Modelling the visual response to an OUReP retinal prosthesis with photoelectric dye coupled to polyethylene film.

Objective.Retinal prostheses have been developed to restore vision in blind patients suffering from diseases like retinitis pigmentosa.Approach.A new type of retinal prosthesis called the Okayama University-type retinal prosthesis (OUReP) was developed by chemically coupling photoelectric dyes to a polyethylene film surface. The prosthesis works by passively generating an electric potential when stimulated by light. However, the neurophysiological mechanism of how OUReP stimulates the degenerated retina is unknown.Main results.Here, we explore how the OUReP affects retinal tissues using a finite element model to solve for the potential inside the tissue and an active Hodgkin-Huxley model based on rat vision to predict the corresponding retinal bipolar response.Significance.We show that the OUReP is likely capable of eliciting responses in retinal bipolar cells necessary to generate vision under most ambient conditions.

Development of highly durable retinal prosthesis using photoelectric dyes coupled to polyethylene film and quantitativein vitroevaluation of its durability.

Retinal prostheses have been developed to restore vision in blind patients suffering from such diseases as retinitis pigmentosa. In our previous studies, we developed a retinal prosthesis called dye-coupled film by chemical coupling of photoelectric dyes, which absorb light and then generate electrical potential, with a polyethylene film surface. The dye-coupled film is nontoxic, and we recovered the vision of a monkey with macular degeneration. The amount of dye on the dye-coupled film, however, decreased to one-third after five months in the monkey's eye. The photoelectric dye consists of a cation with photoresponsivity and a bromide ion (Br-). Therefore, an anion-exchange reaction could be applied to the dye-coupled film to improve its durability. In this study, the anion-exchange reaction was conducted using bis(trifluoromethanesulfonyl)imide ion (TFSI-), which has lower nucleophilicity than Br-. First, the long-term durability was examined without using animal subjects and in a short period. Subsequently, an elemental analysis was performed to confirm the exchange between Br-and TFSI-, and chemical properties, such as photoresponsivity and durability, before and after the anion exchange, were evaluated. It was quantitatively confirmed that the long-term durability of dye-coupled films can be evaluated in anin vitroenvironment and in a short period of one-thirtieth by utilizing a saline solution at 60 °C, compared with anin vivoenvironment. In addition, the durability of the dye-coupled film with TFSI-was improved to 270%-320% compared with that of the dye-coupled film with Br-.

Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery.

Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.

Vision evaluation by functional observational battery, operant behavior test, and light/dark box test in retinal dystrophic RCS rats versus normal rats.

BACKGROUND: Vision plays a key role in some behavior tests for rats. Okayama University-type retinal prosthesis (OUReP) is a photoelectric dye-coupled polyethylene film which generates electric potential in response to light and stimulates nearby neurons. This study aims to assess vision in retinal dystrophic (RCS) rats, in comparison with normal rats, by selected behavior tests. We also examined whether the tests could detect vision changes in RCS rats with dye-coupled film implantation. METHODS: Data sets were 5 normal rats, 4 untreated RCS rats, 7 RCS rats with dye-coupled films implanted at the age of 7 weeks after excluding unsuccessful implantation at autopsy. Behavior tests chosen were landing foot splay and visual forelimb-placing response in the menu of functional observational battery, operant-conditioning lever-press response and light/dark box test. RESULTS: Normal visual placing response was significantly less frequent in untreated RCS rats at the age of 9 and 11 weeks, compared with normal rats (P = 0.0027, chi-square test) while normal response was significantly more frequent at the age of 9 weeks in RCS rats with dye-coupled film implantation, compared with untreated RCS rats (P = 0.0221). In operant-conditioning lever-press test, the correct response rate was significantly lower in untreated RCS rats than in normal rats at the age of 9 weeks (P < 0.05, Tukey-Kramer test) while the rate was not significantly different between normal rats and RCS rats with dye-coupled film implantation. In light/dark box test, the time to enter dark box was significantly shorter in normal rats, compared with untreated RCS rats or RCS rats with dye-coupled film implantation (P < 0.05, Tukey-Kramer test). CONCLUSIONS: Behavior tests of functional observational battery, operant-conditioning lever-press response and light/dark box test discriminated vision between normal rats and RCS rats. The visual placing response and operant-conditioning lever-press test might have sensitivity to detect vision recovery in RCS rats with OUReP implantation.

Visual Evoked Potential Recovery by Subretinal Implantation of Photoelectric Dye-Coupled Thin Film Retinal Prosthesis in Monkey Eyes With Macular Degeneration.

