cells cultured in zinc-reduced medium are more susceptible to oxidative insult.

Purpose: We have previously shown that an experimental, low-zinc environment decreased catalase activity in cultured human fetal retinal pigment epithelial (RPE) cells. The purpose of this study was to investigate the effect of zinc supplementation on catalase expression in cultured human fetal RPE cells., Methods: Confluent fetal RPE cells incubated in Coon's modified Ham's F12 (CMF-12) were treated (18 h) with zinc chloride (ZnCl2) (15, 30, or 100 microM) to assess changes in catalase enzyme activity or for 6 h to assess the induction of catalase mRNA by Northern analysis and in situ hybridization. RPE cells were also treated with 30 microM ZnCl2 for 2, 6, 24, 48 and 72 h to assess the time course of changes in catalase enzyme activity, changes in mRNA levels and status of the Sp1 transcription factor., Results: Catalase activity was increased above control by the addition of 15, 30 and 100 microM ZnCl2. Catalase gene expression was induced by 30 microM zinc in 6 h, but decreased to non-treated control levels by 24 h. The transcription factor Sp1 was also activated by zinc treatment (30 microM) which peaked at 2 h and declined to non-treated control levels by 24 h. Catalase enzyme activity peaked at 24 h and decreased to control levels by 72 h., Conclusions: Our results demonstrate that zinc treatment of RPE cells increases catalase expression and activates the transcription factor Sp1. The results suggest zinc may play a role in the transcriptional regulation of catalase in RPE cells.

The short-chain fatty acid butyrate has been shown to elevate fetal hemoglobin (HbF) by inducing expression of the gamma-globin gene. Regulation of gene expression by butyrate is thought to proceed via inhibition of the enzyme histone deacetylase, leading to elevated levels of core histone acetylation which affect chromatin structure and transcription rates. To determine whether changes in histone acetylation are critical for the regulation of the gamma-globin gene, we tested three potent and specific inhibitors of histone deacetylase, the cyclic tetrapeptides trapoxin and Helminthsporium carbonum toxin (HC toxin), and the antifungal antibiotic trichostatin A for their ability to induce fetal hemoglobin expression in erythroid cells. These compounds induced fetal hemoglobin in both primary erythroid cell cultures and human erythroleukemia (K562) cells. A butyrate-responsive element spanning the duplicated CCAAT box region of the gamma-globin promoter has been identified in transient transfection assays using a reporter construct in K562 cells, and we show that the same promoter region is required for response to trapoxin and trichostatin. Mutational analysis of the gamma-globin promoter indicates that the distal CCAAT box and 3' flanking sequence (CCAATAGCC) is critical for activation by butyrate, trapoxin, and trichostatin, whereas the proximal element (CCAATAGTC) plays a less important role. These results show that inhibition of histone deacetylase can lead to transcriptional activation of gamma-globin promoter reporter gene constructs through proximal promoter elements, and suggest that butyrate induces gamma-globin expression via such changes in histone acetylation.

Purpose: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells., Methods: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay., Results: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS., Conclusions: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coon's modified Ham's F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbecco's modified Eagle's medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.

Purpose: Reactive oxygen intermediates have been implicated in the aging process and degenerative diseases of the eye, including retinopathy of prematurity, cataractogenesis, and macular degeneration. The purpose of this study was to investigate the effect of phagocytosis of photoreceptor outer segments and the addition of exogenous H2O2 on catalase and metallothionein expression in human retinal pigment epithelial cells., Methods: Confluent RPE cells were treated with bovine photoreceptor outer segments or H2O2 for either 6 or 18 hours. Slot blot hybridization was used to assess catalase and metallothionein gene expression after 6 hours. Catalase enzyme activity and metallothionein content were measured after 18 hours., Results: Phagocytosis or the addition of H2O2 increased catalase enzyme activity and metallothionein twofold above control levels. The addition of n-acetyl cysteine abrogated the inductive effect caused by either stress. Catalase and metallothionein gene expression, measured by slot blot hybridization, also were measurably induced by either stress. Phagocytosis of photoreceptor outer segments increased extracellular H2O2 concentration nine times above control., Conclusions: The response of the retinal pigment epithelial cells to phagocytosis was indistinguishable from the response observed after the addition of exogenous H2O2. The generation of H2O2 during phagocytosis may act as an intracellular signal in retinal pigment epithelial cells that leads to increased levels of key antioxidant enzymes and other proteins important for protecting the cells from oxidative damage.

