Thesis (Ph. D.)--University of Hawaii at Manoa, 1991. / Includes bibliographical references (leaves 213-225) / Microfiche. / xvii, 225 leaves, bound ill. 29 cm

A 7-9 fold enriched preparation of bovine retinal pigment epithelial cell plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight retinal pigment epithelial cellular adhesion to the rod photoreceptor cells. The contaminating rhodopsin was partially removed by anti-rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or silver. Monoclonal antibodies raised against the retinal pigment epithelial plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was classified as an IgG₃ kappa light chain immunoglobulin. It was shown to be specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins electrophoretically transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper localized the binding site of this antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic site to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 solubilised discs indicating a detergent solubilisation dependence for antigenic site accessibility. Cultures of bovine retinal pigment epithelial cells were started by initial enzymatic isolation followed by recovery in RPMI-1640 culture medium. The retinal pigment epithelial cells established a doubling time of 52 hours until the cells reached culture confluency . The cells also maintained many of their in vivo characteristics such as a high degree of pigmentation and an abundance of microvilli. Cell surface glycoproteins labelled with FITC-Con A, FITC-WGA, and FITC-RCA showed dense and random surface labelling patterns. Fluorescent labels were induced to redistribute to central spots and clear from the cell surface by incubating the labelled cells in buffer for 60 minutes at 37°C. Treatment by the appropriate saccharide inhibitors indicated that the labelled sites had undergone endocytosis by the cell. Continuous labelling experiments indicated that redistribution and internalization is constantly occurring so that previously unlabeled receptors become accessible for labelling. As a result a dense pattern of label on the cell surface was maintained. The protein actin, with apparent Mr =46,000, was detected with rabbit anti-actin antisera labelling of the RPE plasma membrane preparation proteins electrophoretically transferred to nitrocellulose paper. Immunofluorescent labelling using the rabbit anti-actin antisera confirmed biochemical studies that actin was a major component of the bovine RPE cell. The activation of actin filaments may play an important role in the phagocytosis of bovine rod outer segments by retinal pigment epithelial cells in tissue culture. Scanning electron microscopy and transmission electron microscopy have shown that 2 week old bovine retinal pigment epithelial cells in vitro can be induced to recognize, attach, and engulf dark adapted sealed rod outer segments. In summary a monoclonal antibody was raised against a bovine retinal pigment epithelial cell plasma membrane preparation, however, the antibody raised proved to be specific for rhodopsin, a contaminating ROS protein found in the preparation. The monoclonal antibody, designated Rho-5A3, was fully characterized and its antigenic site determined. Finally, bovine retinal pigment epithelial cells grown in tissue culture acted as a model system for studying cell surface components and rod outer segment phagocytosis at the levels of fluorescence, scanning electron microscopy, and transmission electron microscopy. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves 181-186. / Microfiche. / lMaster negative: Microfiche MS33173. / xiii, 186 leaves, bound ill. 29 cm

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2006. / Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

