Electrochemical evidence on the molten globule conformation of cytochrome c.

To explore a new approach for characterizing the molten globule conformation, cyclic voltammetric studies of salt induced transitions at acidic pH of cyt c have been carried out. The use of modified electrodes has made the observation of direct electrochemistry in native cyt c possible. However, most of these electrodes do not show reversible responses at acidic pH, due to the fact that, for this system, a deprotonated electrode surface is needed. In these studies, we have used a 6-mercaptopurine and cysteine-modified gold electrodes which are effective for direct rapid electron transfer to cyt c, even in acid solutions. The change in the absorption bands of cyt c are used to monitor the conformational states and, hence, to compare the voltammetric results. Under the experimental conditions where the A state of cyt c is obtained, a reversible voltammetric signal is observed. The midpoint peak potentials are found to be very close to the formal potential of native cyt c. Results are discussed in terms of a cooperative two-state transition between the acid unfolded and the globular acidic states of cyt c. This finding establishes, for the first time, the similarity of both the native and the molten globule-like conformations in terms of its redox properties.

Autosomal dominant retinitis pigmentosa in a large family: a clinical and molecular genetic study.

PURPOSE: To characterize the pedigree, visual function phenotype, and responsible mutation in a large family with autosomal dominant retinitis pigmentosa. METHODS: Pedigree data were obtained by personal interviews and corroborated with community records. One hundred twenty-eight members of the family were examined clinically, and a subset of 12 affected subjects was further studied with dark- and light-adapted static perimetry and electroretinography. The coding region of the rhodopsin gene was polymerase chain reaction (PCR) amplified and resolved by denaturing gradient gel electrophoresis. Genomic DNA samples from nine affected and five unaffected family members were analyzed by PCR amplification and restriction enzyme digestion. RESULTS: A 14-generation pedigree was identified in which retinitis pigmentosa (RP) was inherited in an autosomal dominant fashion. Affected individuals reported early night blindness and showed vessel attenuation and bone spicule-like pigmentary changes. In these individuals, the rod electroretinogram (ERG) was not detectable, and the cone ERG was reduced in amplitude and delayed in timing. With dark-adapted perimetry, rod function could be detected in only one young patient, and it was markedly abnormal. Light-adapted perimetry indicated that cone sensitivity could be relatively well preserved in the central field, but it was diminished in the periphery even in the most mildly affected subjects. A valine345-to-leucine mutation was identified in the rhodopsin gene and shown to cosegregate in the heterozygous condition with the disease. CONCLUSIONS: The natural history of RP in this family begins with a loss of rod function, progresses to involve the cone system, and leads eventually to a severe loss of visual function. The invariance of valine345 in all functional vertebrate visual pigments sequenced to date, and the unusually conservative nature of the valine345-to-leucine mutation suggests that the carboxy terminus of rhodopsin is involved in a highly specific interaction with one or more rod proteins.

X-linked retinitis pigmentosa: functional phenotype of an RP2 genotype.

Rod- and cone-mediated function was studied with psychophysics and electroretinography in members of an X-linked retinitis pigmentosa pedigree with the RP2 genotype. An asymptomatic hemizygote with an early stage of the disease had cone dysfunction in the mid-periphery and an abnormal cone electroretinogram (ERG); rod function was normal. Hemizygotes with more advanced disease had cone and rod dysfunction in the mid-peripheral retina and cone dysfunction in the far periphery; cone and rod ERGs were abnormal. At very advanced stages, there was an absolute mid-peripheral scotoma and marked cone and rod dysfunction in the far peripheral and central retina. Cone and rod ERGs were severely abnormal or not detectable. Heterozygotes showed tapetal-like reflexes, patches of pigmentary retinopathy, and a range of functional findings from no detectable abnormalities to moderate levels of retinal dysfunction. There were regions of normal function adjacent to dysfunctional patches that had greater cone than rod sensitivity losses or comparable cone and rod losses. The results suggest that the phenotype of this RP2 genotype of X-linked retinitis pigmentosa, unlike other forms of retinitis pigmentosa, is first expressed as a cone photoreceptor system dysfunction, and as the disease progresses, both rod and cone systems are involved.

