Kinetic profiles of photocurrents in cells expressing two types of channelrhodopsin genes.

Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550 nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293 cells stably expressing ChR2, photocurrents were elicited at stimuli of 400-550 nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600 nm. However, the photocurrent at 550 nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.

Single-incision Laparoscopic Appendectomy for acute Appendicitis using a 10-mm Laparoscope and the Glove Port Technique.

OBJECTIVE: To evaluate the single incision laparoscopic appendectomy (SILA) using existing instruments, the 10-mm laparoscope, and glove port technique. METHODS: SILA was performed on 16 patients (8 male cases, 8 female cases) between June 2012 and September 2015. A 20-mm incision was made in the umbilicus and a wound retractor was placed. A 10-mm trocar for the laparoscope and two 5-mm trocars were fixed to the three fingers of the latex gloves and it was attached to the wound retractor. Another thin forceps were inserted from right low abdomen. RESULTS: Average age of patients was 32.6 ± 17.7 years. Preoperative average white blood cell was 13,325 ± 4,584 /mm3, and average CRP was 1.81 ± 3.70 mg/dL. Preoperative body temperature was 36.8 ± 0.5°C. The mean appendix size was 9.6 ± 2.3 mm and none of the patients had an abscess on preoperative CT. The CT also revealed a fecal pellet in 5/16 (31%) of patients. Mean operation time was 66.4 ± 25.4 minutes, and minimal intraoperative bleeding was observed in all patients. Average hospital stay was 5.3 ± 1.9 days and none of the patients had complications. CONCLUSION: SILA using the 10-mm laparoscope and glove port technique may be a safe and feasible operation for mild to moderate appendicitis.

Restoration of the majority of the visual spectrum by using modified Volvox channelrhodopsin-1.

We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468-640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

Different anti-oxidant effects of thioredoxin 1 and thioredoxin 2 in retinal epithelial cells.

Age-related macular degeneration (AMD) affects the retina and is the most common cause of blindness in elderly persons in developed countries. The retina is constantly subjected to oxidative stress; to avoid the effects of oxidative stress, retinal pigment epithelial (RPE) cells possess potent anti-oxidant systems. Disruption of these systems leads to dysfunction of RPE cells, which then accelerates the development of AMD. Here, we investigated the role of thioredoxins (TRXs), scavengers of intracellular reactive oxygen species, by assessing the effect of TRX overexpression on cell viability, morphology, NF-κB expression, and mitochondrial membrane potential, in RPE cells. TRX-overexpressing cell lines were generated by infection of an established human RPE cell line (ARPE) with adeno-associated virus vectors encoding either TRX1 or TRX2. We showed that overexpression of TRXs reduced cell death caused by 4-hydroxynonenal (4-HNE)-induced oxidative stress; TRX2 was more effective than TRX1 in promoting cell survival. 4-HNE caused perinuclear NF-κB accumulation, which was absent in TRX-overexpressing cells. Moreover, overexpression of TRXs prevented depolarization of mitochondrial membranes; again, TRX2 was more effective than TRX1 in maintaining the membrane potential. The difference in the protective effects of these TRXs against oxidative stress may be due to their expression profile. TRX2 was expressed in the mitochondria, while TRX1 was expressed in the cytoplasm. Thus, TRX2 may directly protect mitochondria by preventing depolarization. These results demonstrate that TRXs are potent antioxidant proteins in RPE cells and their direct effect on mitochondria may be a key to prevent oxidative stress.

Establishment of monocular-limited photoreceptor degeneration models in rabbits.

