Alligator rhodopsin: sequence and biochemical properties.

We sequenced selected peptides of alligator rhodopsin that accounted for about half of the total protein. These sequences were confirmed when the total primary structure of alligator rhodopsin was deduced from the cDNA sequence. Differences in the amino-terminal region, compared to that of bovine rhodopsin, account for failure of cross-reactivity of several anti-bovine rhodopsin monoclonal antibodies. Differences in the carboxyl-terminal region give rise to limited antibody cross-reactivity and may also account for a slightly reduced ability of alligator rhodopsin to be phosphorylated by bovine rhodopsin kinase. Alligator rhodopsin regenerates much faster than bovine rhodopsin. The pseudo-first-order rate constant for alligator rhodopsin regeneration is approximately 25 times that of bovine. Phylogenetic analysis of 17 rhodopsin sequences indicates that the alligator is more closely related to the chicken than to the other species examined.

X-ray microanalysis of ocular melanin in pigs maintained on normal and low zinc diets.

Retinal-choroidal tissues were collected from older sows that were fed either control (100% Zn), intermediate zinc (59% Zn) or low zinc (21% Zn) diets for a 6-month period. The tissues were prepared for elemental energy dispersive X-ray analysis and examined by scanning transmission electron microscopy. Melanosomes of the retinal pigment epithelium and choroidal melanocytes were specifically analysed. The elemental spectra of a specific type of melanosome within a sample region were consistently similar. The elemental spectra of choroidal melanosomes differed slightly from that of RPE melanosomes, having greater levels of calcium, iron and zinc. Changes of the elemental content of both types of melanosomes were observed in the animals that were maintained on low zinc nutrition. Iron and zinc decreased in level, while copper increased. Calcium decreased in the RPE melanosomes of intermediate and low zinc diet animals. However, calcium only decreased in choroidal melanosomes of the low zinc-fed sows, having increased substantially in the intermediate zinc-fed group. Abnormal melanosomes were concomitantly seen in the melanocytes of both the intermediate and low zinc groups.

Neurotransmitter-specific identification and characterization of neurons in the all-cone retina of Anolis carolinensis. II: Glutamate and aspartate.

Immunocytochemical and autoradiographic methods were used to identify neurons in the pure cone retina of the lizard (Anolis carolinensis) that are likely to employ glutamate (GLU) or aspartate (ASP) as a neurotransmitter. GLU immunocytochemistry demonstrated high levels of endogenous GLU in all cone types and numerous bipolar cells. Moderate GLU levels were found in horizontal and ganglion cells. Müller cells and most amacrine cells had very low GLU levels. GLU immunoreactivity (GLU-IR) in the cones was present from the inner segment to the synaptic pedicle. A large spherical cell type with moderate GLU-IR was identified in the proximal inner plexiform layer (IPL). These cells also contain ASP and have been tentatively identified as amacrine cells. Uptake of [3H]-L-GLU labeled all retinal layers. All cone types and Müller cells sequestered [3H]-D-ASP, a substrate specific for the GLU transporter. Anti-ASP labeling was observed in cones, horizontal cells, amacrine cells, and cells in the ganglion cell layer. ASP immunoreactivity (ASP-IR) in the cones was confined to the inner segment. One ASP-containing pyriform amacrine cell subtype ramifying in IPL sublamina b was identified. Analysis of GLU-IR, ASP-IR, and GABA-IR on serial sections indicated that there were two distinct populations of horizontal cells in the Anolis retina: one containing GABA-IR, GLU-IR, and ASP-IR; and another type containing only GLU-IR and ASP-IR. Light GLU-IR was frequently found in GABA-containing amacrine cells but ASP-IR was not. The distinct distributions of GLU and ASP may indicate distinctly different roles for these amino acids. GLU, not ASP, is probably the major neurotransmitter in the cone-bipolar-ganglion cell pathway of the Anolis retina. Both GLU and ASP are present in horizontal cells and specific subpopulations of amacrine cells, but it is unclear if GLU or ASP have a neurotransmitter role in these cells.

Neurotransmitter-specific identification and characterization of neurons in the all-cone retina of Anolis carolinensis, I: Gamma-aminobutyric acid.