Mammalians process their photoreceptions through lateral eyes; however, non-mammalian vertebrates and invertebrates possess additional extraretinal photoreceptors over their bodies to detect light stimuli. Chromatophores, i.e. dermal specialized pigment cells, play important roles in the regulation of body patterns. Since chromatophores derive from neural crest, they share the common embryonic origin with retina. Recent evidence shows that they are light-sensitive due to opsin expression. In the present study, the expression of seven cone opsins was detected in tilapia caudal fin tissues. Moreover, distinct photoresponses were found in two chromatophore types. Regardless of stimulating wavelengths, melanophores tend to disperse and maintain cell shape at dispersion stage by shuttling pigment granules. Conversely, erythrophores respond to light in a wavelength-dependent manner. The opsin expression profiles of melanophores and erythrophores imply SWS1 and RH2 group genes may play important roles in chromatophore photoresponses. Through measuring photosensitivity, I suggest the two opsins play opposite roles in light-induced translocations of pigment granules within erythrophores: SWS1 for aggregations at UV and short wavelength regions and RH2b for dispersion in middle/long wavelengths. An antagonistic interaction occurs in the overlapping of the absorbance spectra of the two opsins. I also found that the photoresponses take place along with the occurrence of the change of cell membrane potential. In addition, the effect of different light backgrounds (broad spectrum, short wavelength-rich, and red-shifted light conditions) on the photosensitivity of tilapia erythrophores was investigated. I found that the major opsin classes (SWS1 and RH2b) responsible for photoresponses remain constant in three groups of erythrophores. Together, I postulate that melanophores may serve as a light filter in integumentary tissues, and the chromatically antagonistic mechanism enables tilapia erythrophores to sense the subtle change of environmental photic condition and to fine-tune pigmentation. I also investigated the ontogenetic change of photoresponses of rainbow trout melanophores. Distinct photoresponses were found in parrs and smolts. Furthermore, smolt melanophores responded to light in a wavelength-dependent manner. Since the change of coloration and visual system during smoltification of salmonids is regulated by thyroid hormone (TH), I suggest that the development of melanophore photosensitivity is associated to TH as well. / Thesis (Ph.D, Biology) -- Queen's University, 2013-08-27 09:57:22.907

Mammalians process their photoreceptions through lateral eyes; however, non-mammalian vertebrates and invertebrates possess additional extraretinal photoreceptors over their bodies to detect light stimuli. Chromatophores, i.e. dermal specialized pigment cells, play important roles in the regulation of body patterns. Since chromatophores derive from neural crest, they share the common embryonic origin with retina. Recent evidence shows that they are light-sensitive due to opsin expression. In the present study, the expression of seven cone opsins was detected in tilapia caudal fin tissues. Moreover, distinct photoresponses were found in two chromatophore types. Regardless of stimulating wavelengths, melanophores tend to disperse and maintain cell shape at dispersion stage by shuttling pigment granules. Conversely, erythrophores respond to light in a wavelength-dependent manner. The opsin expression profiles of melanophores and erythrophores imply SWS1 and RH2 group genes may play important roles in chromatophore photoresponses. Through measuring photosensitivity, I suggest the two opsins play opposite roles in light-induced translocations of pigment granules within erythrophores: SWS1 for aggregations at UV and short wavelength regions and RH2b for dispersion in middle/long wavelengths. An antagonistic interaction occurs in the overlapping of the absorbance spectra of the two opsins. I also found that the photoresponses take place along with the occurrence of the change of cell membrane potential. In addition, the effect of different light backgrounds (broad spectrum, short wavelength-rich, and red-shifted light conditions) on the photosensitivity of tilapia erythrophores was investigated. I found that the major opsin classes (SWS1 and RH2b) responsible for photoresponses remain constant in three groups of erythrophores. Together, I postulate that melanophores may serve as a light filter in integumentary tissues, and the chromatically antagonistic mechanism enables tilapia erythrophores to sense the subtle change of environmental photic condition and to fine-tune pigmentation. I also investigated the ontogenetic change of photoresponses of rainbow trout melanophores. Distinct photoresponses were found in parrs and smolts. Furthermore, smolt melanophores responded to light in a wavelength-dependent manner. Since the change of coloration and visual system during smoltification of salmonids is regulated by thyroid hormone (TH), I suggest that the development of melanophore photosensitivity is associated to TH as well. / Thesis (Ph.D, Biology) -- Queen's University, 2013-08-27 09:57:22.907

