Melanopsin signalling in mammalian iris and retina.

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCß4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCß4-mediated pathway that nonetheless diverges in the two locations.

Detection of c-myc amplification in uveal melanoma by fluorescent in situ hybridization.

PURPOSE: Genetic abnormalities of chromosomal arm 8q have been reported by many studies in uveal melanoma. To better understand the role of 8q abnormalities in uveal melanoma development, copy number anomalies of the c-myc oncogene (located on 8q24.1) have been investigated. METHODS: Forty-three uveal melanomas were analyzed by fluorescent in situ hybridization (FISH) with probes for c-myc and the chromosome 8 centromere. Results of the FISH analysis were compared with genetic changes previously detected by microsatellite analysis on chromosomes 3 and 6p. RESULTS: Thirty uveal melanomas (70%) had extra copies of c-myc, 2 tumors (5%) had loss of c-myc, and 11 tumors (25%) had no abnormalities in c-myc copy number. Of those with extra copies of c-myc, 13 tumors (43%) had amplification of the c-myc gene, 14 tumors (47%) had an intermediate relative increase in the c-myc copy number, and 3 tumors (10%) had a simple gain of chromosome 8. An association between larger tumor size and c-myc amplification was found (P < 0.01). Although extra copies of c-myc were seen in tumors with retention of chromosome 3, remarkably only tumors with monosomy 3 showed amplification of c-myc (P = 0.03). CONCLUSIONS: The specific amplification of the c-myc oncogene detected in at least 30% of primary uveal melanomas cannot be explained by the simple 8q abnormalities observed in cytogenetic studies. The striking association between c-myc amplification and monosomy 3 suggests a unique pathway of genetic progression in a subset of uveal melanomas.

Prevention of prolapsed silicone stents in lacrimal intubation using an intrasac fixation suture.

Silicone stents are routinely used for the maintenance of patent mucosal passages in patients with nasolacrimal disorders. A common complication associated with the use of silicone stents is lateral migration or displacement of the tubes, which can be difficult to correct. This report describes a modified Quickert-Dryden approach with fixation of the tubes by an intrasac suture. From 1990 to 1996, 53 patients had silicone stents placed by this method with no complications related to tube displacement. The intrasac fixation suture has distinct advantages over other fixation methods.

Allelotype of posterior uveal melanoma: implications for a bifurcated tumor progression pathway.

To further elucidate the somatic genetic alterations leading to acquired choroidal and ciliochoroidal melanoma, we screened every autosomal arm and the X chromosome of 50 primary posterior melanomas (31 choroidal tumors and 19 ciliochoroidal tumors). A minimum of two microsatellite markers were used to achieve at least 90% informativity (excluding X). Twenty-eight of 47 informative tumors (59%) showed allelic loss of all informative markers on chromosome 3, consistent with monosomy 3 (M3). Allelic imbalance of 8q was observed in 60% of tumors. A total of 28% of tumors displayed allelic loss of 6p. We then compared these genetic alterations with the status of chromosome 3 and found a relative absence of 6p alteration in tumors with M3 (P = 0.0005). Additionally, all observed 8q imbalance was associated with either M3 or alteration of 6p, suggesting that 8q alterations occur later in tumor progression. The mutual exclusivity of M3 and 6p alterations suggests a bifurcated tumor progression model. In this model, M3 or 6p loss identify distinct pathways, both followed by 8q loss in tumor progression.

Analysis of p16 (CDKN2/MTS-1/INK4A) alterations in primary sporadic uveal melanoma.

PURPOSE: To define more clearly the role of the tumor suppressor gene p16 in uveal melanoma by determining the relative contribution of all known mechanisms of p16 inactivation in this tumor. METHODS: A comprehensive genetic analysis of the p16 gene was performed in 33 primary sporadic ciliochoroidal and choroidal melanomas. Fourteen highly polymorphic microsatellite markers surrounding the p16 locus on chromosome 9p21 were used for the microsatellite analysis. Sequence analysis of the p16 gene was performed on those tumors with 9p21 loss of heterozygosity. To investigate methylation as an alternative mechanism of inactivation of p16, methylation-specific polymerase chain reaction was performed on all tumor DNA samples. RESULTS: Loss of heterozygosity (LOH) was found in 8 of 33 (24%) uveal melanomas. No evidence of a second region of LOH that did not include the p16 locus was found. Four cases had hemizygous losses including markers both distal and proximal to p16. Homozygous deletion of the p16 gene was detected in the 4 remaining cases by microsatellite analysis. Sequence analysis revealed no p16 mutations in the tumors with hemizygous loss of p16. Methylation of the 5' CpG island of p16 was found in one tumor with 9p21 LOH and in another without LOH. CONCLUSIONS: p16 inactivation by HD or methylation occurs in 27% of uveal melanomas, representing the most common molecular genetic alteration identified thus far in uveal melanoma.

Optic nerve avulsion from a diving injury.

