Three novel mutations of the RPGR gene exon ORF15 in three Japanese families with X-linked retinitis pigmentosa.

We describe three new mutations in a recently identified exon, ORF15, of the retinitis pigmentosa GTPase regulator gene (RPGR) in three unrelated Japanese families (Families 1-3) with X-linked retinitis pigmentosa (XLRP). The affected males had typical retinitis pigmentosa (RP), whereas the obligate carrier females showed a wide clinical spectrum, ranging from minor symptoms to severe visual disability. Some carrier females in Families 1 and 2 showed typical RP, most carriers manifested high myopia and astigmatism, and their corrected visual acuity was insufficient. They showed an impairment of cone function following the rod dysfunction and accompanied by refractive errors. Microsatellite analysis of Family 1 revealed that the RP in the family was linked to the RP3 locus. Although one patient in the family had no mutation in the previously published exons 1-19 including exon 15a, he had a single-nucleotide insertion in exon ORF15 (g.ORF15 + 753-754 insG). Likewise, patients in Families 2 and 3 had two-base insertion/deletion in the exon, i.e., g.ORF15 + 833-834delGG and g.ORF15 + 861-862insGG, respectively. These insertional/deletional mutations observed in the three families are all different and new, and are predicted to lead to a frameshift, resulting in a truncated protein. These findings may support the previous hypothesis that RPGR-ORF15 is a mutational hot spot.

Flavonol glycosides from Epimedium sagittatum and their neurite outgrowth activity on PC12h cells.

The MeOH extract of Epimedium sagittatum was found to show neurite outgrowth activity on cultured PC12h cells. Bioassay-guided fractionation of the MeOH extract yielded six prenylated flavonol glycosides, ikarisoside A (1), icarisid II (2), epimedoside A (3), icariin (4), epimedin B (5), and epimedokoreanoside-I (6) as the active ingredients.

Effects of bicarbonate ion on chick retinal pigment epithelium: membrane potentials and light-evoked responses.

The purpose of this study was to determine how changes in [HCO3-] alter the electrical properties of the retinal pigment epithelium (RPE). Experiments were conducted on the isolated chick retina-RPE-choroid preparation. The chamber holding the preparation allowed independent perfusion of the retinal and the choroidal surfaces. The light-evoked trans-tissue potential (TTP), the trans-epithelial potential (TEP), the trans-retinal potentials, and the intracellularly-recorded apical and basal membrane potentials were studied. Increasing the [HCO3-]0 in the choroidal bath from 25 to 40 mEq/1 led to an increase in the TTP and TEP. The same change in the retinal bath decreased the TTP because of a biphasic change of the RPE membrane potentials. There was also an increase in the amplitudes of the TEP, the c-wave and the slow PIII. The light-evoked subretinal K+ decrease was greater which is consistent with an increase in the photoreceptor light response. These observations indicated that the decrease of TTP resulted from a basal membrane hyperpolarization followed by an apical membrane depolarization induced by an increase in retinal [HCO3-]0. The relationship of these potential changes to the human bicarbonate responses is discussed.

[Multi-focal electroretinograms in normal subjects].

The multi-focal electroretinogram (multi-focal ERG), developed by Sutter (1992), is a method of recording the spatial distribution of focal ERG in a short time using multi-input stimulation. Using this technique, we can detect the spatial extent and severity of damage to the macula. In this study, we recorded multi-focal ERGs from 20 eyes of 20 normal subjects and analyzed the topographical property of responses. In every subject, a negative wave followed by a positive wave could be recorded and we named them the N1-wave and the P1-wave, respectively. The amplitudes of the N1-wave and the P1-wave were the largest in the fovea and they became smaller with eccentricity. In P1-wave amplitude, the greatest inter-subject variability was observed at the fovea. The N1 and P1 latencies were shorter in the upper retina than in the lower retina. The amplitude was larger in the upper retina than in the lower retina, which suggests functional superiority of the upper retina. There was no statistical difference of latency and amplitude between nasal and temporal retina. We found no statistical difference between the responses of the papillomacular bundle and those of the temporal retinal area. The mapping obtained by multi-focal ERG was useful as objective perimetry.

Autosomal dominant cone-rod dystrophy associated with a Val200Glu mutation of the peripherin/RDS gene.

OBJECTIVE: Mutations of the peripherin/RDS gene have been reported in several kinds of retinal dystrophy, and they show a variety of manifestations. The authors identified a novel Val200Glu mutation of the peripherin/RDS gene in a Japanese family with autosomal dominant cone-rod dystrophy (CRD). This report describes a genotype-phenotype correlation of the Val200Glu mutation. PATIENTS AND METHODS: Fifteen members of one Japanese family with autosomal dominant CRD were screened for mutations in the peripherin/RDS and ROM 1 genes. Clinical features were identified by visual acuity, visual field testing, fundus examination, and electroretinography. RESULTS: A Val200Glu mutation was found in all of the affected family members examined and was segregated with the disease. No patient had a mutation in the ROM 1 gene. Phenotypic characteristics of each affected member in this family showed intrafamilial similarity. Characteristic features included cone function more severely impaired than rod function and degenerative change in the macular region associated with peripheral retinal degeneration. CONCLUSION: The mutation at codon 200 of the peripherin/RDS gene causes both cone and rod degeneration. The Val200Glu mutation results in a type of autosomal dominant CRD.

