Cleavage of lens protein-GSH mixed disulfide by glutathione reductase

Mixed disulfide complexing the bovine lens erystallins and GSH was prepared by incubating the soluble proteins with GSSG. GSH could be released from the mixed disulfide in the presence of NADPH or NADH. Mixed disulfide was purified using DEAE-Sephadex A-50 column chromatography and was found to be free of glutathione reductase. In such preparation of mixed disulfide NADPH failed to release GSH. However, addition of glutathione reductase along with NADPH released substantial amounts of GSH from the mixed disulfide. Bovine serum albumins also form mixed disulfide with GSH and the mixed disulfide could be cleaved by glutathione reductase. Alkylation of protein sulfhydryl groups by N-ethylmaleimide prior to the addition of GSSG for the preparation of mixed disulfide, almost completely prevents the formation of mixed disulfide indicating that the formation of mixed disulfide involves protein sulfhydryl groups. Evidence is presented that the cleavage of mixed disulfide is not mediated by GSH with cyclic reduction of GSSG by glutathione reductase, but rather proceeds enzymically through glutathione reductase.Using the borohydride method, small amounts of GSH were found to be bound to normal human lens proteins and a substantial amount of GSH was found to be bound to lens proteins from senile cataracts. Only a very small amount of GSH was bound to bovine lens proteins. Glutathione reductase failed to release a significant amount of GSH bound to lens proteins in human cataractous and normal lens. The significantly greater increase in the protein thiols after the borohydride treatment of lens proteins from normal and cataractous human lens and from bovine lens cannot be accounted for by release of GSH bound to proteins. This indicates significant intramolecular and/or intermolecular disulfide bridges in lens proteins.     

Destabilization of lens protein conformation by glutathione mixed disulfide

Mixed disulfide between lens crystallin and glutathione has been observed in human cataracts and could be formed in vitro by thiol-disulfide exchange reaction. The glutathionyl crystallins have been reported to become partially unfolded. The present paper reports the conformational destabilization by the mixed disulfide formation in calf alpha- and gamma-II crystallin. The conformational stability was studied by the denaturants urea and guanidine hydrochloride (Gdn-HCl), and by proteolytic degradation. The denaturation curves of both urea and Gdn-HCl shift to lower denaturant concentration for crystallins of glutathione mixed disulfide. The decrease in conformational stability is estimated to be 0.22- and 0.92 kcal mol-1 for modified alpha- and gamma-II crystallin, respectively. Proteolytic digestion also shows a faster rate of degradation for the modified crystallins. These results indicate that mixed disulfide destabilizes the crystallin conformation. The destabilization may make crystallins more susceptible to changes as observed in aging lenses.     

A new method to perform the cover test

A new method is presented to perform the cover/uncover test and the alternate cover test. With the subject's head in a chin rest, he fixates an object at either near or far distance, and one eye is illuminated from above with a collimated infrared beam. A high-sensitivity infrared TV camera observes the first and fourth Purkinje images from below. The examiner covers the nonilluminated eye and, viewing a TV monitor, compares the relative positions of the two images before and after covering. By changing the incident angle of the beam, the amount of deviation can be measured. To perform the alternate cover test, the illuminated eye is covered with an infrared transmitting filter.     

Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione

Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.     

A new method for oxygen supply to acute ischemic retina

This paper introduces a new method for supplying oxygen directly to ischemic inner retina, using an oxygen source in the vitreous. Acute retinal vascular occlusion was created in cat eyes by direct pressure on the optic disk and its margins with a glass probe. The satisfactory occlusion of the retinal vessels was documented by direct observation, and functionally by recording the ERG. The vascular occlusion caused a large decrease in the size of the ERG b wave, with no change in the a wave amplitude. The oxygen source was a catheter made of strands of an oxygen-permeable membrane which was inserted into the vitreal cavity. After successful vascular occlusion was documented, 100% gaseous oxygen was perfused through the catheter while recording the ERG. In response to the perfused oxygen the b wave partially recovered. Ventilating the animal with 100% oxygen when the retinal vessels were occluded also caused recovery of the b wave amplitude. Termination of the vitreal oxygen source caused a decrease in b wave amplitude to the level previously observed after the occlusion of the retinal vessels. When the retinal circulation was restored by removal of the glass probe the b wave recovered. The results show that it is possible to supply adequate oxygen to the inner retina via the vitreous to replace the oxygen normally supplied by the retinal circulation. Modification of this method may be useful for the treatment of recent and incomplete retinal vascular occlusion.     

Comparison of Eyemetrics and Orbscan automated method to determine horizontal corneal diameter

Purpose:

To compare horizontal corneal diameter measurements using the Orbscan Eyemetrics function and Orbscan corneal topographer.

Materials and Methods:

Seventy-three eyes of 37 patients were included in the study. In all cases, the automated white-to-white (WTW) measurements were obtained using Orbscan by two observers. Using the Eyemetrics function, the WTW was measured manually by the same observers from limbus to limbus using the digital caliper passing through the five point corneal reflections on the Orbscan real image. The data was analyzed using SPSS software for correlation, reliability and inter-rater repeatability.