As corujas formam um grupo diversificado, estando presentes em diversos habitats ao redor do globo e têm diferentes padrões de atividade, com espécies diurnas, noturnas e crepusculares. Os fotorreceptores encontrados em corujas são os bastonetes e três classes de cones, levando potencialmente à tricromacia, e as demais camadas da retina mantém a mesma organização de outras aves. O gene LWS tem sido estudado em aves e o pico de absorção espectral da opsina expressa por esse gene está entre 560-570nm. Exceções foram reportadas no melro-preto (P557), pinguim Humboldt (P543) e na corujado- mato (Strix aluco). Entre esses três gêneros, somente as corujas apresentam espécies com diferentes hábitos circadianos. Dessa forma é possível que diferentes adaptações visuais possam ser encontradas em associação com o padrão circadiano. Neste trabalho foi investigada a morfologia da retina e a genética do pigmento visual LWS de cinco espécies de corujas com diferentes ritmos circadianos: Asio clamator, Megascops choliba, Tyto alba (noturnas), Athene cunicularia e Glaucudium brasilianum (diurnas). Um indivíduo de cada espécie foi utilizado nos experimentos. Foi realizada a extração de RNA a partir de uma retina homogeneizada de cada espécie e o RNA mensageiro (mRNA) foi convertido em DNA complementar (cDNA). Partes do gene LWS foram amplificadas utilizado a reação em cadeia da polimerase (PCR) e sequenciadas utilizando a metodologia de Sanger. Cinco sítios importantes para o ajuste espectral da opsina LWS (164,181, 261, 269 e 292) foram analisados e comparados com a sequência de outras aves e da rodopsina bovina, a qual foi referência para determinar as posições dos aminoácidos. No estudo morfológico, foram realizados cortes transversais em criostato de uma retina de cada espécie de coruja. Para a reação de imunohistoquímica foi utilizado o anticorpo Rabbit anti opsin (AB5405) para marcar cones L/M e DAPI marcando núcleos celulares. Também foi realizada a coloração de Hematoxilina-Eosina (HE) para visualizar a organização da retina. A partir das análises morfológicas foi possível observar a presença de cones nas retinas das cinco espécies de corujas, bem como uma organização laminar semelhante a de outros vertebrados. Para todas as espécies estudadas, os resultados da análise de sequência da opsina LWS foram: A164, H181, Y261, T269 e A292. Ao menos para o gene LWS, não foram encontradas diferenças entre espécies diurnas e noturnas de corujas / The owls forms a diverse group present in many habitats around the world and they have different activity patterns, with diurnal, nocturnal and crepuscular species. Photoreceptors found in owls are the rods and three classes of cones that potentially provide trichromacy, and the other retinal layers maintain the same organization of other birds. The LWS gene has been studied in birds and the peak spectral absorption of opsin expressed by this gene is between 560- 570nm. Exceptions were reported on blackbird (P557), Humboldt penguin (P543) and tawny owl (Strix aluco). Among these three genera, only owls have species with different circadian habits. It is therefore possible that different visual adaptations can be found in association with the circadian pattern. In this study the retinal morphology and the genetics of LWS visual pigment of five owl species with different circadinan habits were investigated: Asio clamator, Megascops choliba, Tyto alba (nocturnal), Athene cunicularia e Glaucudium brasilianum (diurnal). One individual of each species was used in the experiments. RNA extraction was performed from a homogenized retina of each species and messenger RNA (mRNA) was converted into complementary DNA (cDNA). Parts of the LWS gene were amplified using the polymerase chain reaction (PCR) and sequenced using the methodology of Sanger. Five important sites for the spectral tuning of the LWS opsin (164, 181, 261, 269 and 292) were analyzed and compared to the sequence of other birds and bovine rhodopsin, which was referenced to determine amino acid positions. In the morphological study, cross - sections were performed in cryostat of a retina of each owl species. For the immunohistochemistry reaction, the rabbit anti-opsin antibody (AB5405) was used to label L / M cones and DAPI labeling cell nuclei. Hematoxylin-Eosin (HE) staining was also performed to visualize the organization of the retina. From the morphological analyzes it was possible to observe the presence of cones in the retinas of the five species of owls, as well as a laminar organization similar to that of other vertebrates. For all species studied, the results of LWS opsin sequence analysis were: A164, H181, Y261, T269 and A292. At least for the LWS gene, no differences were found between diurnal and nocturnal species of owls

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