Effect of vitrectomy on epiretinal membranes after endogenous fungal endophthalmitis.

Pars plana vitrectomy was used to remove proliferative membranes caused by endogenous fungal endophthalmitis in 3 patients (3 eyes). Tractional retinal detachment was present in all. In 2 eyes, a fibrovascular membrane originated in the optic nerve head and was connected to a full-thickness chorioretinal scar. In the other, a thick preretinal membrane was found in the macula, with total retinal detachment. In all eyes, the epiretinal membrane was successfully removed and the retina reattached. Visual outcome depended on the site of the chorioretinal scar. We advocate pars plana vitrectomy as the method of choice for tractional retinal detachment following fungal endophthalmitis.

The normal c-wave amplitude in rabbits.

We examined some of the problems arising when c-waves are employed in pharmacological experiments with pigmented rabbits. The cyclic variations of the c-wave are smaller in rabbit than in other species. Under our experimental conditions the c-wave amplitudes in some rabbits reached an equilibrium under intermittent stimuli, while in others they increased with time and did not reach a constant level within 3 h. The c-wave amplitude varied with the standing potential during dark adaptation. However interindividual variations made it difficult to standardize the c-waves by this change. Since the c-wave amplitudes of the right and left eyes were similar, the contralateral eye should be used as a control when drugs are administered topically. Interindividual variations of the c-wave amplitude were smaller under dim light. This method is useful in chronic pharmacological experiments when drugs are administered systemically.

Effect of glucose added to intraocular irrigating solutions on the rabbit electroretinogram in vitro.

The most adequate concentration of glucose in intraocular irrigating solution for the retina was studied electrophysiologically in the pigmented rabbit in vitro. Comparison of b-wave amplitudes demonstrated that glucose at a concentration of 20 mM/l yielded the highest values. A 40 mM/l glucose solution also increased the b-waves but to a lesser extent than did a solution of 20 mM/l. This result might be due to the detrimental effects of osmolarity. However, under hypoxic conditions the amplitudes of the b-wave obtained with 40 mM/l glucose solution was greater than those with 20 mM/l. It thus appears that slight hyperglycemic concentrations might have favorable effects on the ischemic retina.

New disposable ERG electrode made of anomalous polyvinyl alcohol gel.

We developed a disposable electrode for the electroretinogram made of anomalous polyvinyl alcohol gel. This new hydrogel is a rubber-like elastic with water content 80-90 wt%, but insoluble in water. The elastic electrode plate is approximately 0.3-1.0 mm thick, sterile, flexible, and easily cut to the desired shape. Its low cost permits it to be discarded after use.

Studies on the reconstitution of macromomycin a d auromomycin from the chromophore and protein moieties.

The chromophores extracted from macromomycin (MCR) and auromomycin (AUR) with methanol had identical ultraviolet absorption spectra, antibacterial spectra and analytical profiles in high pressure liquid chromatography. The chromophore content of AUR was about 8 times higher than that of MCR. MCR reconstituted from the chromophore and protein fraction was identical with native MCR by Sephadex G-50 chromatography, ultraviolet absorption spectrum and antibacterial spectrum. The antibacterial activity of MCR and AUR was due to the chromophore; the protein moiety had no activity. However, the protein moiety enhanced the activity of the chromophore against Gram-positive bacteria, while it suppressed the activity against Gram-negative organisms. It also protected the chromophore from heat-inactivation.

Studies on auromomycin.

A new antitumor antibiotic, named auromomycin, was isolated from the culture broth of Streptomyces macromomyceticus, a macromomycin-producing strain. The antibiotic was recovered from the culture filtrate by salting out with ammonium sulfate and further purified by successive application of ion-exchange chromatography on Amberlite IRA-93 (Cl form) and DEAE-Sephadex (OH form), Gel filtration on Sephadex G-50 and hydrophobic chromatography on Octyl-Sepharose CL-4B. The antibiotic is an acidic polypeptide with a molecular weitht of 12,500 and an isoelectric point of pH 5.4 and consists of 16 different amino acids. It has characteristic absorption maxima at 273 nm and 357 nm in the ultraviolet spectrum and two minima at 280 nm and 350 nm in the optical rotatory dispersion spectrum. Auromomycin exhibits antibacterial activity not only against Gram-positive bacteria, but also Gram-negative bacteria. Antitumor activities of auromomycin were revealed against EHRLICH ascites carcinoma, ascites sarcoma 180, L1210 leukemia and LEWIS lung carcinoma. Auromomycin was found to be converted into macromomycin by adsorption chromatography on Amberlite XAD.

Further purification and characterization of macromomycin.

The antitumor polypeptide, macromomycin (MCR) produced by Streptomyces macromomyceticus, was purified by ammonium sulfate precipitation, ultrafiltration and chromatographic techniques using Amberlite IRA-410, DEAE Sephadex A-25 (CI- and OH-type) and Sephadex G-50. Purified MCR was obtained as a white powder by lyophilization. MCR, thus purified, exhibited a single peak on Sephadex G-50 chromatography with no detectable contaminant by ultracentrifugation and polyacrylamide gel electrophoresis. MCR is an acidic polypeptide having an isoelectric point of pH 5.4. It contains no arginine and methionine. The molecular weight was 11,700, 12,500 and 11,400 by amino acid composition, gel filtration and analytical ultracentrifugation, respectively. MCR is labile as a lyophilized powder but is successfully stabilized by the addition of maltose.