Results:

The mean horizontal corneal diameter was 11.74 ± 0.32mm (SD) with the Orbscan and 11.92 ± 0.33mm (SD) with Eyemetrics Software-based measurement. A good positive correlation (Spearman r = 0.720, P = 0.026) was found between these two measurements. The coefficient of inter-rater repeatability was 0.89 for the Orbscan and 0.94 for the Eyemetrics software measurements on the anterior segment images. The Bland and Altman analysis showed large limits of agreement between Orbscan WTW and Eyemetrics WTW measurements. The intra-session repeatability scores for repeat measurements for the Orbscan WTW and Eyemetrics measurements were good.

Conclusion:

Eyemetrics can be used to measure WTW and the Eyemetrics measured WTW was longer than the WTW measured by Orbscan.     

A new statistical method to evaluate the significance of clinical data

The application of the Bayesian statistical method proves that gradient A, which takes into account the real and theoretical values of the intravascular and intraocular pressures is highly significant to evaluate the stage of deterioration of the visual field.Moreover, the vascular factors, pressure, flow, and condition of the vessel wall, may constitute a correcting explanation in cases of pseudoincompatible correlation between visual field deterioration and normal intraocular pressure, but rather low intravascular pressure.     

A new method for computer-assisted orbitotomies

BACKGROUND: Surgical planning and tumor margins from preoperative image data sets can be transferred to the surgical site by use of computer-assisted navigation systems. Usually registration markers are placed prior to image data acquisition in order to correlate the patient's preoperative image data set with his/her position in the operating room. Pointers are commonly used for navigation, and the position of the pointer within the surgical site is compared to its position within the image data set. With a new navigation system, patient registration can be performed without registration markers. Tumor margins and osteotomy lines can be projected onto the surgical site. MATERIALS AND METHODS: With the new navigation system, which has been developed by the Sonderforschungsbereich 414 of the Deutsche Forschungsgemeinschaft, markerless patient registration is performed using the contours of the patient's face. A scanner is mounted above the operating table, which detects the surface of the surgical site intraoperatively. Osteotomy lines and tumor margins are projected onto this surface using an intense beamer. The procedure is illustrated for a patient with a meningeal tumor of the orbit. RESULTS: Patients can be registered with an accuracy of 1 mm. Pointers are not required to transfer the data on the surgical site. Furthermore, monitors are no longer required, since the information is projected directly on the patient's surface. CONCLUSIONS: Surgical planning and tumor margins can exactly be visualized within the surgical site. Ergonomics are especially improved for navigated orbitotomies via a coronal approach.     

A new method to test vertical ocular deviations using perilimbal light reflexes

BACKGROUND: To develop a new diagnostic technique to determine vertical ocular deviations when the center of the pupil is covered by swollen eyelids in up- and downgaze. PATIENTS AND METHODS: In upgaze (downgaze) the reflex of a diagnostic lamp held at about 50 cm distance from the patient is observed on the lower (upper) limbus. In the case of an asymmetric reflex, prisms are used to obtain symmetrical reflexes. The amount of prisms indicates the size of the vertical misalignment. In five healthy volunteers, the angles of vertical changes of gaze position were plotted against the prism size needed to recenter the perilimbal reflex. RESULTS: There was a linear correlation between the amount of upgaze changes in degrees and the strength of prisms used for compensation in degrees. This linear correlation was also found in downgaze. For both the correlation coefficient was r = 0.98 +/- 0.01. In upgaze the slope of the average regression line was 0.55 +/- 2.3 degrees, in downgaze - 4.1 +/- 0.8 degrees. A prism of 1 degrees corresponds in upgaze to a vertical deviation of about 1.3 +/- 0.14 degrees, in downgaze to a deviation of about 1.1 +/- 0.07 degrees. CONCLUSIONS: These results demonstrate that the perilimbal light reflex test is suitable for measuring simulated vertical ocular deviations. Therefore, the test may also be used in patients with vertical deviations who cannot be measured with classical methods. The method is more exact for measurements in upgaze.     

A new method to evaluate retinal adhesive force in living rabbit eye

A new method to evaluate the retinal adhesive force in rabbit eye in vivo is described. A small dome-shaped retinal detachment (bleb) was made in the posterior pole fundus of Dutch rabbit by injecting balanced salt solution into the subretinal space through a micropipette. The transmural pressure was directly measured with a micropressure measuring system, utilizing the resistance servo-nulling methods when the bleb started to extend again. According to Laplace's law, the retinal adhesive force of normal Dutch rabbits was calculated to be (1.8 +/- 0.2) x 10(2) dyn/cm (n = 84). This value was about 5 times larger than that previously obtained by peeling methods in vitro. Our newly developed method might resolve several problems encountered in the previous methods and allow for the measurement of the retinal adhesive force more physiologically and precisely.