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PubMed2001

Retinal dysfunction in cancer-associated retinopathy is improved by Ca(2+) antagonist administration and dark adaptation.

PURPOSE: It was recently found that recoverin acts as an autoantigen recognized by sera of patients with cancer-associated retinopathy (CAR), and that CAR-like retinal dysfunction is produced by intravitreous administration of anti-recoverin antibody in Lewis rat eyes. To examine the pathologic molecular mechanism of CAR, and to elucidate an effective therapy for CAR, the function and morphology of CAR were compared with those of phototoxic retinal damage, another form of photoreceptor dysfunction, and the effect of nilvadipine, a Ca(2+) antagonist, on the retinal degenerations was studied, using these models. METHODS: Under different illumination conditions and/or medication with nilvadipine, the functional and morphologic properties of the retinas were evaluated after intravitreous injection of anti-recoverin antibody into Lewis rat eyes (six rats, 12 eyes in each experimental condition), using electroretinogram (ERG), rhodopsin phosphorylation, and light microscopy. RESULTS: Anti-recoverin antibody administered into the vitreous of Lewis rat eyes induced a significant decrease and increase of ERG responses and rhodopsin phosphorylation levels, respectively, under cyclic or continuous light. Similar changes were observed in eyes of rats bred under continuous illumination that did not receive anti-recoverin antibodies. However, anti-recoverin antibody-induced retinal dysfunctions were not observed in rat eyes under dark conditions. Administration of nilvadipine, a Ca(2+) antagonist, to the anti-recoverin antibody-treated rats and rats with phototoxic retinal dysfunction caused significant improvement of the deterioration of ERG and normalization of rhodopsin phosphorylation. CONCLUSIONS: The present data indicate that anti-recoverin antibody-induced retinal dysfunction was functionally similar to phototoxic retinal dysfunction and was markedly suppressed under dark conditions or by systemic administration of a Ca(2+) antagonist.

Animals↗
PubMed2002

Depletion of cholinergic amacrine cells by a novel immunotoxin does not perturb the formation of segregated on and off cone bipolar cell projections.

Cone bipolar cells are the first retinal neurons that respond in a differential manner to light onset and offset. In the mature retina, the terminal arbors of On and Off cone bipolar cells terminate in different sublaminas of the inner plexiform layer (IPL) where they form synapses with the dendrites of On and Off retinal ganglion cells and with the stratified processes of cholinergic amacrine cells. Here we first show that cholinergic processes within the On and Off sublaminas of the IPL are present early in development, being evident in the rat on the day of birth, approximately 10 d before the formation of segregated cone bipolar cell axons. This temporal sequence, as well as our previous finding that the segregation of On and Off cone bipolar cell inputs occurs in the absence of retinal ganglion cells, suggested that cholinergic amacrine cells could provide a scaffold for the subsequent in-growth of bipolar cell axons. To test this hypothesis directly, a new cholinergic cell immunotoxin was constructed by conjugating saporin, the ribosome-inactivating protein toxin, to an antibody against the vesicular acetylcholine transporter. A single intraocular injection of the immunotoxin caused a rapid, complete, and selective loss of cholinergic amacrine cells from the developing rat retina. On and Off cone bipolar cells were visualized using an antibody against recoverin, the calcium-binding protein that labels the soma and processes of these interneurons. After complete depletion of cholinergic amacrine cells, cone bipolar cell axon terminals still formed their two characteristic strata within the IPL. These findings demonstrate that the presence of cholinergic amacrine cells is not required for the segregation of recoverin-positive On and Off cone bipolar cell projections.

Acetylcholine↗
PubMedDate not supplied

[An immunohistochemical study of localization of the calcium-binding protein recoverin in retina of the newt Pleurodeles waltl].

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studied of retina regeneration in newts.

Animals↗
PubMed2002

The canine Recoverin (RCV1) gene: a candidate gene for generalized progressive retinal atrophy.

PURPOSE: We describe the cloning, sequence, and mutation analysis of the canine Recoverin (RCV1) gene, a candidate gene for generalized progressive retinal atrophy (PRA). METHODS: The gene was isolated from a genomic lambda Fix II library using an exon 1 probe of the human RCV1 cDNA. Canine RCV1 sequences were identified by subcloning, polymerase chain reaction (PCR), and sequence analysis. Furthermore, selected DNA samples of 22 dog breeds (including all PRA-affected and several representative unaffected dogs from the pedigrees) were screened for mutations and polymorphisms using PCR-SSCP (single strand conformation polymorphism) and sequence analysis. RESULTS: The canine RCV1 gene revealed 3 exons and an open reading frame of 606 bp, potentially coding for a protein of 202 amino acids. The deduced amino acid sequence of the canine RCV1 gene shares 89% identity with the homologous human, 94% with bovine, and 91% identity with the mouse genes. The protein sequence reveals two typical Ca2+-binding EF-hand motifs. In the ORF (open reading frame) of the RCV1 gene a C272A (exon 1) and a C4275A transversion (exon 3) were discovered. These exchanges result in amino acid substitutions (N3K and P202H), but they do not segregate with PRA in the breeds investigated. Additionally, two sequence variations were identified in the 5'-UTR, one in intron 1 and thirteen variations in intron 2 as well as one in the 3'-UTR. CONCLUSIONS: Using intragenic polymorphisms, we excluded the RCV1 gene as a candidate gene for autosomal recessively transmitted (ar) PRA in 16 dog breeds. In addition the RCV1 gene was excluded for presumedly autosomal dominant (ad) PRA in 8 out of the 22 dog breeds investigated.

