Ahuva Dovrat

Computer assisted scanning laser monitor of optical quality of the excised crystalline lens

We describe a technique, involving the projection of low power laser beams through excised lenses in solution, that makes it possible to measure photographically lens optical aberrations. Preliminary work indicates that the laser method can be used in combination with lens culture experiments as a means of monitoring changes in studies of lens biology. The goal of this research is the development of an effective in vitro optical monitor of ocular lenses in order to assist the study of the biochemistry of lens aging and cataractogenesis and be used as a sensitive toxicity measuring device.

Rat lens superoxide dismutase and glucose-6-phosphate dehydrogenase: studies on the catalytic activity and the fate of enzyme antigen as a function of age.

The specific activities of superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase(G-6PD) in the rat lens decline as a function of animal age. Inactive or partially active molecules of SOD, as shown by immunotitration with monospecific antibody, were detected in newborn lens when compared to SOD from rat liver preparations. The proportion of these inactive molecules appeared to increase with age. G-6PD showed reduction in total activity in the lens which was not accompanied by the appearance of catalytically altered but antigenically intact G-6PD molecules. G-6PD activity could be found only in the cortex of the 6- and 27-month-old lens. Neitheractivity nor antigenically recognized form of the enzyme could be discerned in the nucleus of these lenses. A similar but less pronounced loss of enzyme antigen in the nucleus was also found for SOD. A possible explanation for this phenomenon is that inactive molecules are either degraded or altered to such a degree that they lose their antigenic determinants and consequently cannot interact with the antiserum.

Glyceraldehyde 3-phosphate dehydrogenase activity in rat and human lenses and the fate of enzyme molecules in the aging lens☆

The specific activity of the enzyme glyceraldehyde 3-phosphate dehydrogenase declines as a function of age. Immunotitration with monospecific antibodies demonstrated that with age there is an increase of catalytically defective, but antigenically reactive enzyme molecules in the lens. Antiserum, produced against denatured enzyme, removed the inactive molecules from lens homogenates, without effects on the levels of enzyme activity. These studies suggest that inactive enzyme molecules in aging lenses are totally devoid of catalytic activity, and are at least partially denatured.

Defective superoxide-dismutase molecules accumulate with age in human lenses

The specific activity of superoxide dismutase (SOD) in human transparent lenses declines as a function of age. Immunotitration using monospecific antibodies showed that, with increasing age, lenses exhibit an accumulation of catalytically inactive, but antigenically reactive, enzyme molecules. Antiserum produced against denatured enzyme removed the inactive molecules from the lens homogenates without affecting the enzyme activity. These aberrant molecules are at least partially denatured and are totally devoid of catalytic activity.

Superoxide Dismutase Molecules in Human Cataractous Lenses

Specific activity of superoxide dismutase (SOD) in cataractous lenses was examined. Immunoprecipitation with monospecific antibodies showed an accumulation of catalytically inactive, but antigenically reactive enzyme molecules in lenses with various types of cataracts. The inactive enzyme molecules can be demonstrated in all kinds of cataracts, including dense, mature cataracts with very low or nondetectable enzyme activity. In cataracts from diabetic patients, the mean enzyme activity was higher than in nondiabetic patients. This was especially marked in nuclear cataracts, in which the specific activity of SOD was higher than in similar cataracts from nondiabetics.

Studies on the fate of aldolase molecules in the aging rat lens

It was found that aldolase activity declined considerably in the lens of adult animals with increasing age. Immunoassay showed that defective aldolase C molecules were accumulated. In addition, antibody prepared against denatured enzyme preferentially removes inactive molecules from lens homogenates without affecting active molecules. It is concluded that defective aldolase molecules encountered in aging lenses are at least partially denatured and are inactive.

Effects of UV-A irradiation on lens morphology and optics

Epidemiological studies have indicated that ultraviolet radiation (UVR) is one of the main factors leading to senile cataract formation. We investigated morphological changes in the eye lens caused by UVR-A. Twenty three pairs of lenses obtained from 23 one-year-old calves were used for this study. For each pair, one lens was exposed to 44 J/m(2) UVR in the 365 nm wavelength region while the contralateral lens was not exposed and served as a control. The lenses were placed in specially designed organ culture containers for pre-incubation. Lenses were exposed to UVR after one day in culture. After irradiation, lens optical quality was monitored throughout additional 15 days of the culture period and lenses were taken for morphological analysis by scanning electron microscopy. Damage to lens optical quality was evident as early as day 8 after the irradiation and increased with time in culture. We found irregularity of fiber morphology in lenses exposed to UV-A irradiation (but not in control lenses), similar to that reported previously for aged lenses. At the end of the culture period (day 16), lens fiber membranes also showed holes in fiber membranes. We conclude that UVR-A caused damage to cell membranes of the lens and alterations in lens optics, which may subsequently lead to senile cataract formation.