BACKGROUND: Numerous rodent models of photoreceptor degeneration have been developed for the study of visual function. However, no viable model has been established in a species that is more closely related to Homo sapiens. Here, we present a rabbit model of monocular photoreceptor degeneration. METHODS: We tested 2 chemicals, verteporfin and sodium nitroprusside (SNP), for developing a 1-eye limited photoreceptor degeneration model in pigmented rabbits. After the intravenous injection of verteporfin, the retina was exposed to light from a halogen lamp for 0, 10, 30, or 60 min. Alternately, 100 µL of various concentrations of sodium nitroprusside (0.1 mM, 0.5 mM, and 1 mM) were intravitreously injected into the rabbit eye. Retinal degeneration was evaluated by fundus photography, electroretinogram (ERG), and histological examinations. RESULTS: Fundus photographs of animals in the verteporfin- or SNP-treated groups showed evidence of retinal degeneration. The severity of this degradation depended on the duration of light exposure and the concentration of SNP administered. The degeneration was clearly limited to the light-exposed areas in the verteporfin-treated groups. Extensive retinal atrophy was observed in the SNP-treated groups. The a- and b-wave amplitudes were dramatically decreased on the ERGs from SNP-treated groups. Histological examination revealed that either verteporfin or SNP induced severe photoreceptor degeneration. High-dose SNP treatment (1 mM) was also associated with inner retinal layer degeneration. CONCLUSIONS: Both SNP and verteporfin clearly caused photoreceptor degeneration without any effect on the contralateral eye. These compounds therefore represent valuable tools for the empirical investigation of visual function recovery. The findings will inform guidelines for clinical applications such as retinal prostheses, cell-based therapy, and gene therapy.

Differentiation of neuronal cells from NIH/3T3 fibroblasts under defined conditions.

We attempted to test whether the differentiated NIH/3T3 fibroblasts could be differentiated into neuronal cells without any epigenetic modification. First, a neurosphere assay was carried out, and we successfully generated neurosphere-like cells by floating cultures of NIH/3T3 fibroblasts in neural stem cell medium. These spheres have the ability to form sub-spheres after three passages, and express the neural progenitor markers Nestin, Sox2, Pax6, and Musashi-1. Second, after shifting to a differentiating medium and culturing for an additional 8 days, cells in these spheres expressed the neuronal markers ß-tubulin and neurofilament 200 and the astrocytic marker glial fibrillary acidic protein (GFAP). Finally, after treating the spheres with all-trans retinoic acid and taurine, the expression of ß-tubulin was increased and the staining of photoreceptor markers rhodopsin and recoverin was observed. The present study shows that NIH/3T3 fibroblasts can generate neurosphere-like, neuron-like, and even photoreceptor-like cells under defined conditions, suggesting that the differentiated non-neuronal cells NIH/3T3 fibroblasts, but not pluripotent cells such as embryonic stem cells or induced pluripotent stem cells, may have the potential to be transdifferentiated into neuronal cells without adding any epigenetic modifier. This transdifferentiation may be due to the possible neural progenitor potential of NIH/3T3 fibroblasts that remains dormant under normal conditions.

A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice.

PURPOSE: To characterize an N-ethyl-N-nitrosourea-induced dominant mouse mutant, M-1156, that exhibits progressive retinal degeneration and to investigate the pathogenesis of the retinal phenotype in the mutant. METHODS: A positional candidate gene approach was used to identify the causative gene in the M-1156 mutant. Funduscopic examination, light microscopy, transmission electron microscopy, and electroretinography were performed to analyze the M-1156 phenotype. Real-time quantitative PCR, immunohistochemistry, and western blotting were also performed. RESULTS: Linkage analysis enabled the mutant gene to be mapped to a region of chromosome 19 containing Rom1, which encodes rod outer segment membrane protein 1. Sequence analysis demonstrated that the mutation consisted of a single base T-->C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed. A putative missense mutation designated Rom1(Rgsc1156) that was identified in the M-1156 mutant mouse causes a Trp to Arg substitution at position 182 in the translated protein. Rom1(Rgsc1156) heterozygotes were found to have a mottled retina and narrowed arteries in the fundus oculi. Photomicrographs of the retina revealed significant differences among the genotypes in the thickness of the outer nuclear layer and in the length of the outer segments of the photoreceptors. The alterations were more marked in the homozygotes than in the heterozygotes. Electron micrographs showed that the diameters of the discs varied in the heterozygotes and that the discs were more compactly stacked than in the wild type. There were significant differences among the genotypes in the amplitude of the a-wave in single-flash electroretinograms, but there were no significant differences among the photopic electroretinograms. Real-time quantitative PCR showed that there were no significant differences among the genotypes in Rom1 or peripherin/rds (Prph2) mRNA levels relative to the rhodopsin (Rho) mRNA level. Rom1 and Prph2 immunoreactivity were decreased in the retinas of the Rom1(Rgsc1156) mutants. Semiquantitative western blot analysis of retinas from 3-week-old Rom1(Rgsc1156) mutants demonstrated significant decreases in Rom1, Prph2, and Rho protein levels in all of the genotypes. CONCLUSIONS: The Trp182Arg substitution in Rom1(Rgsc1156) mutants causes retinal degeneration. The results suggested that Trp182Arg mutant Rom1 causes a decrease in the levels of wild-type Prph2 and Rom1, which in turn cause a reduction in the level of Prph2 containing tetramers in the disc rim region and ultimately result in unstable, disorganized outer segments and photoreceptor degeneration.