Amino Acid Sequence↗
PubMed2003

Retarded outer segment development in TrkB knockout mouse retina organ culture.

PURPOSE: To determine the effects of trkB deficiency in the mouse retina on photoreceptor development and retinal organization, in the absence of confounding systemic effects. METHODS: Newborn mice that carried two null trkB alleles (trkB-/-) and their wild type (WT) littermates were used for retinal organ cultures. On Day 21, rod development was assessed histologically in plastic sections (outer segment length) and retinal organization was analyzed using retinal cell-type specific antibodies. Anatomical data obtained from the organ cultures were compared to previously published histological results from in vivo data. RESULTS: (1) Rod outer segment length was significantly shorter in retinas from trkB-/- mice in the presence of normal numbers of rods. (2) No dopaminergic amacrine cells were observed in the knockout retina. (3) Unlike in the in vivo condition, recoverin-positive OFF-cone bipolar cells were present in trkB-/- retinas grown in culture. CONCLUSIONS: (1) These results demonstrate that rod outer segment development is compromised in the absence of trkB in the retina. (2) This study further supports our previous conclusion that the elimination of trkB expression alters rod development, because the presence of trkB receptors within the retina is essential for normal rod maturation and not because of confounding systemic effects. (3) More generally, this study stresses the importance of investigating complex phenotypes in gene knockout mice under conditions that isolate the organ under investigation from unrelated systemic variations.

Animals↗
PubMed2003

Ectopic photoreceptors and cone bipolar cells in the developing and mature retina.

An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.

Animals↗
PubMedDate not supplied

[An organotypic culture of the newt retina together with other tissues of the posterior eye segment as a model for studying the effects of the cell adhesion glycoproteins].

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins and the localization of acetylcholinesterase activity were found to appear during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.

Acetylcholinesterase↗
PubMed2003

Overlapping microarray profiles of dentate gyrus gene expression during development- and epilepsy-associated neurogenesis and axon outgrowth.

Neurogenesis and axon outgrowth are features shared by normal nervous system development and certain forms of epileptogenesis. This observation has led to the hypothesis that some aspects of normal development and epileptogenesis have common molecular mechanisms. To test this hypothesis, we have used DNA microarray analysis to characterize gene expression in the dentate gyrus and identify genes exhibiting similar patterns of regulation during development and epileptogenesis. Of more than 8000 sequences surveyed, over 600 were regulated during development or epileptogenesis, and 37 of these were either upregulated or downregulated during both processes. In situ hybridization analysis of a subset of these "commonality genes" confirmed the patterns of regulation predicted by the microarray data in most cases and demonstrated various spatial and temporal patterns of commonality gene expression. Of the 25 named commonality genes in which some functional characteristics are known, 11 have been implicated in cell morphology and axon outgrowth or cellular proliferation and fate determination. This enrichment for candidate plasticity-related genes supports the concept that developmental mechanisms contribute to network alterations associated with epileptogenesis and offers a useful strategy for identifying molecules that may play a role in both of these processes.

Analysis of Variance↗
PubMed2003

Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin.

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.

Anilino Naphthalenesulfonates↗
PubMed1992

The NH2 terminus of retinal recoverin is acylated by a small family of fatty acids.

Recoverin is a recently identified Ca(2+)-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.

Acylation↗
PubMed1992

The cancer-associated retinopathy antigen is a recoverin-like protein.

Cancer-associated retinopathy (CAR) is a rare form of retinal degeneration that occurs in association with certain forms of cancer. CAR patients typically possess high titers of autoantibodies against a specific photoreceptor protein--the 23 kD retinal CAR antigen. The mechanisms involved in the vision loss experienced by CAR patients are not understood, but serologic studies indicate the process could include a series of autoimmune reactions directed at specific components of the retina. Because the retinal CAR antigen is the principal ocular autoantigen involved in the antibody response of CAR patients, characterizing it would contribute to the understanding of putative autoimmune involvement. Serum antibodies from CAR patients have been used to isolate the gene encoding the CAR antigen from a cDNA library of human retina. Nucleotide sequence analysis suggests that the CAR antigen shows approximately 90% homology to the published amino acid sequence of bovine recoverin.

Amino Acid Sequence↗
PubMed2003

Growth of the postnatal rat retina in vitro: quantitative RT-PCR analyses of mRNA expression for photoreceptor proteins.