Pseudohyperkalaemia was detected in four members of a family all of whom have hereditary spherocytosis with normal white blood cells and platelets counts. The degree of pseudohyperkalaemia was related to the time between sampling and cell separation, and inversely related to the temperature in which the sample was left to stand before cell separation. A fifth member of this family was free from both conditions. The association suggests linkage at a membrane level.

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.

This study investigates the mechanism by which insulin stimulates phagocytosis in cultured human retinal pigment epithelium, using a flow cytometric assay of retinal outer segment uptake. RPE cells were isolated from adult human donors and grown in culture. Retinal outer segments were isolated from fresh bovine eyes and covalently labeled with the fluorescent dye carboxy-SNAFL-2. Retinal outer segment binding and uptake was quantified by direct visualization and the increase in cellular fluorescence measured using a flow cytometer and the methods compared. Uptake measured by flow cytometry was further characterized by concentration, time, temperature and serum stimulation, and shown to be comparable to other published methods. Uptake of retinal outer segments was acutely stimulated by insulin in the absence or presence of serum with half maximal stimulation occurring between 0.1 and 1.0 microgram ml-1. Theophylline, forskolin and cholera toxin all reduced retinal outer segment uptake by RPE cells, but had no effect upon insulin stimulation. Measurements of cAMP showed that insulin did not change intracellular cAMP concentration compared to controls in the absence or presence of added retinal outer segments. One hundred nanomolar okadaic acid also inhibited retinal outer segment uptake but not insulin-stimulated uptake, nor did 100 microM genistein have an effect upon insulin-stimulated uptake. Preincubation of RPE cells with 25 microM ZnCl2 overnight stimulated retinal outer segment uptake and appeared to inhibit the insulin stimulation. Preincubation of the cells in 25 mM glucose overnight also increased the uptake of retinal outer segments over control and reduced the effect of insulin. We conclude that insulin stimulates retinal outer segment phagocytosis by an as yet unknown process which may involve specific tyrosine phosphatases.

We studied the use of human retinal pigment epithelial cells cultured on a collagen support as a potential transplantation therapy to replace diseased or damaged retinal pigment epithelium. Using a transvitreal approach, we transplanted human retinal pigment epithelial cells attached to either a sheet of noncross-linked or cross-linked type I collagen into the subretinal space of New Zealand white rabbits, whose eyes lack pigment. Animals were killed after six weeks, and the eyes were fixed for light microscopy. The results demonstrated that, in eyes receiving the noncross-linked collagen support, a layer of pigmented donor retinal pigment epithelium was visible within the subretinal space, with a normal-appearing retina and no evidence of proliferative vitreoretinopathy or graft rejection. We believe this method may be applicable to replace dysfunctional retinal pigment epithelial cells in humans.

Objective: Hyaluronic acid (HA) has a key role in the structure and organization of the extracellular matrix. We sought to identify the distribution of HA in human eye tissue with regard to age using a biotinylated HA-binding protein., Methods: Fetal and adult (from donors ranging from 28 to 94 years of age) eye tissues were fixed less than 24 hours post mortem and embedded in JB-4 medium (Polysciences, Warrington, Pa). Sections of 2-microns thickness were used. Control sections were pretreated either with Streptomyces hyaluronidase or HA-binding protein inactivated by HA. The binding of the protein to HA was detected with avidinbiotin alkaline phosphatase and developed by incubation with naphthol as-mx phosphate and Texas Red Salt (Pierce, Rockford, Ill)., Results: Specific staining for HA was observed in fetal eyes in the choroid, Bruch's membrane, sclera, retinal pigment epithelium, and developing retina from the vitreoretinal interface to the inner plexiform layer. Specific staining decreased with age in the choroid, retinal pigment epithelium, and Bruch's membrane. Hyaluronic acid-specific staining was undetectable in tissues from donors over 50 years of age., Conclusions: The localization of HA in the chorioretinal complex and its disappearance after the fifth decade of life may play a role in aging and age-related retinal disorders.