Channelrhodopsin-2 gene transduced into retinal ganglion cells restores functional vision in genetically blind rats.

To test the hypothesis that transduction of the channelrhodopsin-2 (ChR2) gene, a microbial-type rhodopsin gene, into retinal ganglion cells of genetically blind rats will restore functional vision, we recorded visually evoked potentials and tested the experimental rats for the presence of optomotor responses. The N-terminal fragment of the ChR2 gene was fused to the fluorescent protein Venus and inserted into an adeno-associated virus to make AAV2-ChR2V. AAV2-ChR2V was injected intravitreally into the eyes of 6-month-old dystrophic RCS (rdy/rdy) rats. Visual function was evaluated six weeks after the injection by recording visually evoked potentials (VEPs) and testing optomotor responses. The expression of ChR2V in the retina was investigated histologically. We found that VEPs could not be recorded from 6-month-old dystrophic RCS rats that had not been injected with AAV2-ChR2V. In contrast, VEPs were elicited from RCS rats six weeks after injection with AAV2-ChR2V. The VEPs were recorded at stimulation rates <20Hz, which was the same as that of normal rats. Optomotor responses were also significantly better after the AAV2-ChR2V injection. Expression of ChR2V was observed mainly in the retinal ganglion cells. These findings demonstrate that visual function can be restored in blind rats by transducing the ChR2V gene into retinal ganglion cells.

BDNF increases the phagocytic activity in cultured iris pigment epithelial cells.

To investigate the effect of brain derived neurotrophic factor (BDNF) on the phagocytic activity in iris pigment epithelial (IPE) cells, purified porcine photoreceptor outer segments (POS) were applied to cultured IPE cells for three hours. To measure phagocytic activities, bound and total POS were differentially stained using a double immunofluorescence staining method. BDNF increased the binding of POS in IPE cells in a dose-dependent manner. Ingestion of POS, however, was not affected throughout the concentrations used in this study. To investigate the signal transduction pathways of BDNF, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and MAPK/ERK kinase (MEK) inhibitor, PD98059, were used for this study. LY294002 had no effect on the binding and ingestion of POS in BDNF-treated IPE cells. On the other hand, PD98059 completely inhibited the increase of POS binding in BDNF-treated cells and also decreased the ingestion of POS. These results indicate that increased POS binding activity by BDNF and the decreased ingestion of POS were mediated through the MAPK pathway.

Presence of myocilin sequence variants in Japanese patients with open-angle glaucoma.

PURPOSE: To examine the myocilin (MYOC) gene for mutations in Japanese patients with primary open-angle glaucoma (POAG) and to determine the phenotypes of the patients with the mutations. METHODS: One-hundred thirty-eight unrelated Japanese patients with POAG were studied. Genomic DNA was extracted from leukocytes of peripheral blood, and the three coding exons including the intron-exon boundaries were amplified by polymerase chain reaction (PCR) and directly sequenced bi-directionally. RESULTS: Two sequence variants were identified, one novel non-synonymous amino acid change (p.Gln297His) and one reported synonymous amino acid change (p.Ala363Thr). These mutations were not detected in the 118 ethnically-matched controls. p.Gln297His was found in a 70-year-old man, who developed POAG at a late age, and his intraocular pressure was high. p.Ala363Thr was found in two cases, and both patients developed POAG at an early age and had high intraocular pressures that responded poorly to medical treatment. CONCLUSIONS: Two non-synonymous variants, p.Gln297His and p.Ala363Thr, indicate that they are involved in the pathogenesis of POAG. p.Ala363Thr has been found in another Japanese population and would be useful in genetic testing.