Simulation of heat exposure and damage to the eye lens in a neighborhood bakery.

Epidemiological studies indicated a link between high temperature environment and cataract. The purpose of the study was to investigate if the high temperature in neighborhood bakeries can cause damage to the eye lens. Measurements were done to determine the temperature and exposure time in the neighborhood bakeries during a workday. Thermal analysis was done using finite volume and finite element Computational Fluid Dynamics (CFD) codes in order to determine the temperature in the eye lens when exposed to environmental temperature fluctuations. A simulation of heat exposure was carried out using a bovine lens organ culture system. Two-hundred and seventy bovine lenses were divided into five groups. (1) Control group kept in culture for 11-14 days (2) Lenses exposed to 39.5 degrees C, 6h daily starting on the second day of the culture and kept in culture for 13 days (3) Lenses exposed to 39.5 degrees C, 4h daily starting on the second day of the culture and kept in culture for 11 days (4) Lenses exposed to 39.5 degrees C, 2h daily for 3 days starting on the second day of the culture and kept in culture for 12 days (5) Lenses exposed to 39.5 degrees C, 1h on the second day of the culture and kept in culture for 14 days. Lens optical quality was assessed during the culture period. At the end of the culture lens damage was demonstrated by inverted microscopy. Lens epithelial samples were taken for analysis of Catalase activities. Control lenses maintained their optical quality throughout the 14 days of the culture. Exposure to heat caused optical damage to the cultured lenses. The damage appeared earlier in the 6h exposure group and progressed from the lens anterior suture to its center. Optical damage was recovered in lenses exposed 1h to 39.5 degrees C, but the damage remained in the lens epithelial cells. Our study indicates that exposure to heat in bakeries can cause damage to the eye lens and that the damage is dependent on the length of exposure.

Non-thermal electromagnetic radiation damage to lens epithelium.

High frequency microwave electromagnetic radiation from mobile phones and other modern devices has the potential to damage eye tissues, but its effect on the lens epithelium is unknown at present. The objective of this study was to investigate the non-thermal effects of high frequency microwave electromagnetic radiation (1.1GHz, 2.22 mW) on the eye lens epithelium in situ. Bovine lenses were incubated in organ culture at 35°C for 10-15 days. A novel computer-controlled microwave source was used to investigate the effects of microwave radiation on the lenses. 58 lenses were used in this study. The lenses were divided into four groups: (1) Control lenses incubated in organ culture for 10 to15 days. (2) Electromagnetic radiation exposure group treated with 1.1 GHz, 2.22 mW microwave radiation for 90 cycles of 50 minutes irradiation followed by 10 minutes pause and cultured up to 10 days. (3) Electromagnetic radiation exposure group treated as group 2 with 192 cycles of radiation and cultured for 15 days. (4) Lenses exposed to 39.5ºC for 2 hours 3 times with 24 hours interval after each treatment beginning on the second day of the culture and cultured for 11 days. During the culture period, lens optical quality was followed daily by a computer-operated scanning laser beam. At the end of the culture period, control and treated lenses were analyzed morphologically and by assessment of the lens epithelial ATPase activity. Exposure to 1.1 GHz, 2.22 mW microwaves caused a reversible decrease in lens optical quality accompanied by irreversible morphological and biochemical damage to the lens epithelial cell layer. The effect of the electromagnetic radiation on the lens epithelium was remarkably different from those of conductive heat. The results of this investigation showed that electromagnetic fields from microwave radiation have a negative impact on the eye lens. The lens damage by electromagnetic fields was distinctly different from that caused by conductive heat.

Analysis of UVA-Related Alterations upon Aging of Eye Lens Proteins by Mini Two-Dimensional Polyacrylamide Gel Electrophoresis

This study is a first approach to identify UVA-related alterations in situ of bovine eye lens proteins from the water-soluble and urea-soluble fractions upon aging. The fractions were obtained from irradiated long-term organ culture lenses and analyzed by mini two-dimensional gel electrophoresis. This micropreparative method followed by computer analysis allows high resolution and separation of microgram quantities of proteins and to detect spots which arose as a consequence of irradiation. To facilitate the analysis we first separated the water-soluble fraction into the major crystallin classes by gel filtration. Moreover, we immunoblotted the gel of the urea-soluble fraction with a specific antibody against the intermediate filament protein vimentin. Upon irradiation of young and adult lenses, αA-crystallin and vimentin showed obvious modifications. During aging the susceptibility to irradiation increased when vimentin started to degrade, whereas deamidation of αA-crystallin seems to occur.