Purpose: To investigate the possible role of zinc-metallothionein in human retinal pigment epithelium with regard to age-related changes., Methods: A cadmium/heme assay was used to quantitate metallothionein in isolated macular and peripheral retinal pigment epithelium from donors ranging in age from 28 to 91 yr (n = 16, mean age = 68.6 yr)., Results: It was found that peripheral retinal pigment epithelium contained significantly more metallothionein and zinc than macular retinal pigment epithelium. Macular retinal pigment epithelium cells contained 17.6 +/- 2.2 micrograms metallothionein/mg cytosolic protein in donors younger than 70 yr, compared to 5.6 +/- 0.9 in macular retinal pigment epithelium from donors older than 70 yr, a 68% decline (P = 0.0007). In cultured retinal pigment epithelium, when we lowered the zinc concentration in the medium, metallothionein was reduced by 72% after 1 wk of incubation., Conclusions: It is suggested that lower levels of metallothionein in retinal pigment epithelium are caused by reduced metallothionein gene activity or a faster rate of protein degradation, both of which are known to be regulated, at least partly, by bioavailable zinc.

Human retinal pigment epithelium (RPE) contains two genetically distinct forms of superoxide dismutase (SOD) enzymes that scavenge harmful superoxide anions. Biochemical and immunochemical techniques were used to compare levels of copper-zinc- and manganese-containing forms of SOD (CuZn-SOD and Mn-SOD) in human adult and fetal RPE cells. It was found that Mn-SOD activity was higher in adult than fetal RPE cells, both in vivo and in vitro. Immunolocalization of Mn-SOD in cultured RPE cells showed a greater reactivity in the mitochondria of the adult cells. Primary cultures of adult RPE contained cells with various patterns of mitochondria as shown by immunolabeling for Mn-SOD. Adult RPE cells were more resistant to the effects of a superoxide generator, paraquat, which appeared to disrupt mitochondrial integrity as judged by staining with rhodamine 123. These results suggest that high levels of Mn-SOD protect mitochondria from oxidative damage that probably occurs with aging in the RPE.

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

The retinal pigment epithelium (RPE) plays several important roles in the continual support and renewal of photoreceptor outer segments. In the present study, we have demonstrated that RPE cells contain a low molecular weight protein with a high capacity for zinc binding that is dependent on available sulfhydryl groups. This protein is inducible by a 24 hour incubation of cultured RPE in medium supplemented with zinc, cadmium, or dexamethasone. The induction of this protein is correlated with an increased capacity for zinc-65 uptake into cultured RPE. Analysis with a cDNA probe specific for the human metallothionein II gene corroborated the existence and induction of metallothionein gene products in RPE cells. Based on these properties, we have identified this protein as metallothionein. The induction of metallothionein likely has a critical influence on the zinc economy of the RPE.

Retinal pigment epithelium (RPE) isolated from 13 of 49 eyes of donors with insulin-dependent diabetes produced viable, proliferating cultures. No difference was found in baseline glucose uptake and lactate production, as measured by 13C and 1H nuclear magnetic resonance, between the RPE cells from diabetic donors and those cultured from normal donors. Insulin-mediated stimulation of glucose uptake and lactate production was decreased significantly in the RPE cells from diabetic donors compared with those from normal controls. Oxygen consumption and the percentage of endogenous oxygen consumption from fatty-acid oxidation, determined by using a specific inhibitor, were similar in both groups. Cellular proliferation and endocytosis, measured by liposome uptake, also were stimulated by insulin similarly in both types of cells. These results show that at least one or more mechanisms of insulin action in the RPE, thought to be a noninsulin-dependent tissue, may be altered permanently by chronic insulin-dependent diabetes. This finding may have implications for the pathogenesis of disease in noninsulin-dependent tissues even in patients with tight glycemic control.

