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PubMed1996

Examining rhodopsin folding and assembly through expression of polypeptide fragments.

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.

Amino Acid Sequence↗
PubMed2005

Sp4 is expressed in retinal neurons, activates transcription of photoreceptor-specific genes, and synergizes with Crx.

To investigate the molecular mechanisms of photoreceptor-specific gene transcription, we examined the role of the neuronal-enriched Sp4 nuclear protein in transcription from the rod-specific beta-PDE and rod opsin gene promoters and compared it to the ubiquitous members of the Sp family, Sp1 and Sp3. Sp4 activates both the rod opsin and beta-PDE promoters, whereas Sp1 activates only the rod opsin promoter and Sp3 activates neither promoter. Interestingly, Sp1 and Sp3 competitively repress Sp4-mediated activation of the beta-PDE promoter. In addition, Sp4, Sp1, and Sp3 each show functional synergy with the photoreceptor-enriched Crx transcriptional regulator on the rod opsin promoter but not the beta-PDE promoter, although Sp4-mediated activation was the most significant. Sp4, Sp1, and Sp3 bind Crx in co-immunoprecipitation experiments, and their zinc finger domains as well as the Crx homedomain are necessary and sufficient for these interactions. Chromatin immunoprecipitation showed that the rod opsin and beta-PDE promoters are targets of both Sp4 and Crx, which further supports Sp4-Crx interactions in vivo in the context of retinal chromatin environment. In situ hybridization and immunohistochemistry demonstrated that Sp4 is abundantly expressed in various neurons of all retinal layers, and thus co-localizes or overlaps with multiple retina-restricted and -enriched genes, its putative targets. Our results indicate that photoreceptor-specific gene transcription is controlled by the combinatorial action of Sp4 and Crx. The other Sp family members may be involved in photoreceptor-specific transcription directly or through their competition with Sp4. These data suggest the potential importance of Sp4 in retinal neurobiology and pathology.

Animals↗
PubMed1988

The spectral properties and photosensitivities of analogue photopigments regenerated with 10- and 14-substituted retinal analogues.

Analogues of 11-cis- and 9-cis-retinal with substitutions at positions 10 and 14 were used to regenerate analogue photopigments with two opsins: that of the transmuted (cone-like) 521-pigment of Gekko gekko and that of the rhodopsin of Porichthys notatus. The spectral absorbances and photosensitivities of the regenerated photopigments were determined and compared, first, between the two systems of analogue photopigments, and second, in the responses to the two opsins. Unlike the 10-fluoropigments, the comparable 14-compounds were significantly red-shifted by 19-30 nm and their sensitivity to light was similar to that of the parent 11-cis- and 9-cis-pigments. These were the results for both analogue pigments. In contrast, the 10-pigments were spectrally located close to the wavelengths of the parent compounds and the photosensitivity was significantly reduced, especially in the case of the 9-cis-analogues. Evidence was obtained for a steric hindrance effect at position 14, for no regeneration was obtained when methyl or ethyl groups were at this carbon. In the 10-substituted retinals, steric hindrance was noted only for the gecko; only the fluorosubstituted, but not the chloro-, the methyl- or the ethyl-substituted, retinals reacted. With the fish opsin, pigments were regenerated with all but the ethyl-substituted retinal. The gecko opsin appears to have a more restricted binding site. Another feature of the gecko was related to the chloride bathochromic and hyperchromic effects, in which the 521-pigment prepared in a chloride-deficient state has a blue-shifted spectrum compared with the spectrum obtained after the addition of chloride, and its extinction is raised by the addition of chloride to give a mean ratio of 1.23 for the two extinctions, one with, the other without, added chloride. The 11-cis-10-F-analogue pigment gave both chloride effects and the hyperchromic ratio was the same as that recorded for the native visual pigment. In contrast, the pigment formed with 11-cis-14-F-retinal gave a hyperchromic ratio significantly greater than 1.23. A similar contrast in the responses to chloride was obtained with the analogue photopigments regenerated with the 9-cis-10-F- and 9-cis-14-F-chromophores. This difference between the two systems is interpreted as the result of a specific configurational feature of the gecko opsin when in the chloride-deficient state that is relevant to the binding of the retinal analogue.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Genetic and environmental variation in the visual properties of bluefin killifish, Lucania goodei.