Iris pigment epithelial cell transplantation for degenerative retinal diseases.

The transplantation of different types of cells into the eye to treat retinal diseases has advanced in the past 20 years. One of the types of cells used for transplantation is the iris pigment epithelial (IPE) cell, because autologous IPE cells are easily obtained and their properties are similar to those of retinal pigment epithelial (RPE) cells and retinal cells. IPE cells are transplanted as; freshly isolated or cultured cells to replace defective or diseased RPE cells, genetically modified IPE cells for delivering target molecules to the retina or RPE, and retinal progenitor cells. IPE cells have also been transplanted for non-retinal disorders. The survival of the transplanted cells in the host is an important factor for the success of transplantation. Autologous IPE cells have been found in the transplanted subretinal space and were able to phagocytose rod outer segments even 6 months after transplantation. Allogeneic and xenogenic cells will not remain in the region longer than autologous cells. Allogenic cells transplanted into the subretinal space are rejected in humans. Thus, we have transplanted cultured autologous IPE cells in 56 patients with age-related macular degeneration. The long-term results (more than 2 years with a maximum of 8 years) showed that the visual acuity (VA) was significantly improved over the pre-transplantation VA, although a slight decrease of VA was observed 2 weeks after the transplantation. One patient showed a vasculitis-like lesion. IPE cells that were transduced with neurotrophic factors by plasmid or viral vectors have also been transplanted in animals. We have transduced several neurotrophic factor genes into IPE cells with a plasmid vector, adeno-associated virus, or adenovirus. Transplantation of these transduced IPE cells into the subretinal space rescued photoreceptor cells from several types of photoreceptor toxicities. In addition, transduction of a gene into the IPE cells suppressed the systemic dissemination of the viral genome. The neuroprotective effects of the IPE cells were different for the different types of neurotrophic factor, and some of the neurotrophic factors may enhance systemic immune reaction after transplantation. IPE cells have also been used as retinal progenital cells because they originate from the same cell lines that give rise to the neural retina and RPE cells. The transduction of the photoreceptor-related homeobox gene was reported to induce photoreceptor phenotypes in IPE cells. Furthermore, transplantations of IPE cells have been performed to treat central nervous system disorders. In this review, we summarize recent progress on IPE transplantation.

Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.

PURPOSE: To investigate whether the channelopsin-2 (Chop2) gene would restore visual responses in 10-month-old dystrophic Royal College of Surgeons (aged RCS; rdy/rdy) rats, the authors transferred the Chop2 gene into the retinal cells of aged RCS rats using the adenoassociated virus (AAV) vector. METHODS: The N-terminal fragment (residues 1-315) of Chop2 was fused to a fluorescent protein, Venus, in frame at the end of the Chop2 coding fragment. The viral vector construct (AAV-Chop2V) for the expression of the Chop2V in the retina was made by subcloning into an adenoassociated virus vector, including the CAG promoter. To evaluate the expression profile of Chop2V in the retina, the rats were killed and the eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Retinal wholemount specimens and cryosections were made. Under anesthetized conditions, electrodes for the recording of visually evoked potentials (VEPs) were implanted onto the visual cortex in aged-RCS (rdy/rdy) rats. AAV-Chop2V vectors were then injected into the vitreous cavity of the left eyes. As a control, AAV-Venus vectors were applied to the right eyes. VEPs were evoked by the flash of a blue, white, or red light-emitting diode (LED) and were recorded from the visual cortex of the rats at various time points after the AAV vector injection. RESULTS: Chop2V fluorescence was predominantly observed in retinal ganglion cells (RGCs). Some fluorescence was observed in the inner nuclear layer and the inner plexiform layer neurites. A tendency of recovery was observed in the VEPs of aged RCS (rdy/rdy) rats after the AAV-Chop2V injection but not after the AAV-Venus injection. The visual response of AAV-Chop2V-injected aged RCS (rdy/rdy) rats was less sensitive to the blue LED flash than that of nondystrophic RCS (+/+) rats. The AAV-Chop2V-injected aged RCS (rdy/rdy) rats were insensitive to the red LED flash, which evoked a robust VEP in the RCS (+/+) rats. CONCLUSIONS: The visual response of aged RCS (rdy/rdy) rats was partially restored by transduction of the Chop2 gene through AAV into the inner retinal neurons, mainly RGCs. These results suggest that the transduction of Chop2 would provide a new strategy to treat some retinitis pigmentosa (RP) symptoms independent of their etiology.

