[Retinal hazard from blue light emitting diode].

PURPOSE: To compare the effect of exposure time from a blue(460 nm) light emitting diode(LED) on the morphology of the outer retina and determine conditions where damage occurs. MATERIALS AND METHODS: Young adult rhesus monkeys were anesthetized, and received blue LED exposure from a modified slit-lamp. A 3 mm beam of 0.85 mW was imaged onto the retina through a lens positioned before the cornea and exposure damage was determined at time intervals for 12 to 90 min. Fundus photography, fluorescein angiography(FAG), retinal tomography(HRT), and s-cone electororetinogram(S-ERG) were recorded at baseline, 2, and 30 days. RESULTS: Two days after 40 min exposure, there was a grey, discolored region, which was over-fluorescent in FAG, and an incresse in HRT and S-ERG corresponding to the site which was exposed to LED light. In histological examination at 30 days, the LED had caused produced a marked disruption of the disks of photoreceptor cells, damaged retinal pigment epithelium(RPE) apical villi, and a loss of RPE melanin after 90 min exposure. CONCLUSION: A threshold level was found around 40 min. This morphological damage may impair function and continuous exposure to blue light is potentially dangerous to vision.

Local fundus response to blue (LED and laser) and infrared (LED and laser) sources.

Light damage research began during the early years of laser light exploration. There is a clear and significant literature that identifies an easily demonstrated retina-pigment epithelium pathology which is associated with short wavelength exposures below 520 nm. Recent interest has expanded because of the growing evidence for a blue light contribution to the retina aging process by way of a poorly understood chemical process(es) that involve circulation, oxidative reactions and the spectral absorption properties of the pigment epithelium. New powerful sources of relatively inexpensive blue energy have become available as a family of light emitting diodes. In this experiment, we examined funduscopic, angiographic and scanning laser tomographic measures of the retinal-pigment epithelium response to LED and laser spectral blue and infrared emissions closely matched in wavelengths and delivered under carefully matched circumstances. Ten retinas in normal young rhesus monkeys were locally exposed to various energy density values at 458 nm (Argon laser) ranging from 5 to 54 J cm(-2). Eight rhesus eyes were exposed to LED irradiation with a peak wavelength of 460 nm ranging from 9 to 62 J cm(-2). Similarly, a matched infrared (IR) laser and IR LED pair were used to expose an additional ten eyes for comparison of the long wavelengths. IR irradiance ranged from 21 to 306 J cm(-2). There was no response to IR exposure in any of the eyes. Blue light exposure results were measured from the color fundus photographs, scanning laser tomographs and early- and late-phase fluorescein angiogram responses at 2 and 30 days after the exposure. Results scores were accumulated for the four measures at the two time periods. The resulting lesion scores when plotted against the exposure in J cm(-2)showed no demonstrable effect at irradiance lower than 10 J cm(-2)and near 100% effectiveness for irradiance greater than 30 J cm(-2). The most sensitive and enduring indicator of change was the late fluorescein angiograms. Nonparametric statistical analysis of the scores from the two samples support the conclusion that there is no difference in the consequences of LED and laser light exposures under these matched conditions.

Effect of Antioxidants on Radical-initiated Peroxidation of Retinal Homogenate.

Purpose: To compare the effect of antioxidants on radical-initiated peroxidation of retinal homogenate.Methods: Lipid peroxides in bovine retinal homogenate were induced by 5 mM FeNO(3) (Fe), 25 mM 2, 2'-azobis(2, 4'-dimethylvaleronitrile) (lipid-soluble, AMVN) or 50 mM 2, 2'-azobis (2-amidinoprpane) dihydrochloride (water-soluble, AAPH) and the preventive effects of antioxidants were measured. Phosphatidylcholine hydroperoxide (PC-OOH) was analyzed with high performance liquid chromatography (HPLC) as the endpoint biomarker.Results and Conclusion: Troglitazone, an oral hypoglycemic agent, inhibited PC-OOH production by Fe and AMVN. Therefore, it may be effective for protecting against oxidative stress on the inner surface plasma membranes and subcellular organelle. Quercetin has radical scavenging effects on both sides of the membrane, because it prevents PC-OOH production by AMVN or AAPH. These results demonstrate the usefulness of an in vitro screening test that can accurately and rapidly determine the capacity of an antioxidant against lipid peroxidation or oxidative stress. (Jpn Ophthalmol Soc 104:466-70, 2000)

[Effect of antioxidants on radical-initiated peroxidation of retinal homogenate].