The inhibitory amino-acid neurotransmitter, gamma-aminobutyric acid (GABA), was localized in the pure cone retina of the lizard Anolis carolinensis by autoradiographic and immunocytochemical techniques. Uptake of [3H]-GABA labeled horizontal cells, amacrine cells, numerous cells in the ganglion cell layer, both plexiform layers, and the nerve fiber layer. Label in the inner plexiform layer showed distinct lamination. The pattern of GABA immunoreactivity was similar to the pattern of [3H]-GABA uptake, although some differences, particularly in labeling of amacrine and ganglion cells, were observed. Immunocytochemistry revealed endogenous stores of GABA in a set of horizontal cells, amacrine cells, and cells in the ganglion cell layer. Both plexiform layers were labeled by the GABA antisera. Labeling in the inner plexiform layer (IPL) was highly stratified and GABA-immunoreactive strata were present in both sublaminae a and b. Six subtypes of conventionally placed GABA-immunoreactive amacrine cells and one displaced amacrine cell subtype were identified. Three of the six conventional amacrine cell subtypes were of pyriform morphology and three subtypes were of multipolar morphology. GABA-immunoreactive interstitial cells also were observed. Under certain conditions the GABA antiserum labeled the cones. Etching the resin eliminated cone labeling, suggesting that GABA in the cones is present in a labile pool, unlike GABA in horizontal or amacrine cells, or the observed labeling was not due to endogenous GABA. Cones did not demonstrate [3H]-GABA uptake.

Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina.

Differentiation and maturation of the photoreceptor outer segments are key steps in the development of the visual system. Morphological studies presented here show that the cow and human are nearly identical in the timing of outer segment appearance during fetal development, implying that the bovine retina is a good model system for the final stages of human photoreceptor development. To study photoreceptor maturation, rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) were quantified by ELISA in a developmentally staged series of fetal bovine retinas. In addition, their localization within these retinas was determined by immunogold electron microscopy. Rhodopsin, as detected by antibodies directed against either the N- or C-terminal portions of the molecule, is first found at about 5.5 months gestation. It is first detected on the plasma membrane of the immature cilia and on the earliest emergent outer segment membrane, even before organized disk membranes are apparent. In contrast, whereas rhodopsin levels and outer segments are nearly undetectable before 5 months gestation, IRBP accumulates to a significant level (4-5% of the adult) as early as 3 months gestation. Immunogold electron microscopy confirmed this finding, with localization of IRBP predominantly in the subretinal space.

A primate model for age related macular drusen.

A closed colony of semi-free-ranging rhesus monkeys maintained in isolation since 1938 by the Caribbean Primate Research Center (CPRC) is being studied as a model for age related macular drusen. Of examined colony animals 57.7% of the monkeys and 47.3% of their eyes have drusen. The prevalence and severity of drusen are linearly related to increasing age and are significantly higher in specific maternal lineages (matrilines). An electrophysiological estimate indicates loss of function associated with drusen. Prevalence of drusen in CPRC females is almost twice that of males, while the prevalence among CPRC animals in general appears to be several times that of monkeys from continental US facilities. Evidence suggests that the frequency of endstage lesions is also similar to that in human populations. The CPRC matriline monkeys appear to provide the best model yet reported for human age related macular drusen.

A reliable two-dimensional gel electrophoresis procedure for separating neural proteins.

A facile two-dimensional gel electrophoresis procedure has been developed for the analysis of neural tissue proteins which eliminates the serious problems associated with protein insolubility at the point of sample application onto polymerized first-dimension isoelectric focusing gels. This was accomplished by combining the methods of two previously published procedures. Our procedure provides an alternative method to the complex gel systems often employed for less soluble proteins, and yields very reproducible, high resolution separations. This procedure, which is in routine use in our laboratories for the analysis of total proteins extracted from retina and brain, produces protein patterns that are easily compared using both visual and computer-assisted image analysis techniques. Presented here are the results of a set of experiments designed to identify proteins unique to retina. This procedure should be useful to investigators studying protein changes resulting from genetic mutation, development, drug treatment or disease, in neural tissue as well as in virtually all other tissues.

Excitatory amino acid involvement in retinal degeneration.

Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells.

Immunocytochemical identification of outer segment proteins in the rd chicken.

Immunoreactivities of two monoclonal antibodies (MAbs) that recognize cone photopigments were tested in the retinas of congenitally blind retinal degenerate (rd) chicks and compared to normally sighted carrier chicks, heterozygous for the mutation. MAb OS-2 had been previously determined to label rod and most cone outer segment membranes in normal chick retinas and is believed to bind to an epitope that is common to several photopigments in chickens. MAb COS-1 labels specifically middle-to-long-wavelength-sensitive cone photopigments in a number of vertebrate species. In rd chicks MAb OS-2 labeled the same number of rod outer segments at the same densities as carrier chicks. However, cone outer segments were less frequently and significantly less heavily labeled with this MAb at all ages tested (1 day, 1 week and 2 weeks post hatching). MAb COS-1 labeled the same number of cone outer segments in both rd and carrier retinas at 1 day of age, however, those outer segments that were labeled in rd specimens had significantly fewer gold particles on them. At both 1 week and 2 weeks of age, rd chick retinas had a significant reduction in numbers of cone outer segments labeled by COS-1. These findings support the hypothesis that the cone photopigment protein is abnormal in the rd chick model of hereditary blindness and retinal degeneration.