The antioxidant enzymes catalase and superoxide dismutase have integral roles in controlling reactive oxygen radicals that can harm cells. In the present study, we quantitated catalase activity in retinal pigment epithelium, retina, iris, and vitreous from human donors. To our knowledge, our results represent the first quantitation of catalase activity in human retinal pigment epithelium and show six-fold greater catalase activity in retinal pigment epithelium than in other ocular tissues analyzed (P less than .0001). To investigate whether aging or macular degeneration affects retinal pigment epithelium catalase or superoxide dismutase activities, we measured enzyme levels in retinal pigment epithelium from donors 50 to 90 years of age with and without evidence of macular degeneration. Superoxide dismutase activity showed no significant correlations with aging or macular degeneration, while catalase activity decreased with age (P less than .02) and macular degeneration (P less than .05) in both macular and peripheral retinal pigment epithelium.

Superoxide dismutase (SOD) molecules occur in all cells exposed to an oxygen-containing environment, including retinal pigment epithelial (RPE) cells. Previous studies of nonhuman RPE have either probed specifically for copper-zinc-containing SOD (CuZn-SOD) or have not distinguished between CuZn-SOD and the SOD molecule that contains manganese (Mn-SOD). The authors used specific enzymatic assays and immunologic probes, both in vivo and in vitro, to show that human RPE cells contain both CuZn-SOD and Mn-SOD. The CuZn-SOD had a diffuse cytosolic distribution, whereas the Mn form was located primarily in the mitochondria. The role of SODs in protecting the chorioretinal complex against oxidative damage and with regard to aging processes is not well understood and warrants further investigation, and the two cellular forms of SOD should be considered in future studies.

The flash evoked ERG is used extensively as a measure of retinal function. To understand better the relationship between extent of retinal damage and the ERG we created multistage laser lesions in one eye each of three Macaca fascicularis monkeys. An argon-dye laser was used to deliver 630 nm energy to cumulative quarter sectors of the temporal hemiretina and the foveal region at two week intervals. Latencies of the a- and b-waves and amplitude from the trough of the a-wave to peak of the b-wave were measured preoperatively and one week after each new lesion. Following the last recording session the eyes were removed and prepared for histological examination. In general, but depending on stimulus condition, the maximum decrease in ERG amplitude (50-70% of normal) occurred following the third or fourth sector lesions. These lesions accounted for a loss of the outer nuclear layer in 18-25% of the entire retinal surface area extending from the edge of the fovea into the anterior equatorial region. Surprisingly, subsequent lesions of the remaining sector and fovea did not result in further reduction of ERG amplitudes. Significant increases in amplitudes were even observed for some stimulus conditions following these final lesions. Latencies of the a- and b-waves were not affected. The fact that in the present study ERG amplitudes recovered, suggest that such values may be tenuous evidence of amount of functional retina in laser-lesioned eyes.

Retinal pigment epithelium (RPE) was isolated from human donors with no known eye disease and normal-appearing fundi and was grown in culture. Population doubling times were measured for fifth passage RPE cells; the mean was 2.25 +/- 0.75 days and showed a correlation with the age of the donor. Metabolic studies were performed by perfusing the cells with 1-[13C]-D-glucose and monitoring the perfusate with magnetic resonance spectroscopy. Glucose uptake was 10.5 +/- 3.3 nmoles/min/mg protein. Lactate production from glucose was 20.0 +/- 5.00 nmoles/min/mg protein. The percentage of glucose through the hexose monophosphate shunt was 21 +/- 2%. Oxygen (O) consumption was measured at 20.1 +/- 4.1 nmoles O/min/mg endogenous and 14.5 +/- 4.8 nmoles O/min/mg with 10 mM glucose. These cells exhibit high rates of lactate production concomitantly with high oxygen consumptions and high flux through the hexose monophosphate shunt.