Relatively enhanced S cone function in the Goldmann-Favre syndrome.

Using electrophysiologic and psychophysical tests that measure rod, midspectral, and S (blue) cone function, we studied four patients with the Goldmann-Favre syndrome, an autosomal recessive vitreoretinal degeneration. With spectral electroretinography, the predominant signal was from the S cones. With dark-adapted perimetry, all patients had severely reduced rod sensitivities and subnormal midspectral cone sensitivities. With S cone perimetry, the patients had normal or subnormal S cone function. Sensitivity differences between S and midspectral cones were significantly different from normal; there was relatively higher sensitivity to S cones compared to midspectral cones throughout the visual field. This relationship of dysfunctional cone mechanisms in the Goldmann-Favre syndrome is similar to that in the enhanced S cone syndrome, a recently identified retinal degeneration with S cone hypersensitivity. The results suggest that the Goldmann-Favre and the enhanced S cone syndromes are linked by a common pattern of retinal dysfunction.

Abnormal rod dark adaptation in autosomal dominant retinitis pigmentosa with proline-23-histidine rhodopsin mutation.

We studied rod and cone function in 13 patients from four families with autosomal dominant retinitis pigmentosa and the proline-23-histidine rhodopsin mutation. In patients with early stages of this disease, rod sensitivity was mildly abnormal throughout the retina and cone sensitivity was normal. In more severely affected patients, sensitivity loss varied with retinal region, some regions showing mild rod loss only and other regions having pronounced rod and cone dysfunction. Rhodopsin levels were decreased below normal by amounts that indicated the rod sensitivity loss was determined by the reduced ability to absorb light. The most characteristic abnormality of this genotype was a slowed rod branch of dark adaptation, which was present regardless of the extent or severity of disease. The time required for recovery of rod sensitivity was more than twice the normal time. These findings with dark-adapted perimetry, fundus reflectometry, and dark adaptometry showed intrafamilial and interfamilial consistency.

Enhanced S cone syndrome: evidence for an abnormally large number of S cones.

The cellular basis of the hypersensitivity of the S (blue) cone system in patients with enhanced S cone syndrome was examined by analyzing ERGs from three patients. The patients had large alpha-waves in response to the blue and white flashes. These alpha-waves were shown to be driven nearly entirely by the S cones. Although these S cone alpha-waves were 4-6 times the size of the normal L/M cone alpha-wave, they are of the same form, and could be quantitatively described with the same model previously shown to fit cone alpha-waves. We propose that the retina of these patients has many more S cones than the normal retina and that these cones replace some of the normal L/M cones and many of the rods.

Genetic heterogeneity in autosomal dominant retinitis pigmentosa with low-frequency damped electroretinographic wavelets.

PURPOSE: To define molecular and ophthalmic features of a rare phenotype in autosomal dominant (ad) retinitis pigmentosa (RP). METHODS: A 32-year-old woman (proband) with adRP and the low-frequency damped electroretinographic (ERG) wavelet phenotype and her mother were studied with optical coherence tomography (OCT), chromatic perimetry and ERG. A previously reported adRP patient with this ERG phenotype (Lam et al) was also studied with OCT. Genotype in the two families was determined with DNA sequencing. RESULTS: ERGs from the proband were identical to those reported previously. Chromatic perimetry and ERG stimulus intensity series indicated that there can be severely reduced rod function in addition to substantial cone dysfunction. A heterozygous deletion in peripherin/RDS (Met152del3 atGAA) was present in the patient and the affected mother. There were foveal cystoid changes and pericentral splitting of the inner nuclear layer. ONL thickness and vision tapered with eccentricity, and 'blind' regions without discernible ONL showed a thickened, delaminated inner retina. Similar OCT findings were present in the reported adRP patient with this ERG; the patient was heterozygous for a 4-bp deletion (Leu107del4 ctGAGT) in PRPF31. CONCLUSIONS: The low-frequency damped ERG wavelet phenotype is genetically heterogeneous. Inner retinal structural abnormalities are also present in this rare disease expression.