Distributions of elements in the human retinal pigment epithelium.

Distributions of elements above the atomic number of sodium were mapped in the retinal pigment epithelia of eight human eyes. X-ray energy spectra and maps were collected from cryofixed, freeze-dried, and epoxy-embedded tissues using energy-dispersive x-ray microanalysis. All eyes had high concentrations of phosphorus in the nuclei of retinal pigment epithelial cells. Melanosomes were rich in sulfur, zinc, calcium, and iron. Lipofuscin and cytoplasm contained only phosphorus and sulfur in detectable amounts. Drusen, when present, contained phosphorus and calcium. Six eyes had a prominent aluminum peak recorded from melanosomes, nuclei, and Bruch's membrane. In one pair of 90-year-old eyes, small, electron-dense deposits surrounded many melanosomes and contained mercury and selenium. Retinal pigment epithelial melanosomes may bind and accumulate metals and other potentially toxic ions over time, preventing them from reaching the neural retina.

Lipid crystals in malignant melanoma of the choroid.

The crystallization of extracellular lipid (other than cholesterol) in histologic preparation is a rarely observed phenomenon. The following case report describes lipid crystals within a necrotic uveal melanoma. Ultrastructural findings suggest that lipids released from necrotic lipidized melanoma cells were the source of the extracellular deposits. No unique lipids were identified from extracts of the tumor using thin layer chromatography. Energy dispersion microanalysis revealed elemental concentrations within the crystals comparable to intracellular organelles.

Analysis of proteins in developing rd (retinal degeneration) chick retina using two-dimensional gel electrophoresis.

The rd (retinal degeneration) chick possesses an autosomal recessive mutation which results in behavioral and electrophysiological blindness at hatch. Using the technique of two-dimensional gel electrophoresis, we have identified two groups of proteins whose expression in the retina/pigment epithelium/choroid (RET/PE/CH) of +/+, +/rd and rd/rd chicks is related to genotype. The two proteins within Group 1 had an apparent mass (Mr) of 63 kDa and isoelectric points (pI) of 6.48 and 6.55. The four proteins in Group 2 had an apparent Mr of 98 kDa and pI values ranging from 6.08 to 6.25. Quantities of the Group 1 proteins in RET/PE/CH of each of the three types of animals were found to be related to genotype; the amounts of each did not change with development. The expression of the Group 2 proteins in PET/PE/CH was found to change during retinal development. Proteins P98-6.08 (Mr-pI) and P98-6.13 were found in all rd/rd and +/rd RET/PE/CH and in most +/+ embryonic tissues. Proteins P98-6.19 and P98-6.25, which were present in the majority of +/+ embryonic and in all +/+ E21 (hatch RET/PE/CH, did not appear in +/rd tissues until hatch. P98-6.19 and P98-6.25 were never observed in rd/rd RET/PE/CH.

Adult-onset macular degeneration in the Cayo Santiago macaques.

Since 1985 a group from the University of Florida has examined 136 rhesus monkeys from the Cayo Santiago colony. From the sample, 97 are older than nine years (approximately 30 human years) and 39 are younger. Drusen were found in 17% of the younger eyes and in 46% of the older eyes. All animals over 25 years of age had drusen in the central fundus. The incidence of drusen varied from 19-77% between five social groups. Incidence reported in random-source colonies in the continental U.S.A. is about six percent. Compared to near-age matched controls without drusen, selected rhesus exhibited visual resolution losses amounting to two Snellen-lines or more. The end-stage disciform changes and ultrastructural similarities are comparable with human macular disease. Future prospective studies may include therapies, surgical intervention, environmental manipulation and genetic research.

Axonal transport and central visual projections of ganglion cells in congenitally blind chickens.

The rd (retinal degenerate) strain of chicken is an example of a recessively inherited mutation characterized by blindness at the time of hatching, as defined by behavioral and electrophysiological tests. Paradoxically, blind mutants have normal retinal morphology, even at the ultrastructural level. Eventually, however, the entire retina degenerates in this strain, perhaps as a result of disuse atrophy. Results of preliminary studies imply that a defect in the visual transduction cascade in photoreceptor cells is responsible for the lack of vision. As well as being an important animal model for studies on photochemistry and transduction, the rd chicken may afford a paradigm for studies on inner retinal physiology and pathology, as electrical input to this inner neuronal system appears to be absent. In the current study we examined axonal transport (both retrograde and anterograde) in rd retinal ganglion cells and connectivity of ganglion cells to visual centers in the brain and compared these to normally sighted chicks. All visuorecipient nuclei were present in rd animals and appeared normal at the light microscopic level. When 3H-proline was injected into one eye of a blind chicken on the day of hatching, labeled polypeptides or proteins were transported via a fast transport mechanism to the same visual centers in roughly the same quantities as in normally sighted chicks. When horseradish peroxidase (HRP) was injected in the optic tectum of blind and normal 1 day old chicks, this label was transported retrogradely to the soma of retinal ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Macular disease in related rhesus monkeys.