PURPOSE: To report a patient with optic nerve avulsion caused by forceful rotation of the globe that occurred when his thumb penetrated the orbit while he was diving. METHODS: A 17-year-old boy was initially examined for sudden loss of vision after jumping feet first from a bridge 50 feet above a river. Upon hitting the water, he felt his right thumb push into his right globe. The patient underwent ophthalmologic and imaging examination. RESULT: Examination disclosed a tear of the optic nerve head from the sclera temporally in the right eye. CONCLUSION: Optic nerve avulsion occurs secondary to forceful rotation of the eye with tearing of the optic nerve as it exits the globe.

In-frame linker insertion mutagenesis of yeast transposon Ty1: phenotypic analysis.

A plasmid bearing a transpositionally functional GAL1::Ty1 fusion was mutagenized by insertion of four or five codons semirandomly throughout the plasmid. This collection of mutant plasmids was introduced into yeast cells and studied with regard to the properties of the mutant Ty1-encoded proteins and the transposition phenotypes observed. All of the transposition-inactivating mutations were previously found to be recessive with the exception of a single mutation in TYA. In this mutant, TYA protein of normal abundance is produced, but the virus-like particles containing this protein are unstable and have aberrant behavior. The effects of mutations in noncoding regions, as well as the capsid protein coding region TYA, and the regions encoding the protease, integrase and reverse transcriptase proteins are described. Effects on gene expression, types of proteins produced, proteolysis of precursor proteins, virus-like particle structure, and biochemical activities of the encoded proteins are summarized. In addition, we show that one of the mutations in the 3' LTR represents a new nonessential site into which foreign marker DNA can be inserted without compromising transposition.

Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments.

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for serine at position 180, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.

Cloning and expression of goldfish opsin sequences.

Five opsin cDNA clones were isolated from a goldfish retina cDNA library and sequenced. On the basis of homology to previously characterized visual pigments, one clone was identified as goldfish rod opsin and a second as a goldfish red cone opsin. Two rhodopsin-like clones were found to be similar to the chicken green opsin, a pigment which shares properties with both rod and cone pigments. A fifth clone was equally homologous to human blue cone opsin and human rod opsin. In order to characterize the spectral properties of the encoded pigments, the five clones were expressed in tissue culture cells and the apoproteins reconstituted with 11-cis-retinal. The wavelength of maximal absorption for goldfish rhodopsin is 492 nm and for the fifth pigment, identified as the goldfish blue pigment, 441 nm. Pigments encoded by the two rhodopsin-like clones absorb at 505 and 511 nm and are likely to correspond to the goldfish green pigment previously characterized by microspectrophotometry. The putative red cone opsin cDNA may encode a pigment that is a polymorphic variant of goldfish red since it absorbs maximally at 525 nm.

Photobleaching difference absorption spectra of human cone pigments: quantitative analysis and comparison to other methods.

Four human cone pigment apoproteins were expressed by transfection of human tissue culture cells with the corresponding complementary DNA clones. Following reconstitution of the cone pigments by incubation with 11-cis retinal, photobleaching difference absorption spectra were obtained for the blue pigment, the green pigment, and two polymorphic variants of the red pigment. These spectra were analyzed to determine the wavelengths of maximal absorbance and the bandwidths. The recombinant cone pigment spectra were compared to human cone spectral sensitivities and cone pigment absorption spectra determined by microspectrophotometry, single-cell electrophysiology, reflection densitometry, electroretinography, and psychophysical color and brightness matching.

Absorption spectra of the hybrid pigments responsible for anomalous color vision.

Unequal homologous recombination events between green and red cone pigment genes produce the red-green or green-red hybrid pigment genes found in many individuals with variant color vision. Photobleaching difference absorption spectroscopy of hybrid pigments produced in cultured cells shows that the spectral sensitivity of each hybrid pigment is intermediate between the parental green and red pigment sensitivities. Amino acids encoded by exons 2, 3, 4, and 5 produce spectral shifts at the wavelength of maximal absorbance of 0 to 4, 0 to 4, 3 to 4, and 15 to 21 nanometers, respectively, the exact value depending on the identities of amino acids elsewhere in the hybrid.

A locus control region adjacent to the human red and green visual pigment genes.

Deletion of sequences 5' of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a beta-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5' of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5' region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Absorption spectra of human cone pigments.

Human colour vision is mediated by three light-sensitive pigments, each found in a different cone-cell type. The absorption spectra of the human cone pigments have been sought for over a century using techniques such as psychophysical colour matching, reflection densitometry, electroretinography, single-cell action spectra and, most directly, microspectrophotometry. We report here a direct determination of the human cone pigment photobleaching difference absorption spectra after the production of each cone pigment apoprotein in tissue culture cells transfected with the corresponding complementary DNA clones. The mean values for the wavelength of maximal absorption are 426 nm for the blue pigment, 530 nm for the green pigment, and 552 nm and 557 nm for two polymorphic variants of the red pigment.