Animals use their sensory systems to detect information about the external environment in order to find mates, locate food and habitat and avoid predators. Yet, there is little understanding of the relative amounts of genetic and/or environmental variation in sensory system properties. In this paper, we demonstrate genetic and environmental variation in opsin expression in a population of bluefin killifish. We measured expression of five opsins (which correlates with relative frequency of corresponding cones) using quantitative, real-time polymerase chain reaction for offspring from a breeding study where offspring were raised under different lighting conditions. Sire (i.e. genetic) effects were present for opsin found in yellow photopigment. Dam effects were present for opsins that create violet, blue and red photopigment. Lighting conditions affected expression of all opsins except SWS2A and mimicked the pattern found among populations. These results highlight the fact that sensory systems are both plastic and yet readily evolvable traits.

Analysis of Variance↗
PubMed1991

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.

Animals↗
PubMed1958

Human rhodopsin.

Human rhodopsin in aqueous solution has lambda(max). of 493 mmicro and is lower in the spectrum than the rhodopsins of all other known vertebrates, with the exception of certain deep-sea fishes. Its molar extinction is 40,000 +/- 800. Like other rhodopsins, it bleaches to a mixture of opsin and all-trans retinene and is resynthesized by incubating opsin with neo-b (11-cis) retinene. The regenerated rhodopsin has the same lambda(max). as the extracted pigment; this is due, therefore, not to an unusual retinene but to a characteristic human opsin. The regeneration in solution from opsin and neo-b retinene is a second-order reaction with a half-time, at 29.5 degrees C, of about 2.5 minutes. This is much faster than the synthesis of rhodopsin in the living human eye, and faster than human rod dark-adaptation; the rate of both processes in vivo must be limited by reactions which precede the union of neo-b retinene with opsin, the final step in rhodopsin synthesis. In the rods, rhodopsin is virtually in the solid state-highly oriented in close relation with other highly oriented molecules. In this situation its spectrum is displaced toward the red (lambda(max) 500 mmicro) and is narrower than in solution. For light entering the rods axially, rhodopsin has also a considerably increased extinction, some 1.5 times higher than when randomly oriented. The spectrum of rhodopsin in rods agrees well in form and position with the spectral sensitivity of human rod vision, measured at the retinal surface.

Animals↗
PubMed2004

Transgenic mice expressing Cre-recombinase specifically in M- or S-cone photoreceptors.

PURPOSE: To establish lines of transgenic mice that express Cre-recombinase in M- or S-cone photoreceptors for generating cone photoreceptor-specific (conditional) mutants. METHODS: Five kilobases of 5' upstream sequence of the mouse red-green (M) opsin gene or 0.5 kb of the mouse blue (S) opsin gene was cloned into a Cre-expression plasmid. Transgenic mice were generated and characterized, and appropriate lines were established. The Cre-transgenic mice were crossed with ROSA26-lacZ mice (containing floxed beta-galactosidase gene) and analyzed to determine Cre-recombinase activity. RESULTS: Immunofluorescence study showed successful targeting of Cre-recombinase expression to cone photoreceptors. Double staining with anti-Cre antibody and anti-M- or anti-S-opsin antibody revealed specificity of Cre expression in M-opsin- and/or S-opsin-positive photoreceptors. Mating with ROSA26-lacZ mice demonstrated that Cre-recombinase was functionally active in M- or S-cones. CONCLUSIONS: Lines of transgenic mice that specifically express functional Cre-recombinase in M- or S-cones were established in this study. Because mutations in several widely expressed genes lead to photoreceptor degeneration, these transgenic mice should be valuable in generating conditional mutants to investigate the function of various genes specifically in cone photoreceptors.

Animals↗
PubMed2005

Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species.

Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.

Amino Acid Sequence↗
PubMed2005

The primordial, blue-cone color system of the mouse retina.

