OBJECTIVE: Changes in fundus autofluorescence (AF) are observed in various retinal disorders. Lipofuscin accumulation within the retinal pigment epithelium (RPE) is a source of fundus AF (FAF); however, the causes of short-term increases in FAF observed in inflammatory conditions or after laser treatment are unknown. Here, we describe an RPE cell culture model that is useful for investigations of FAF.
METHODS: ARPE-19 cells were cultured in 2-well chamber slides. Cells were exposed to isolated rabbit photoreceptor outer segments (POS) to mimic in vivo phagocytic activity. The AF of RPE cells exposed to POS was measured before and after focal coagulation of the cultures. AF was measured over a period of 4 weeks. Cell lysates were examined by two-dimensional (2D) gel electrophoresis and mass spectrometry analysis.
RESULTS: The exposure of ARPE cells to POS did not lead to increased AF; however, after coagulation, cells exposed to POS showed a statistically significant increase in AF (p < 0.05). 2D electrophoresis of the cell lysates revealed changes in 3 proteins. One of these proteins, identified by mass spectrometry as ezrin-radixin-moesin-binding phosphoprotein 50, was reduced in the coagulated cell population.
CONCLUSIONS: We have established an in vitro model of RPE cells in culture that can be used to evaluate the development of AF and changes in cellular proteins that accompany laser photocoagulation.

X-ray diffraction patterns were recorded from isolated single rod outer segments of frog. The outer segments in Ringer\'s solution were exposed to a 6 µm microbeam (15 keV) at the BL40XU beamline, SPring-8. The diffraction pattern demonstrated a remarkable regularity in the stacking and flatness of the disk membranes. The electron density profile calculated from the intensity of up to tenth-order reflections showed a pair of bilayers that comprise a disk membrane. The structure of the disk membrane and the changes in the profile on swelling generally agreed with previous reports. Radiation damage was significant with an irradiation of 5 × 10(5) Gy which is much lower than the known damaging dose on proteins at the liquid-nitrogen temperature.

OBJECTIVE: To determine the anatomical morphology of Stage 1-A impending macular holes characterized by a yellow spot using spectral-domain optical coherence tomography to study the fellow eyes of patients diagnosed with a full-thickness macular hole or eyes with a typical yellow spot with metamorphopsia.
METHODS: Prospective observational case series of 45 patients with a full-thickness macular hole or a foveolar yellow spot with metamorphopsia. Visual acuity assessment, biomicroscopic examination, and spectral-domain optical coherence tomography were performed. Clinical ophthalmologic examinations were repeated, including spectral-domain optical coherence tomography, in the eyes with a foveolar yellow spot at 1-month interval.
RESULTS: We examined 43 fellow eyes of 43 patients with a macular hole and 2 eyes of 2 patients with a foveolar yellow spot. Five fellow eyes had a yellow spot. Spectral-domain optical coherence tomography clearly showed the reflectivity of the perifoveal posterior vitreous detachment with vitreofoveal adhesion in the 7 eyes with a yellow spot; the foveal microstructure had a triangular foveolar detachment of the cone outer segment tip line, of which only 2 eyes (29%) had foveal splits during the observational period, 3 eyes (43%) had a foveal perpendicular line, and 4 eyes (57%) showed progression to vitreofoveal separation before the development of inner splits or pseudocysts. In those 7 eyes, the 2 eyes (29%) with foveal splits progressed to a full-thickness macular hole. The 38 fellow eyes without a yellow spot had no foveolar detachment.
CONCLUSIONS: The foveolar detachment of the cone outer segment tip line might be responsible for the yellow spot seen in Stage 1-A macular holes.

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

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Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (Gt) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of Gt following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent KD of the Gt/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.

The experimental strategies developed in kinetic studies of interactions between RGS9 isoforms with G proteins of the Gi subfamily provide a useful framework for conducting similar studies with essentially any regulator of G-protein signaling (RGS) protein-G-protein pair. This article describes two major kinetic approaches used in the studies of RGS9 isoforms: single turnover and multiple turnover GTPase assays. We also describe pull-down assays as a method complementary to the kinetic assays. The discussion of the strengths and limitations of each individual assay emphasizes the importance of combining multiple experimental approaches in order to obtain comprehensive and internally consistent information regarding the mechanisms of RGS protein action.

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45-75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25-2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.

OBJECTIVE: To determine relative susceptibility to, and regional variation of, light-induced retinal damage in two rhodopsin-mutant rat models of retinitis pigmentosa, using slow- and fast-degenerating lines.
METHODS: Transgenic S334ter (lines 4 and 9) and P23H (lines 2 and 3) rats were reared in dim cyclic light or darkness and then exposed to intense green light for 1 to 8 hours. Sections along the vertical meridian were collected for retinal morphology and photoreceptor morphometry 2 weeks later. Unexposed transgenic and normal Sprague-Dawley rats served as the control. Mean outer segment lengths and outer nuclear layer thicknesses were analyzed as a function of position along the vertical meridian and as averages across that vector.
RESULTS: Rapidly degenerating S334ter-4 retinas, reared in dim cyclic light, exhibited no light-induced damage, whereas retinas in the other sublines sustained damage within a sensitive region in the superior hemisphere. Light-induced damage always involved loss of outer segment membrane and photoreceptors. In some cases, the retinal pigment epithelium and inner nuclear layer were also affected. Potentiation of light-induced damage by dark-rearing was increased by at least a factor of three, and in some sublines the sensitive region was enlarged to include the entire vertical meridian.
CONCLUSIONS: A complex pattern of light-induced damage outcomes was identified in S334ter (sublines 4 and 9) and P23H (sublines 2 and 3) rats. The relative susceptibilities of each subline to damage by light were different, even within the same transgene, but consistent factors included a sensitive region in the superior hemisphere and potentiation by dark-rearing.

Monomolecular films of the membrane protein rhodopsin have been investigated in situ at the air-water interface by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and X-ray reflectivity in order to find conditions that retain the protein secondary structure. The spreading of rhodopsin at 0 or 5 mN m(-1) followed by a 30 min incubation time at 21 degrees C resulted in the unfolding of rhodopsin, as evidenced from the large increase of its molecular area, its small monolayer thickness, and the extensive formation of beta-sheets at the expense of the alpha-helices originally present in rhodopsin. In contrast, when spreading is performed at 5 or 10 mN m(-1) followed by an immediate compression at, respectively, 4 or 21 degrees C, the secondary structure of rhodopsin is retained, and the thickness of these films is in good agreement with the size of rhodopsin determined from its crystal structure. The amide I/amide II ratio also allowed to determine that the orientation of rhodopsin only slightly changes with surface pressure and it remains almost unchanged when the film is maintained at 20 mN m(-1) for 120 min at 4 degrees C. In addition, the PM-IRRAS spectra of rod outer segment disk membranes in monolayers suggest that rhodopsin also retained its secondary structure in these films.