PURPOSE: To investigate whether previously reported changes in protein expression of middle and long (M/L) and short (S) wavelength cone opsin pigments in cultured retina are correlated with changes in their gene expression. Additionally, to elucidate the importance of a functional retinal pigment epithelium for the development of photoreceptor outer segments. METHODS: Neonatal rat retinas were maintained in culture for 11 days and either fixed in 4% paraformaldehyde for immunohistochemistry or prepared for RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR. S-cone and M/L-cone photoreceptors as well as rod photoreceptors were immunohistochemically identified using specific antibodies. Peanut agglutinin (PNA)-lectin histochemistry was used to identify interphotoreceptor matrix associated with cone photoreceptors. Immunolabeling for ED-1 and RPE65 was performed in combination with PNA-lectin staining to examine interactions between photoreceptor cells and the retinal pigment epithelium. Relative estimates of mRNA expression levels for M/L-opsin, S-opsin, recoverin, and rhodopsin in normal and cultured retina were determined by using quantitative RT-PCR. RESULTS: Strong immunolabeling for recoverin and rhodopsin accumulated in outer segments as well as photoreceptor somata in vitro. Cultured and normal retinas showed similar relative expression levels of recoverin and rhodopsin mRNA. In cultured rat retina, the density of S-cones was high and M/L-cones could not be immunohistochemically detected. However, M/L-cone photoreceptor mRNA was detectable, but at a fourfold lower level in cultured than in vivo retinas. The S-cone photoreceptor mRNA level was almost twofold lower than in vivo. Retinal pigment epithelium cells in cultured specimens showed no RPE65 immunolabeling, but expressed immunolabeling for ED-1 indicating phagocytic activity of these cells in vitro. CONCLUSIONS: We assume that the high density of S-cones and virtually no M/L-cones seen in in vitro retinas might represent an immature stage with numerous S-cones and suppressed transdifferentiation into M/L-cone phenotype. A non-functional relationship between photoreceptor cells and a dysfunctional retinal pigment epithelium may have severe consequences for the development of outer segments.

Animals↗
PubMed1991

Recoverin.

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Calcium-Binding Proteins↗
PubMedDate not supplied

The monoclonal antibody-defined CAR-3 antigen is a serological marker associated with pancreatic carcinoma.

The monoclonal antibody-defined CAR-3 antigen is a new carcinoma associated marker which is expressed on a mucin-like molecule. Serum concentrations of CAR-3 were assayed in 181 patients with carcinomas of different organs, 20 patients with non-carcinomatous malignancies, 123 patients with inflammatory diseases and 150 healthy controls. Serum levels of CAR-3 were significantly increased in 51% of the patients with pancreatic carcinomas, in 60% of patients with biliary tract carcinomas and in about 15% of the patients with carcinomas of the digestive apparatus. Sera from patients with breast carcinomas were negative, as well as sera from patients with melanomas or sarcomas. CAR-3 values in samples from patients with chronic pancreatitis were constantly negative, as were samples from healthy donors. Significant concentrations of CAR-3 were detected in 20% of the sera from patients with acute pancreatitis and in 15% of the sera from patients with cirrhosis. Because of its high specificity for pancreatic carcinomas compared to chronic pancreatitis, CAR-3 seems a promising marker for distinguishing between neoplastic and chronic inflammatory diseases of the pancreas, whose differential diagnosis is difficult.

Adult↗
PubMed1994

Photoreceptor-specific proteins in the mammalian pineal organ: immunocytochemical data and functional considerations.

The mammalian pineal organ contains photoreceptor-specific proteins, whose distribution shows conspicuous variation among different species of mammals. Nevertheless, the following general conclusions can be drawn: immunoreactions for S-antigen and recoverin labeled more pinealocytes than the rod-opsin immunoreaction. The intensity of the recoverin- and S-antigen immunoreactions varied from cell to cell. alpha-Transducin immunoreaction was absent from the pineal organ of all mammals investigated with the exception of the blind mole rat. Immunoreaction for the cyclic GMP-gated cation channel was undetectable in the pineal organ of all mammals investigated. The functional significance of photoreceptor-specific proteins in the mammalian pineal organ remains unknown. It has been speculated that the S-antigen might be involved in adrenergic transduction mechanisms. To test this assumption, we have started to analyze calcium responses of single rat pinealocytes to norepinephrine stimulation using the Fura-2 technique. The cells were subsequently labeled by means of S-antigen immunocytochemistry. These combined investigations showed that variation in S-antigen immunoreactivity is not correlated with differences in the rapid calcium response to stimulation with norepinephrine. It remains to be determined whether cells displaying different intensities of the S-antigen immunoreaction show different cyclic AMP responses to noradrenergic stimulation. Investigations along this line should help to clarify further whether there is indeed a relation between the expression of S-antigen and noradrenergic transduction mechanisms in the mammalian pineal organ.

Animals↗