Humans and old world primates have trichromatic color vision based on three spectral types of cone [long-wavelength (L-), middle-wavelength (M-), and short-wavelength (S-) cones]. All other placental mammals are dichromats, and their color vision depends on the comparison of L- and S-cone signals; however, their cone-selective retinal circuitry is still unknown. Here, we identified the S-cone-selective (blue cone) bipolar cells of the mouse retina. They were labeled in a transgenic mouse expressing Clomeleon, a chloride-sensitive fluorescent protein, under the control of the thy1 promoter. Blue-cone bipolar cells comprise only 1-2% of the bipolar cell population, and their dendrites selectively contact S-opsin-expressing cones. In the dorsal half of the mouse retina, only 3-5% of the cones express S-opsin, and they are all contacted by blue-cone bipolar cells, whereas all L-opsin-expressing cones (approximately 95%) are avoided. In the ventral mouse retina, the great majority of cones express both S- and L-opsin. They are not contacted by blue-cone bipolar cells. A minority of ventral cones express S-opsin only, and they are selectively contacted by blue-cone bipolar cells. We suggest that these are genuine S-cones. In contrast to the other cones, their pedicles contain only low amounts of cone arrestin. The blue-cone bipolar cells of the mouse retina and their cone selectivity are closely similar to primate blue-cone bipolars, and we suggest that they both represent the phylogenetically ancient color system of the mammalian retina.

Animals↗
PubMed1996

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Animals↗
PubMed2003

Organ-specific gene expression in the rhesus monkey eye following intravenous non-viral gene transfer.

PURPOSE: The transfer of exogenous genes to the entire retina and other ocular structures is possible with a vascular route of gene delivery using a non-viral gene transfer method. The present studies examine the extent to which either beta-galactosidase or luciferase expression plasmids are targeted to the retina in the adult rhesus monkey following intravenous administration. In addition, these studies examine the pattern of organ expression of the transgene in the rhesus monkey depending on whether the plasmid is under the influence of a widely expressed promoter, the SV40 promoter, or an ocular-specific promoter, the opsin promoter. METHODS: The plasmid DNA with either the SV40 or opsin promoter is encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs), which are targeted across the blood-retinal barrier and into ocular cells with a monoclonal antibody to the human insulin receptor. Following a single intravenous injection of the PIL carrying the transgene, the animals were sacrificed 2, 7, or 14 days later for the measurement of beta-galactosidase or luciferase gene expression in the monkey eye and peripheral organs. RESULTS: Histochemistry showed expression of the beta-galactosidase gene throughout the entire primate retina including the photoreceptor cells with either an SV40 or a bovine opsin promoter. Whereas the SV40 promoter enables gene expression in other organs of the primate (brain, liver, spleen), the opsin promoter restricted trans-gene expression to the primate eye, as there was no gene expressed in other organs. The retinal luciferase activity at 2 days after administration was 9.6+/-0.4 pg luciferase/mg protein, and at 14 days after administration was still comparable to maximal levels of luciferase gene expression in the mouse or rat. Confocal microscopy with antibodies to the insulin receptor and to beta-galactosidase demonstrated co-localization in the retina, with high expression of the trans-gene and the insulin receptor in the inner segments of the photoreceptor cells. CONCLUSIONS: The PIL non-viral gene transfer technology makes possible adult transgenics in 24 h. Ectopic expression of exogenous genes in organs other than the target organ is made possible with the use of organ specific promoters, and gene expression in the primate is restricted to the eye when the trans-gene is under the influence of the opsin promoter. Plasmid-based gene expression is still in the therapeutic range for 2-3 weeks after a single intravenous administration. Exogenous genes are expressed throughout the entire primate retina following the delivery of the gene to the eye via a trans-vascular route.

Animals↗
PubMed1992

Fibroblast growth factor stimulates photoreceptor differentiation in vitro.