Retinal prosthesis or artificial retina is a promising modality of treatment for outer retinal degeneration, caused by primary and secondary loss of photoreceptor cells, in hereditary retinal dystrophy and age-related macular degeneration, respectively. Okayama University-type retinal prosthesis (OUReP) is a photoelectric dye-coupled polyethylene film which generates electric potential in response to light and stimulates nearby neurons. The dye-coupled films were implanted by vitreous surgery in the subretinal space of monkey eyes with macular degeneration which had been induced by cobalt chloride injection from the scleral side. A pilot 1-month observation study involved 6 monkeys and a pivotal 6-month observation study involved 8 monkeys. Of 8 monkeys in 6-month group, 3 monkeys underwent dye-coupled film removal at 5 months and were observed further for 1 month. The amplitude of visual evoked potential which had been reduced by macular degeneration did recover at 1 month after film implantation and maintained the level at 6 months. Optical coherence tomography showed no retinal detachment, and full-field electroretinograms maintained a-wave and b-wave amplitudes, indicative of no retinal toxicity. Pathological examinations after 6-month implantation showed structural integrity of the inner retinal layer in close apposition to dye-coupled films. The implanted films which were removed by vitrectomy 5 months later showed light-evoked surface electric potentials by scanning Kelvin probe measurement. The photoelectric dye-coupled film (OUReP), which serves as a light-receiver and a displacement current generator in the subretinal space of the eye, has a potential for recovering vision in diseases with photoreceptor cell loss, such as retinitis pigmentosa and age-related macular degeneration.

Visual evoked potential in rabbits' eyes with subretinal implantation by vitrectomy of Okayama University-type retinal prosthesis (OURePTM).

Okayama University-type retinal prosthesis (OURePTM) is a photoelectric dye-coupled polyethylene film which generates electric potential in response to light and stimulates nearby neurons. This study aims to test surgical feasibility for subretinal film implantation and to examine functional durability of films in subretinal space. Dye-coupled films were implanted subretinally by vitrectomy in the right eye of normal white rabbits: 8 rabbits for 1 month and 8 rabbits for 6 months. The implanted films were removed by vitrectomy in 4 of these 8 rabbits in 1-month or 6-month implantation group. The films were also implanted in 4 rhodopsin-transgenic retinal dystrophic rabbits. Visual evoked potential was measured before film implantation as well as 1 or 6 months after film implantation, or 1 month after film removal. The films were successfully implanted in subretinal space of retinal detachment induced by subretinal fluid injection with a 38G polyimide tip. The retina was reattached by fluid-air exchange in vitreous cavity, retinal laser coagulation, and silicone oil injection. The ratios of P2 amplitudes of visual evoked potential in the implanted right eye over control left eye did not show significant changes between pre-implantation and post-implantation or post-removal (paired t-test). In Kelvin probe measurements, 4 pieces each of removed films which were implanted for 1 or 6 months showed proportional increase of surface electric potential in response to increasing light intensity. The film implantation was safe and implanted films were capable of responding to light.

Subretinal implantation of Okayama University-type retinal prosthesis (OURePTM) in canine eyes by vitrectomy.

Okayama University-type retinal prosthesis (OURePTM) is a photoelectric dye-coupled polyethylene film which generates electric potential in response to light and stimulates nearby neurons. This study aims to test surgical feasibility of subretinal implantation and functional durability of dye-coupled films in the subretinal space. The dye-coupled films were implanted subretinally by 25-gauge vitrectomy in the right eye of 11 normal beagle dogs: 2 dogs served for film removal after 5-month film implantation, 3 dogs for film removal after 3-month film implantation, 3 dogs for 3-month film implantation and pathological examination, and 3 dogs for sham surgery. The surface electric potential of the removed dye-coupled films in response to light was measured by the Kelvin Probe system. At surgery, rolled-up dye-coupled films in 5 × 5 mm square size could be inserted into subretinal space of retinal detachment induced by fluid injection with a 38-gauge polyimide tip. Retinal attachment was maintained by silicone oil injection in vitreous cavity. At autopsy, the retina in all dogs maintained the ganglion cell layer, inner and outer nuclear layers while it lost the outer segments in some part. All 5 sheets of removed dye-coupled films maintained the dye color. One sheet of the 5-month implanted film showed proportional increase of surface potential in response to increasing light intensity. Subretinal implantation of OURePTM by vitrectomy was technically feasible in canine eyes, and OURePTM maintained the function of generating light-evoked surface potential after 5 months in subretinal implantation.

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion-shallot monosomic additions and allotriploid-bunching onion single alien deletions.

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion-shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST-SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.

Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH.

A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.