Characteristics of mitochondrial calpains.

Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.

Hypothermia protects cultured human retinal pigment epithelial cells against indocyanine green toxicity.

PURPOSE: The aim of this study was to determine whether indocyanine green (ICG) is toxic to cultured human retinal pigment epithelial (ARPE-19) cells, and whether hypothermia can protect the ARPE-19 cells against the ICG toxicity. METHODS: Cultured ARPE-19 cells were exposed to 0.25, 0.5, 1, 2.5, and 5 mg/mL of ICG dye at 37 and 4 degrees C for 30 min. The percentage of ARPE-19 cells that survived was determined by resazurin 1 day after the exposure. RESULTS: Exposure of the RPE cells to a hypotonic saline solution with an osmolarity equal to 5 mg/mL of ICG did not induce a statistically significant decrease in the percentage of RPE cells that survived. Exposure of the ARPE-19 cells to ICG induced a significant decrease in the percentage of cell survival at all concentrations of ICG (P<0.05), except in 0.25 mg/mL at 37 degrees C. At 4 degrees C, on the other hand, ICG induced a statistically significant decrease in the percentage of RPE cell survival only at 5 mg/mL of ICG (P<0.05). CONCLUSIONS: These results indicate that ICG is toxic to human RPE cells in culture, and that cell death cannot be attributed to the low osmolarity. Hypothermia of 4 degrees C has a protective effect against ICG toxicity.

Visual Responses of Photoreceptor-Degenerated Rats Expressing Two Different Types of Channelrhodopsin Genes.

Optogenetic technologies are expected to be applicable for clinical use in restoring vision. However, the degree of recovered visual function is highly dependent on the function of the chosen optogenetic gene. To investigate the effect on visual function of dual expression of genes with different wavelength sensitivities, we transduced a modified Volvox-derived channelrhodopsin gene (mVChR1) via an adeno-associated virus vector into transgenic rats harbouring the ChR2 gene in retinal ganglion cells. These transgenic rats were given an intraperitoneal injection of N-methyl-N-nitrosourea to induce the degeneration of native photoreceptor cells prior to transduction of mVChR1. Optical coherence tomography images indicated the degeneration of the native photoreceptor cells after the N-methyl-N-nitrosourea injection. Complete loss of function of the native photoreceptor cells was confirmed using electroretinograms. In the ChR2 transgenic rats, visually evoked potentials were clearly detectable in spite of native photoreceptor function abolishment; however the responses were limited to within blue wavelengths. In contrast, the limited wavelength sensitivities were improved by the additional transduction of mVChR1, which exhibited sensitivities to green and red. Thus, the transductions of dual genes encoding channelrhodopsins that exhibit different wavelength sensitivities represents a promising candidate method to expand and to enhance rescued wavelength sensitivities in blind subjects.

Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination.

PURPOSE: To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. METHODS: Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. RESULTS: No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. CONCLUSIONS: AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Screening of the MERTK gene for mutations in Japanese patients with autosomal recessive retinitis pigmentosa.