Dissociated newborn rat retinal cells were maintained in monolayer culture for periods of up to 11 d. When grown in the absence of exogenous growth factors, 1-2% of the total neuronal population expressed opsin (the photopigment that is specific for maturing photoreceptors). Addition of a single dose of 10 ng/ml basic fibroblast growth factor (bFGF) to the culture medium induced an average increase of sixfold in the numbers of neurons expressing opsin. This supplementation had little effect on the total number of differentiated neurons or of glial cells when measured at the same time points. Furthermore, another specific class of retinal neurons, the amacrine cells, showed no changes following exposure to this growth factor. Two other growth factors known to exert neurotrophic effects, epidermal and nerve growth factor, were without effect. The effect of bFGF was dose dependent, with highly significant differences being observed with as little as 100 pg/ml, and with 700 pg/ml eliciting half-maximal stimulation; maximal effects were observed at 10 ng/ml. Induction of opsin expression by low concentrations of bFGF was blocked completely by an antiserum directed specifically against bFGF, but not by preimmune serum immunoglobulins. This increase in the number of photoreceptors expressing opsin following exposure to bFGF could have been due to either increased cell survival, increased proliferation of progenitor cells, or increased differentiation of immature photoreceptors. There was no increase in overall cell survival under the experimental conditions used, and double labeling immunocytochemistry combined with autoradiographic analysis of 3H-thymidine uptake showed that proliferation of neuronal precursors was not enhanced by the addition of bFGF. In contrast to these observations, cultures established from older (postnatal day 3) retina revealed large numbers of opsin-expressing photoreceptors in all culture plates, with or without added growth factors. This reduction in the stimulatory effects of bFGF with increasing postnatal age is consistent with the period of sensitivity being limited to the cycling of neuronal precursors. It is possible that a bFGF-like molecule is secreted by neighboring cells such as the retinal pigmented epithelium, to participate in retinal development and differentiation. To our understanding, this molecule is the first protein identified to influence specifically the differentiation of photoreceptor cells.

Aging↗
PubMed1991

Proper cotranslational insertion of visual rhodopsin into the lipid bilayer occurs in the absence of protein translocation machinery.

The insertion of visual opsin into membranes occurred during in vitro translation of opsin mRNA in wheat germ extract in the presence of either microsomes or liposomes. The rhodopsin that integrated into both types of membranes after regeneration with 11-cis-retinal was functionally active (in contrast to the nonincorporated protein). Opsin either cotranslationally translocated into microsomes or inserted into liposomes had equal sensitivity to proteolysis and yielded the same pattern of peptides, which differed substantially from the set of peptides produced during proteolysis of opsin not incorporated into membranes. Thus visual opsin does not require protein translocation machinery for proper insertion into the lipid bilayer.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Distribution of membrane proteins in mechanically dissociated retinal rods.

Solitary rods were isolated from frog retinas by mechanical dissociation. Typically, the rods cleave sclerad to the nucleus and consist of outer segments with attached partial inner segments with either tapered or rounded profiles. Light and electron microscopy reveal that the outer and inner segments of rods with tapered inner segments, like rods in the intact retina, are joined by a single connecting cilium. In contrast, the outer and inner segments of rods with rounded inner segments are fused, with no extracellular cleft between the two segments. Opsin distribution was studied in both unfused and fused rods by light and electron microscopic immunocytochemistry. Extensive surface labeling is restricted to the outer segments of tapered rods, as observed in vivo. In contrast, both inner and outer segments of rods with rounded inner segments (fused) label heavily with anti-opsin. Thus opsin, a mobile membrane protein, diffuses from the outer to the inner segment of fused rods. Segregated distribution of opsin in unfused rods suggests that the connecting cilium and/or its associated structures may normally act as a diffusion barrier between the outer and inner segments to mobile membrane proteins such as opsin. Immunofluorescence studies demonstrate that Na+/K+ ATPase is restricted in distribution to the inner segment and calycal processes of both fused and unfused isolated rods, as observed in vivo. Maintenance of its restricted distribution in fused cells indicates that Na+/K+ ATPase is not mobile and may be tethered in the surface membrane of the inner segment.

Animals↗
PubMed1994

Acidification of phagosomes and degradation of rod outer segments in rat retinal pigment epithelium.

PURPOSE: The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, and the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined the effect of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, on the degradation of ROS in the RPE cells in vivo. METHODS: A lysosomal enzyme (cathepsin D), a lysosomal membrane protein (LGP107), and opsin were localized in the RPE cells by the immunogold electron microscopic technique. Bafilomycin A1 was injected into the vitreous, and acidification of the phagosomes was measured in vivo by injecting 3-[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vitreous and detecting the accumulation of DAMP in the phagolysosomes using anti-dinitrophenol antibody. RESULTS: Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabeling of opsin rapidly disappeared soon after the appearance of cathepsin D. By double staining with cathepsin D and DAMP, it was shown that the pH of the phagosomes dramatically decreased after fusion with lysosomes. When bafilomycin A1 was injected into the vitreous, many large phagolysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. CONCLUSIONS: Degradation of opsin and acidification proceeded almost parallel with the appearance of cathepsin D in the phagolysosomes. Bafilomycin A1 did not inhibit the fusion of phagosomes with lysosomes, but it increased intraphagosomal pH and markedly inhibited the degradation of ROS in the phagolysosomes. This result indicates that vacuolar-type H(+)-ATPase is essential for acidifying the lumen of phagolysosomes and subsequent protein degradation of ROS in the RPE cells.