Interocular asymmetry of visual function in heterozygotes of X-linked retinitis pigmentosa.

Heterozygotes of X-linked retinitis pigmentosa were studied with full field rod and cone electroretinography and light adapted kinetic perimetry. Twelve parameters from the electroretinograms (ERGs) and two parameters from the kinetic visual fields of both eyes of 22 heterozygotes were measured and statistical comparisons made with results from female control subjects. Rod and cone ERG amplitude parameters were significantly lower and cone timing delayed in the heterozygotes. Most of the ERG parameters that were abnormal in measured value also showed significantly greater interocular differences compared with controls. Kinetic visual fields with both V-4e and I-4e test targets were smaller in heterozygotes than in controls. Only with the I-4e target, however, were interocular differences significantly larger in the heterozygotes. For the I-4e target and many of the ERG parameters, using the interocular difference in conjunction with the measured parameter value from a single eye significantly increased the efficacy of discrimination between heterozygotes and controls; for some ERG parameters, the interocular difference alone provided the best separation of the two groups.

Evaluación de la toxicidad del extracto acuoso de las hojas de yacón (Smallanthus sonchifolius) administrado por vía oral en ratas / Evaluation of the subchronic toxicity of oral admnistration of aqueous extract of yacon leaves (Smallanthus sonchifolium) in rats

El Smallanthus sonchifolius, oriundo de los Andes y llamado vulgarmente yacón, es conocido por su uso como normoglicemiante e hipolipemiante así como por su utilidad como antioxidante. En la presente investigación evaluamos la toxicidad subcrónica del extracto acuoso de sus hojas, luego de su administración oral en ratas albinas Holtzman. Objetivo: Evaluar la toxicidad del extracto acuoso de las hojas de yacón en ratas albinas sanas. Material y método: Se realizó un estudio experimental en 40 ratas Holtzman, distribuidas en dos grupos (hembras y machos), a las cuales se administró, por vía oral, durante 90 días, dosis de 100, 200 y 500 mg/Kg/día, del extracto acuoso de hojas de yacón. Se tomaron muestras de sangre del plexo orbital del ojo a los 0, 45 y 90 días para llevar a cabo análisis bioquímicos y hermatológicos. Se realizaron, además exámenes histopatológicos de cerebro, hígado y riñones. Resultados: No se encontraron variaciones significativas en ninguno de los exámenes en comparación con las ratas controles (p ø 0.05). Conclusión: El consumo del extracto de hojas de yacón, durante 90 días, no produjo signos de toxicidad en los órganos estudiados.

The Smallanthus sonchifolius, vulgarly known as yacon, an Ades's species is well known for its use as by it's normoglycemic, hipolipemic and antioxidant effects. There are only few studies on this plant. At present we have investigations about it's chemical composition, harverts process, and medical use. In this paper we evaluated the toxicity of the aqueous extract of the organic leaves of Smallanthus sonchifolius on Holtzman albin rats subcronic oral administration of the extract during 90 days. Objective: To evaluate the subchronic toxicity of aqueous extract of the yacon leaves on healthy albina rats. Material and method: An experimental study has been performed on 40 Holtzman rats. Male a female rats were grouped separately. They received oral daily doses of 100, 200 y 500 mg/Kg/ yaconÆs leaves aqueous extract. Blood samples were taken from the rat orbital plexus of the rats at 0,45 and 90 days of treatment for biochemical and haematologic analysis. Histological examination of brain, liver and kigneys was also done. Results: Non significant differences were found in the examinations of the three groups in comparison with control (p ø 0.05). Conclusion: The Smallanthus sonchifolius aqueous extract consumption during 90 days did not produce toxicity signs in the organs studied.