Hirokazu Sakaguchi

Evidence That Light Modulates Protein Nitration in Rat Retina

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4–33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.

Intense light exposure changes the crystallin content in retina

Toward a better understanding of light-induced photoreceptor damage, the crystallin content of rat retina was examined following intense light exposure. Nine crystallin species were identified by mass spectrometric analysis of rat retina fractionated by 2D gel electrophoresis. The Coomassie blue staining intensity of all crystallin 2D gel components was 2- to 3-fold greater in light exposed than in control retinas. Following light exposure, anti-alphaB-crystallin immunoreactivity was increased in rod outer segments and retinal pigment epithelium. These findings support a possible role for crystallins in protecting photoreceptors from light damage.

Claudin localization in cilia of the retinal pigment epithelium

Using immunocytochemistry and confocal microscopy we demonstrate that claudin‐immunoreactivity is a novel marker for retinal pigment epithelial cilia. Claudin‐immunoreactivity obtained by polyclonal anti‐claudin 1 antibody, which could crossreact with claudin 3, was colocalized with acetylated tubulin‐immunoreactivity in cultured human retinal pigment epithelial cells. Claudin‐immunoreactivity associated with the retinal pigment epithelium (RPE) cilia was more intense than was claudin‐immunoreactivity in the junctional complex. Approximately two‐thirds of the RPE cells in the rat contain cilia that are immunoreactive with acetylated tubulin on postnatal day 1, and a significant portion of these cilia label with the anti‐claudin 1 antibody. Cilia decrease in frequency over subsequent postnatal days, and are absent by postnatal day 30. As RPE cilia decrease in number during postnatal rat development, claudin‐immunoreactivity is lost earlier than acetylated tubulin, suggesting that the loss of claudin may initiate RPE cilium degeneration. Claudin‐immunoreactivity was not evident in cilia of photoreceptor cells, epithelia of nasal mucosa, small intestine, or colon, suggesting that claudin may be a unique molecule in RPE cilia. These data suggest that cilia of the RPE, unlike cilia on other cell types, contain claudin, and that this molecule may play an important and specific role in the function and/or maintenance of RPE cilia. Anat Rec 267:196–203, 2002.

Amsler grid examination and optical coherence tomography of a macular hole caused by accidental Nd:YAG laser injury

PURPOSE To compare a macular hole from accidental Nd:YAG laser injury with idiopathic macular holes. METHODS Case report. In a 24-year-old man with accidental Nd:YAG laser injury, right eye, Amsler grid testing and optical coherence tomography were performed. RESULTS Nd:YAG laser injury was responsible for a macular hole about 700 microm in diameter. The visual acuity was 20/100. Amsler grid testing displayed a central scotoma with no surrounding distortion. Optical coherence tomography showed a defect in all retinal layers at the macula. CONCLUSION The scotoma caused by Nd:YAG laser injury is not surrounded by distortion; the hole is produced by the defect of all retinal layers. In contrast, idiopathic macular holes generally produce a pincushion pattern on Amsler grid testing and have no tissue loss.

Annexins in Bruch's membrane and drusen.

Annexins (also known as lipocortins) are a family of calcium and phospholipid-binding proteins. At least 20 members of this family are known, and they have a wide range of potential functions, such as vesicular transport and trafficking, endocytosis, exocytosis and cellcell adhesion. Annexins have molecular weights ranging between 30 and 40kDA (the exception is annexin VI which is 66kDA) and possess striking structural features. To qualify as an annexin, a protein must have 1) the presence of a conserved 70 amino acid domain repeated either 4 or 8 times in the overall structure (annexin VI has an 8 repeating amino acid domain; whereas the rest have 4), 2) the ability to bind phosopholipids in the presence of calcium. Annexins are exported from the cytosol to the exterior of cells across the plasma membrane by an unknown mehanism. When located extracellular, some annexins have been shown to function as receptors for other extracellular proteins: annexin II binds to tenascin and tissue plasminogen activator, while annexin V binds to collagen (Kojima, 1997).