Animals↗
PubMed1998

Spatial and temporal expression of AP-1 responsive rod photoreceptor genes and bZIP transcription factors during development of the rat retina.

PURPOSE: The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl. METHODS: Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellular localization of Nrl was examined with in situ hybridization. RESULTS: The mRNAs for opsin, beta-PDE and c-fos were observed at PN0-2, while cGMP-PDE activity and rhodopsin were detected first at PN5: coincident with rod outer segment development. The developmental pattern of cGMP-PDE activity and rhodopsin accumulation paralleled the expression of beta-PDE and opsin mRNA and all reached their maximal levels by PN45. Nrl expression, for all three transcripts found in the rat retina, was low on PN2 and reached its maximal level at PN14. The c-fos and Nrl expression preceded beta-PDE and opsin mRNA expression by 1-2 days. Nrl expression was detected first in the distal post-mitotic retina at PN5 and then in all nuclear layers during retinal development. Maximal expression shifted from the ganglion cells to the outer nuclear layer as the neural retina matured. In contrast, junD expression was highest at PN0 and declined to a stable level by PN10. CONCLUSIONS: Colocalization of Nrl and c-Fos suggests that expression of rod-specific genes, which utilize AP-1 or NRE sites in their promoter, could be regulated through the formation of Nrl-Fos dimers. We hypothesize that Nrl and c-Fos play a fundamental role in the initiation and regulation of the rod-specific gene expression in developing and adult rod photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Photoreceptor differentiation in cerebellar medulloblastoma: evidence for a functional photopigment and authentic S-antigen (arrestin).

The aim of the present study was to evaluate the putative photoreceptor differentiation found in certain cerebellar medulloblastomas. The analyses were focussed on S-antigen, rod-opsin (the apoprotein of the visual pigment rhodopsin) and 11-cis retinal (the prosthetic group of rhodopsin). Fresh frozen and paraffin-embedded biopsy specimens of three medulloblastomas were investigated by means of immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), high-pressure liquid chromatography (HPLC), and immunoblotting. As shown in paraffin sections, one out of the three tumors (tumor A) contained S-antigen- and rod-opsin-immunoreactive tumor cells. The immunoblotting technique revealed in this tumor a single protein band of approximately 48-50 kDa that reacted with the S-antigen antibody and three protein bands of approximately 40, 75 and 110 kDa recognized by the rod-opsin antibody. These bands could not be detected in the two remaining tumors (tumor B and C). The rod-opsin content of tumor A was quantified by the ELISA; 11.7 pmol rod-opsin were calculated for the biopsy. The HPLC demonstrated the presence of 11-cis- and all-trans-retinal in tumor A, but not in tumors B and C. Furthermore, it was shown that 11-cis-retinal was converted to all-trans-retinal upon illumination of the tumor extract. The ratio between 11-cis- and all-trans-retinal was approximately 1:1 before illumination and 3:5 after illumination. A total of 2-3 pmol of retinal was found in the biopsy of tumor A. In addition all-trans-retinol was present in this tumor. The results indicate that certain medulloblastomas express a functional photopigment and S-antigen, another protein of the phototransduction cascade. They strongly support the concept that medulloblastoma cells may differentiate along the photoreceptor cell lineage.

Antigens↗
PubMedDate not supplied

Identification and distribution of photoreceptor subtypes in the neotenic tiger salamander retina.

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the "red" and "green" rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4+/-1.4% and 37.6+/-1.4% of all photoreceptors, respectively. One rod type comprising 98.0+/-0.7% of all rods showed the immunological and morphological characteristics of "red" rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0+/-0.7% of all rods and possessed the immunological and morphological characteristics of "green" rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8+/-1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4+/-1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8+/-1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.

Animals↗