PURPOSE: To compare the effect of antioxidants on radical-initiated peroxidation of retinal homogenate. METHODS: Lipid peroxides in bovine retinal homogenate were induced by 5 mM FeNO3 (Fe), 25 mM 2, 2'-azobis(2,4'-dimethylvaleronitrile) (lipid-soluble, AMVN) or 50 mM 2,2'-azobis (2-amidinoprpane) dihydrochloride (water-soluble, AAPH) and the preventive effects of antioxidants were measured. Phosphatidylcholine hydroperoxide (PC-OOH) was analyzed with high performance liquid chromatography (HPLC) as the endpoint biomarker. RESULTS AND CONCLUSION: Troglitazone, an oral hypoglycemic agent, inhibited PC-OOH production by Fe and AMVN. Therefore, it may be effective for protecting against oxidative stress on the inner surface plasma membranes and subcellular organelle. Quercetin has radical scavenging effects on both sides of the membrane, because it prevents PC-OOH production by AMVN or AAPH. These results demonstrate the usefulness of an in vitro screening test that can accurately and rapidly determine the capacity of an antioxidant against lipid peroxidation or oxidative stress.

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning.

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0-0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6-7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3-9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.

Proteasome from rabbit skeletal muscle: Some properties and effects on muscle proteins.

Rabbit proteasome, likely to be a 20S proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purified enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20000-29000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580 000-800 000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pH for hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) (Suc-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH 5-9 and up to 60 °C at pH 7.2. The enzyme activity was inhibited by diisopropyl fluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaCl(2). Remarkably, SDS increased the V(max) value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, α-actinin, actin, tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS.

Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and the myofibrillar structure.

1. The mode of degradation of myofibrillar proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not alpha-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,000, 150,000, 87,000, 81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,000 and 30,000 Da. Troponin T was degraded into fragments of 21,000, 12,000 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myofibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.

High-performance liquid chromatographic separation and spectral identification of the photoreaction products of methyl 5,8-epoxyretinoate.

Photoreaction of methyl 5,8-epoxyretinoate was investigated. Irradiation of methyl 5,8-epoxyretinoate in acetonitrile with a light from a high-pressure mercury lamp or a daylight fluorescent lamp afforded three new products, which were purified by high-performance liquid chromatography. They were characterized on the basis of spectroscopic data as 11-(Z), 13-(Z), and 11,13-di(Z) isomers, respectively.

Cysteine proteinase inhibitors from rabbit skeletal muscle.

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.

Purification and some properties of cathepsin B from rabbit skeletal muscle.

Cathepsin B was purified from rabbit skeletal muscle by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocellulose, peptide-conjugated Sepharose, DEAE-Toyopearl and Sephadex G-100. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis. The enzyme did not abolish the Ca sensitivity of the ATPase activity of myofibrils. The molecular mass of the enzyme was found to be 27 kDa on gel filtration and SDS/polyacrylamide gel electrophoresis. The optimum pH for the hydrolysis of N alpha-benzoyl-DL-arginine-beta-naphthylamide was 6.5. The enzyme was stable in the range of pH 4.5-5.5. Tetrathionate reacted with thiol groups of the enzyme reversibly so that it stabilized the enzyme. The enzyme was strongly inhibited by iodoacetate, HgCl2, antipain, leupeptin, N alpha-p-tosyl-L-lysine chloromethane and L-tosylphenylalanylchloromethane, but not by pepstatin or trypsin inhibitor.