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [35S]sulfate and either [3H]serine or [3H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 M guanidine-HCl. After dialysis into 8 M urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-beta-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.

Bruch's membrane may provide a selective filtration barrier for nutrients coming from the choriocapillaris to the outer retina. Because proteoglycans have been shown to have structural and filtration properties in other tissues, we have been investigating the deposition of newly synthesized proteoglycans into Bruch's membrane and how these may be affected by aging and pathology. Proteoglycans deposited in human Bruch's membrane were metabolically labelled with 35SO4 and 3H-glucosamine using a whole-eye organ culture system. Labeled proteoglycans were extracted from dissected Bruch's membranes with 4 M guanidine and isolated by ion-exchange column chromatography on DEAE cellulose using a linear salt gradient. These molecules were subsequently chromatographed on Sepharose CL-4B and glycosaminoglycan content characterized by enzymatic and chemical degradation. The elution profiles for proteoglycans remains relatively unchanged with age, although in eyes from donors over age 70 there is a small change in size distribution toward higher molecular weights. However, the proportions of newly synthesized glycosaminoglycans remain unchanged with age, being approximately 75% chondroitin sulfate/dermatan sulfate and 25% heparan sulfate. Bruch's membrane proteoglycans from donors with different retinal pathologies, however, exhibited an increased proportion of heparan sulfate. Considering the structural and filtration properties of proteoglycans, such alterations could result in abnormal functioning of Bruch's membrane that could ultimately affect the maintenance of the outer retina.

The biosynthesis of corneal epithelial proteins and glycoconjugates and their response to vitamin A were measured in vitamin A-deficient and normal rats. The synthesis of specific high-molecular-weight epithelial glycoconjugates was directly related to the levels of vitamin A administered. Protein synthesis, however, remained unaltered. Since vitamin A is known to reverse the keratinization process, the vitamin A-regulated high-molecular-weight glycoconjugates may play a role in modulating the expression of the keratinizing phenotype.

Posterior subcapsular cataracts (PSC) are associated with hereditary retinal dystrophy in the Royal College of Surgeons (RCS) rat model and with human retinitis pigmentosa. The relationship of lens and retinal pathology has never been explained. Previous studies of pink-eyed RCS rats aged 2.5 to 11 months had shown an incidence of cataract of 24% when observed by the unaided eye and 60% by direct ophthalmoscopy, while 40% of rats were considered to have clear lenses. Unlike the retinal degeneration, which appeared in all homozygous animals, cataract seemed not to be predictably associated with the rdy mutation. To test this further, we studied the lenses of rats of different ages with a diagnostic slitlamp. We confirmed that by 8 to 15 months of age, rats fed a diet containing recommended concentrations of all known nutrients for rodents developed cataracts with an incidence of 23% when observed by unaided eye. In addition, opacities were seen in 74% with the indirect ophthalmoscope and 20 D lens; but 100% had at least a "sugar grain" type PSC by slitlamp. The slitlamp-detectable cataract was first seen in some animals by 49 days, and by 56 days all rats examined had bilateral PSC. This is an age at which the rod photoreceptors have degenerated. We concluded that slitlamp-detectable PSC are predictably associated with the retinal dystrophy of the rdy mutation. The RCS rat model may be relevant to a type of retinal degeneration having a constant association of cataract.

The proteoglycans of the Rhesus monkey corneal stroma were characterized by analyzing both radiolabeled proteoglycans synthesized by corneas in organ culture and native corneal proteoglycans obtained by large scale preparations. The analyses indicate that the proteoglycans synthesized in organ culture were similar to, if not identical with, their counterparts in the stroma although they are synthesized in different prportions in vitro than they acumulate in vivo. The corneal stroma contains two proteoglycans. The chondroitin-dermatan sulfate proteoglycan consists of approximately 70% protein and has a Mr = approximately 100,000 to 150,000. It contains one chondroitin-dermatan sulfate side chain of Mr = approximately 55,000. The keratan sulfate proteoglycan consists of approximately 74% protein and has a Mr = approximately -40,000 to 70,000. It contains one or two keratan sulfate side chains with a Mr = approximately 7,000 each. Radiolabeling indicates that both proteoglycans contain glycoprotein-type oligosaccharides as part of their structure.

Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (UV) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with UV light and then incubated with 3HTdR. Autoradiograms were prepared and showed that UV radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process.

Here we compare several methods for predicting oligonucleotide frequencies in 392 kb of yeast DNA. As in previous work on E. coli, a relatively simple equation based on tetranucleotide frequencies can be used in predicting the frequencies of longer oligonucleotides. For example, the mean of observed/expected abundances of 4,096 hexamers was 1.00 with a sample standard deviation of .18. This simple predictor arises by considering each base on the sense strand of yeast to depend only on the three bases 5' to it (a 3rd order Markov chain) and is more accurate in estimating oligonucleotide frequencies than other statistical methods examined. This equation is useful in predicting restriction enzyme fragment sizes, selecting restriction enzymes that cut preferentially in coding vs noncoding regions, and in constructing detailed physical maps of whole genomes. When ranked highest to lowest abundance, the observed frequencies of oligomers of a given length (up to 6 bases) are closely tracked by the predicted abundances of a 3rd or 4th order Markov chain. These ordered abundance curves have a power curve shape with a broad linear range with a sharp break at the top end of the curve. There is also a strong disparity between the most and least abundant oligomer with for example a 79-fold variation between the most and least abundant hexamer. The curves reveal a strong dependence of oligomer frequencies on base composition. Unlike E. Coli, there is no sharp downturn at the low end of the curves and hence, no class of oligomers rare relative to other oligomers of the same length.

Cyclic GMP and cyclic AMP are present in lower concentrations in the central (macular) region of the neural retina of the human and monkey than in other areas. This pattern approximates the distribution of rod photoreceptor cells. Surprisingly, an inverse gradient of cyclic GMP concentration is observed in the pigmented epithelium. Levels in the central region are over fourfold higher than in cells in the periphery, offering the first evidence of biochemical differences in this embryologically uniform cell type.

Cultured human retinal pigmented epithelial cells were studied using phosphorus-31 nuclear magnetic resonance spectroscopy (P-31 NMR). Retinal pigmented epithelial cells from normal human donors were isolated and expanded using roller-bottle culture. The P-31 NMR spectrum of the intact living cells was obtained at 121 MHz by casting the cells in agarose threads and perfusing the threads with culture medium. The cells remained viable at 37 degrees C in the NMR magnet for at least 24 hr, as determined by the stability of the phosphorus spectrum and by trypan blue dye exclusion at the end of the experiments. The intact cell spectrum showed resonances from phosphorylethanolamine, phosphorylcholine, inorganic phosphate, phosphodiesters, phosphocreatine, ATP, NAD+, and UDP-N-acetylglucosamine, with phosphorylethanolamine, phosphorylcholine, and ATP being the major metabolites present. The resonances were assigned by making a perchloric acid (PCA) extract of the intact cells and running under high-resolution conditions. The PCA extract spectrum also detected sugar phosphates, ADP and UTP, with the latter being approximately 20% of the nucleotide pool. These studies provide the basis for the study of normal and diseased human RPE cells by NMR spectroscopic methods.

Sephadex G-25 fractions of chick embryo extract affect the differentiation of cloned embryonic pigmented epithelial cells (PE) in culture. Both tyrosinase activity and amounts of melanin are markedly affected by the extract fractions. 5-Hydroxyindole decreased melanin accumulation in PE cells while alpha-methyl-p-tyrosine had no apparent effect on the process. Melanin disposal as well as biosynthesis may be important in controlling the accumulation of pigment in PE cells under culture conditions. Triiodothyronine greatly increased visible pigmentation and affected the morphology of cultured PE cells.