During (January) 1986-(May) 1988, we examined 272 eyes in 136 rhesus monkeys in the closed Cayo Santiago colony of the Caribbean Primate Research Center of the University of Puerto Rico. Seventy-eight eyes were less than 10 years of age. One hundred and ninety-four were aged 10-28 years. The fundi were examined and photographed. Fluorescein angiography was performed in some eyes. Selected cases were evaluated for 'acuity' loss by recording of pattern-evoked retinal and cortical signals. Light and electron microscopy were used to evaluate the pigment epithelium of some animals. Thirty-eight percent of all eyes had posterior pole drusen. Incidence was highly age-related. When late-stage lesions were found, we did not see neovascularization, but late hyperfluorescence was consistent with degenerative scarring and atrophy. Electrophysiology demonstrated moderately reduced acuity in the presence of numerous macular drusen. Electrooculograms were low normal. Histopathology showed changes identical to those reported in human age-related macular degeneration. No eyes less than 10 years of age had confluent drusen or disciform-like lesions. The incidence of drusen in samples of some social groups was much higher than others.

Degenerative changes in maculas of rhesus monkeys.

In the hope of identifying an animal model for age-related macular degeneration (AMD) we undertook a pilot investigation of aged rhesus monkeys. Twenty-nine monkeys from a seminatural colony were examined at the Caribbean Primate Research Center. Macular drusen were found in 74% of the monkey eyes. Alterations of the retinal pigment epithelium within the macula were noted in 45% of the eyes. Fluorescein angiography in selected animals revealed window defects consistent with drusen. None of this sample showed the exudative form of AMD or disciform scarring. One typical monkey underwent special studies including measurement of visual resolution by electrophysiological study of the retinal and visual cortex. Application of human criteria to this animal supported the diagnosis of early AMD. Histopathologic study of one eye by transmission electron microscopy confirmed the presence of drusen with nearly identical ultrastructural features to those found in the human pigment epithelium in AMD.

Conditioned medium-mediated photoreceptor differentiation in retina from embryonic rd chickens.

Retina cells from 8-day rd (retinal degenerate) chicken embryos were cultured in media supplemented with optic lobe conditioned medium (OLCM). The morphogenesis of rd photoreceptors is being described. Several differentiating photoreceptors depicted a membranous sac protruding from the apical end of the cell as revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Such structures were found mostly in 8-day-old cultures supplemented with OLCM and were absent in controls. They resembled rudimentary outer segments emanating from a cilium at the apical inner segments. TEM showed a few stacks of free floating disks within the membranous structure which was suggestive of a rudimentary outer segment development. The possible neurotrophic effect of OLCM on rd retina photoreceptor differentiation is being suggested.

Rhodopsin, vitamin A, and interstitial retinol-binding protein in the rd chicken.

In order to determine whether blindness in the rd strain of Rhode Island Red chickens is due to a defect in the vitamin A (visual) cycle, spectroscopy, high performance liquid chromatography, and immunochemical techniques were used to compare the amounts of rhodopsin, interstitial retinol-binding protein, and vitamin A compounds in the dark-adapted eyes of homozygous rd and heterozygous carriers. In both groups of chickens, (up to 6 weeks post-hatching) the distribution of stored vitamin A differed from other vertebrates (mammals, amphibians, fish) in that more than half of the retinyl palmitate/stearate occurred in the neurosensory retina. The 11-cis isomer accounted for nearly 100% of the retinyl palmitate/stearate in the neurosensory retinas of both groups. In the pigmented layers (pigment epithelium and choroid) the 11-cis isomer amounted to 70.1 +/- 4.2% in the carrier, and 65.1 +/- 2.9% in the rd birds. With respect to their content of rhodopsin, IRBP, retinyl palmitate/stearate and unesterified retinol, (both 11-cis and all-trans isomers) no significant difference could be demonstrated between the eyes of rd and carrier chickens (3 days and 28 days post-hatching). These results therefore demonstrate that the ocular tissues of rd chickens do not lack IRBP, the putative extracellular transport protein for vitamin A, that these tissues synthesize and store the 11-cis isomer of vitamin A, and that the 11-cis isomer is used to form rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)