Comparison of retinal pigment epithelium cell preparations from the bovine eye

Summary Retinal pigment epithelium (RPE) cells were collected from, bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

Comparison of retinal pigment epithelium cell preparations from the bovine eye

Retinal pigment epithelium (RPE) cells were collected from bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

An electron microscope study of nerves in the corneal epithelium

Zusammenfassung Eine elektronenmikroskopische Untersuchung der Nerven des Hornhautepithels der Mäuse hat ergeben, dass die Nervenfasern in tiefen Rinnen in den basalen Epithelzellen liegen. So scheint es, dass die Nervenfasern innerhalb der Zellen liegen, obgleich in Wirklichkeit die Fasern von der Zellmembran umhüllt sind.     

Multinucleated cells in the retinal pigment epithelium: A scanning electron microscopic study

Summary Multinucleated cells in the retinal pigment epithelium of the albino rat are shown with the scanning electron microscope beside normal mononucleated cells. We suggest that such elements are formed during foetal life, owing to the absence of any mitotic activity in adult rats.     

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium

Summary 11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25°C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9×10^–7 M.A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled trans and cis isomers of Vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.     

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25 degrees C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9 X 10(-7) M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled 'trans' and 'cis' isomers of vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.     

Carbonic anhydrase type II in regenerating retinal pigment epithelium. A histochemical study in the rabbit

Type II carbonic anhydrase (CAII) in the cytoplasm of the retinal pigment epithelium (RPE) may contribute to the transport of water and solutes across the RPE. The activity of this enzyme in RPE during its response to damage, e.g., during regeneration, is therefore of interest in understanding retinal disease. Immuno-histochemistry was used to compare CAII activity of normal RPE and RPE experimentally induced to regenerate. In normal rabbits, the RPE stained intensely with a peroxidase-linked antibody specific for human CAII. Regenerating RPE stained less intensely. Within the regenerating epithelium, staining appeared more intense in mature cells than in immature ones, suggesting that CAII activity gradually returns during RPE regeneration.     

Table of replaceable amino acids in proteins

Zusammenfassung Auf Grund vorausgegangener Experimente, besonders über degenerierte Triplettcodons, wird ein Verfahren entwickelt, aus dem sich die Austauschmöglichkeiten von Aminosäuren in Eiweißstoffe ergeben, dann nämlich, wenn im Codon nur je eine Base des Tripletts ersetzt wird.     

Accumulation of oxidized lipid-protein complexes alters phagosome maturation in retinal pigment epithelium

Lipid peroxidation has been implicated in many age-associated disorders including macular degeneration of the retina. We sought to elucidate the mechanism by which accumulation of oxidized LDL (oxLDL) reduces the ability of retinal pigment epithelium (RPE) to process photoreceptor outer segments (OS) as a model of peroxidation-induced disruption of phagocytosis. OxLDL did not reduce the lysosomal hydrolytic capacity of the RPE, but efficiently inhibited processing of various internalized proteins. OxLDL caused a delay in the acquisition of late lysosomal markers by newly formed phagosomes. At the same time, an excessive accumulation of markers of early phagosomal compartments was also observed. The activity of phosphatidylinositol 3-kinase (PI3K) was reduced in phagosomes of the RPE treated with oxLDL. These results suggest that accumulation of oxidized lipid-protein complexes in the RPE impedes phagosome maturation by blocking PI3K recruitment to the phagosomal membrane, leading to delayed processing of internalized OS.Received 24 February 2004; received after revision 12 April 2004; accepted 4 May 2004     

The action of hydrogen peroxide on amino acids in presence of iron salts and its bearing on photolysis of amino acids

Zusammenfassung Es wurde der Abbau von Aminosäuren durch Wasserstoffsuperoxyd in Gegenwart von Eisensalzen untersucht. Papierchromatographische Analyse zeigte, dass dabei aus-Aminobuttersäure-Alanin und Glycin, aus-Alanin und Serin Glycin entsteht. Die Bildung von-Alanin und Asparaginsäure aus-Aminobuttersäure und von Asparaginsäure aus Glutaminsäure konnte ebenfalls festgestellt werden. Diese Resultate werden in Zusammenhang gebracht mit dem Mechanismus der Photolyse von Aminosäuren in Gegenwart von TiO₂.     

Free amino acids in some tissues of marine crustacea

Summary Free amino acids contained in extracts of crustaceans were separated and determined quantitatively by ion exchange chromatography. In the hepatoprancreas of the brachyuran crustaceanCarcinus maenas, glycine, arginine, proline and alanine constituted 78.7% of the total amino acid pool. InEriphia spinifrons andMaja verrucosa, glycine, alanine, lysine and leucine comprised 54.9–66.9% of the total content. In the muscle tissue of the macruran crustaceaPalaemon, glycine, arginine, proline and alanine were the most common free amino acids and comprised 70–95% of the total content. The high concentrations of some amino acids in tissues of crustaceans may play a role in intracellular osmotic regulation and also in the maintenance of ionic hydrogen concentration.Acknowledgments. The author wishes to thank Mr R. Mari for his skillful help in the amino acid analyses and Miss G. Princivalli for typing the mansucript.     

Production of lesions in various tissues by amino acids

Zusammenfassung Es wird der Einfluss einer Aminosäure auf verschiedene Gewebsarten und auch auf die Haut kontrolliert. Unterschiedlich starke, z.T. schwere Degenerationen, Entzündungen und auch Zellauflösungen waren festzustellen. Teilweise kam es zu Riesenzellbildungen oder zu Fibrosen in der Leber. Knochennekrosen und Blutungen wurden festgestellt.

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Thanks toK. T. Givens for carbamate, Hercules Incorporated, Wilmington, Delaware.

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Thanks toG. Armstrong for histological specimens, and toR. Wilder for photographs.

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Thanks to Dr.L. Kiesow for critical review and suggestions.

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This research was supported by NMRI Research Project No. MR041.20.02-6002A3IJ.

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The assertions and opinions contained herein are those of the author and are not to be construed as reflecting the views of the Navy Department or the naval service at large.     

Retinol and retinyl esters in pigment epithelium of rats with inherited retinal degeneration

Summary A comparative study of the retinol and retinyl ester concentrations was performed in the retinal pigment epithelium of the normal and affected rats. Our findings indicate that in dystrophic rat retinol content increases, whereas the amount of retinyl esters is always lower than normal. An hypothesis can be made on the deficiency of enzymic activities which regulate retinol retinol and retinyl esters levels in the pigment epithelium.