PURPOSE: To determine whether mutations in the MERTK gene are present in Japanese patients with autosomal recessive retinitis pigmentosa (arRP). METHODS: The coding sequence of all 19 exons and the adjacent flanking intron sequences of the MERTK gene were directly sequenced in 96 unrelated Japanese patients with arRP. RESULTS: Seventeen sequence variants were found; six missense changes, three isocoding changes, and eight intron changes were also observed. One arRP patient had a novel homozygous Leu12Pro missense mutation in the MERTK gene. CONCLUSIONS: Mutations in the MERTK gene are relatively rare in Japanese patients with arRP.

Polymorphisms in Complement Factor H and Hemicentin-1 genes in a Japanese population with dry-type age-related macular degeneration.

PURPOSE: To determine whether polymorphisms in the Complement Factor H (CFH) gene and the Hemicentin-1 gene at the ARMD1 locus are associated with dry age-related macular degeneration (AMD) in Japanese patients. DESIGN: Clinically relevant laboratory investigation. METHODS: Eighty unrelated Japanese patients with dry AMD and 196 Japanese control patients were studied. Two exons of the CFH gene and four exons of the Hemicentin-1 gene were amplified by polymerase chain reaction and sequenced directly. RESULTS: For the CFH gene, the frequency of the previously reported Tyr402His variant was not significantly higher in the AMD group than in the control group (P = .31). In the Hemicentin-1 gene, three sequence alterations (Asp5088Val, IVS99-13C/T, and His5245Gln) were detected, and the originally reported Gln5346Arg was not detected. CONCLUSION: The CFH gene and Hemicentin-1 genes do not appear to be involved in a statistically significant fraction of dry AMD cases in the Japanese population.

Hypothermia of 8 degrees C protects cultured retinal pigment epithelial cells and retinal ganglion cells against trypan blue toxicity.

PURPOSE: To determine whether hypothermia of 8 degrees C can protect cultured human retinal pigment epithelial (ARPE-19) cells and rat retinal ganglion cells (RGC-5) against trypan blue (TB) toxicity. DESIGN: Laboratory investigation. METHODS: ARPE-19 cells and RGC-5 were exposed to balanced salt solution as controls, and 0.05% and 0.5% TB at 37 degrees C, and at 8 degrees C for one minute. The percentage of surviving cells was determined by the resazurin test. RESULTS: TB induced a statistically significant decrease in the percentage of ARPE-19 cells surviving at 0.5% TB at 37 degrees C (P < .01). Conversely, TB induced a statistically significant decrease in the percentage of RGC-5 surviving at all conditions except for 0.05% TB at 8 degrees C (0.05% 37 degrees C; P < .05, 0.5% 37 degrees C and 8 degrees C; P < .01). CONCLUSIONS: These results indicate that reducing the temperature to 8 degrees C has a protective effect against the TB toxicity for ARPE-19 cells and RGC-5 in culture.

Nitric oxide-induced accumulation of lipofuscin-like materials is caused by inhibition of cathepsin S.

To determine whether nitric oxide (NO) is involved in accumulation of lipofuscin-like material (LFM) in retinal pigment epithelial (RPE) cells and if this formation is related to NO-mediated modification of cathepsin S (cat S). RPE cell cultures were fed once every day with porcine photoreceptor outer segments (POS) in the presence of NO-donor [S-nitroso-N-acetylpenicillamine (SNAP) or NOC18] for 2 weeks. LFM autofluorescence within the cells was measured by fluorophotometric flow cytometry (FACS). The activity of purified cat S was measured in the presence of NO-donor with or without dithiothreitol (DTT). The following results were observed. SNAP and NOC18 caused LFM accumulation in RPE cells in a dose-dependent manner, and this accumulation was reversed by the addition of NO-scavengers (hydroxycobalamin, carboxy-PTIO). Purified cat S activities were inhibited by NO-donors without DTT, but in the presence of DTT, NO-donors exhibited no inhibitory effect on its activity. Phagocytic challenge of RPE cells increased cat S activity, which was reduced by the addition of NO donors. These results indicated that cat S activity was inhibited by NO-donors and resulted in LFM accumulation in RPE cells. We conclude that NO-mediated inhibition of cat S was caused through protein modification of cat S and resulted in LFM accumulation.