We used specific antibodies to localize a variety of extracellular matrix components by indirect immunofluorescence reactions on cryostat sections of drusen-bearing human donor eye tissue. These included collagen types I through V, fibronectin, laminin, heparan sulfate-containing basement membrane proteoglycan as well as antibodies to human IgG and IgM. The composition of drusen with respect to these specific molecules varied greatly, even within the same region of an eye. Hard or nodular drusen were more likely to show a discrete organized layer of specific basement membrane molecules over their apical surfaces than were diffuse type drusen. Diffuse but not nodular drusen generally contained fibronectin. Immunohistochemical reactivity of Bruch's membrane showed age-related accumulation of type I collagen and localized changes associated with some drusen.

The plasma membranes of retinal pigment epithelial cells are highly specialized organelles with multiple functions including nutritional and metabolic support of the photoreceptor cells. Using purified bovine retinal epithelial cell plasma membranes as antigen, we produced two monoclonal antibodies, MAbD1-C6 and MAbD1-C8, that cross react with the plasma membranes from bovine, rat and human retinal pigment epithelial cells. In radioimmunoassay both MAbD1-C6 and MAbD1-C8 had similar affinities for bovine plasma membranes. Both monoclonal antibodies identified a protein of 72 Kd with an apparent subunit of 32-35 Kd. The protein was localized to the cell surface of human and bovine retinal pigment epithelium by immunocytohistochemistry. In the normal eye the antigen identified by the monoclonal antibodies was strongly associated with the retinal pigment epithelium and weakly associated with lens tissue. Using either monoclonal antibody, components of purified bovine or rat retinal pigment epithelial plasma membranes were precipitated from solution. Based on these results, we conclude that both monoclonal antibodies are closely related and that they may be useful for the isolation and study of retinal pigment epithelial cell structure.

The capacity of cultured human trabecular meshwork (HTM) cells to secrete an extracellular matrix was studied by indirect immunofluorescence. Synthesis of nine extracellular matrix (ECM) proteins known to be present in the normal trabecular meshwork was assessed in three HTM cell lines. Fourteen primary antibodies were used and cultures were labeled two and four weeks after confluence. The HTM cell lines showed consistent labelling patterns for the normal extracellular connective tissue constituents including collagens (types I, III, IV, V and VI), glycoproteins (laminin and fibronectin) and a basement membrane-associated proteoglycan. These antigens were localized to the basal cell surface in an extracellular reticular pattern corresponding to cell margins. Dextran addition at confluence helped to intensify the staining of these components, but ascorbate had no apparent effect. Interestingly, elastin, another normal component of the trabecular meshwork, was not identified under standard conditions, or after addition of ascorbate or dextran. However, elastin could be detected intracellularly following dexamethasone treatment for three days, and extracellularly in punctate deposits when this treatment was used for 1 or 2 weeks. Our findings indicate that HTM cells may be responsible for the secretion and maintenance of all the major ECM constituents of the trabecular meshwork. The elastin results suggest a possible mechanism contributing to obstruction of outflow in steroid glaucoma if increased amounts of elastin are also produced in vivo. This approach can also serve as a useful baseline for comparison with HTM cell lines treated with glaucoma medications or obtained from patients with glaucoma.

Five patients with mild nonproliferative diabetic retinopathy had visual loss associated with parafoveal telangiectasis. Minimal macular edema with characteristic parafoveal plaques of subretinal pigment epithelial hyperplasia was seen in all patients. Fluorescein angiography revealed the presence of ectatic, dilated, leaking perifoveal capillaries. The occurrence of parafoveal telangiectasis in patients with diabetic retinopathy has, to our knowledge, not been previously described. One previous clinicopathologic report described the histologic appearance of parafoveal telangiectasis in a nondiabetic patient to be similar to that seen in diabetic patients. The association of parafoveal telangiectasis and diabetic retinopathy raises interesting speculations into the pathogenesis of the entity of parafoveal telangiectasis. In addition, the importance of obtaining fluorescein angiography prior to therapy of diabetic macular edema is emphasized.