Biochemical features of the grey squirrel lens.

The ocular lens of the grey squirrel (Sciurus carolinensis) is an excellent model for studies of eye-light interactions that apply to the human system. In this diurnal animal, lens size, shape, yellow pigmentation, and light absorption properties have important similarities to those of young children. This article describes the observations of soluble to insoluble protein conversion with chronological aging, and the loss of heavier lens crystallins in the internal as compared to the external layers of the lens. Such changes are related to aging, as the older lens material is present in the nuclear core, while the younger lens material is superficial. It describes the subunit peptides of the soluble crystallins and of the extrinsic and instrinsic proteins associated with fiber cell membranes. Squirrel lens fiber membranes release most of their extrinsic peptides in 8 M urea, as do those of other young animals. Due to the presence of near-UV absorbing species of low molecular weight, the squirrel lens has great potential for studies of the effects of near-UV radiation on the lens.

Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.

The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.

Effects of near-UV radiation on the protein of the grey squirrel lens.

In vivo exposure of grey squirrels to 40W BLB illumination resulted in alterations in the state of the lens crystallins, mainly in the outer layer of the lens. HPLC revealed an increase of the void volume or crosslinked crystallins and an increase in peptides with molecular weights lower than 20,000 d. In vitro exposure of squirrel lens aqueous extracts to Woods lamp radiation (predominantly 365 nm) led to similar but more exaggerated changes as viewed by high performance liquid chromatography. When viewed by polyacrylamide gel electrophoresis (PAGE), soluble protein crosslinking was also observed. The near-UV absorbing chromophores of low molecular weight present in the lens served as photosensitizers that enhanced the protein changes. Sodium azide inhibited the changes, indicating a role for singlet oxygen in the crosslinking.

Lipids of human lens fiber cell membranes.

A study of the membranes of human lens fiber cells revealed a very high protein to lipid ratio, which tended to increase with aging and in brunescent cataract. Phospholipids were more abundant than cholesterol, cholesterol esters, and other neutral lipids. With aging and cataract formation, a marked decrease in membranes phospholipid content occurred. Sphingomyelin was present in highest amount. Phosphatidylethanolamine, phosphatidylcholine, lysophosphatidyl-ethanolamine, phosphatidylserine, and phosphatidylglycerol were also present. Cholesterol represented approximately 40% of the total lipids. Saturated and unsaturated fatty acids having 16 to 24 carbons were present. The lipid composition varied according to the portion of the lens examined and the state of the lens. The results do not support a conclusion that lipid peroxidation represents a major mechanism of membrane damage that contributes to cataract formation, since there is no decrease in unsaturated fatty acids with age or in cataractous lenses. We suggest that the aggregation of soluble proteins, and their association with lens membranes, and altered membrane function due to the loss of phospholipids are important processes leading to loss of transparency.

The nature and properties of squirrel lens yellow pigment.

The low molecular weight yellow pigment in the gray squirrel lens is confirmed to be n-acetyl-3-OH-kynurenine (NAK). This conclusion is based upon the results of studies of the compound's optical, chromatographic, and mass spectroscopic properties. The original tentative identification of this compound was reported by Van Heyningen (1971, 1973). The pigment is distributed equivalently throughout the lens. Its absorbance increases with age, but the increase is related to the growth of the lens and not to an increase in pigment concentration. The function of NAK in the squirrel lens is to enhance vision by eliminating short-wavelength light that causes scattering and chromatic aberration. The pigmented lens also protects the retina from near-UV radiation-associated damage to the photoreceptors (Collier and Zigman, 1987).

Stability of shark and human lens fiber cell membranes.

1. Scanning electron microscopy of minced dogfish, shark, and human lens fiber cell preparations showed that neither 8 M urea or 1% SDS was capable of totally disrupting the fiber cells, but that their membranes were totally disrupted by 1% SDS containing 50 mM dithiothreitol. 2. Fiber cells were purified by sucrose centrifugation and extracted with Tris-buffer, 8 M urea, 1% SDS and 50 mM dithiothreitol plus 1% SDS. 3. A residual 10% of the protein was capable of maintaining membrane integrity. 4. DTT + SDS totally dissolved the membranes, implying that disulfide bonds are involved in maintaining structure. 5. Non-tryptophan fluorescence was nearly totally extracted prior to the SDS-DTT step, indicating that the fluorescence associated with the membrane protein was not serving as crosslinks between extrinsic and intrinsic proteins. 6. Polyacrylamide slab gel electrophoresis revealed that after exhaustive extractions of lens fiber cells with Tris-buffer, 8 M urea and 1% SDS, the addition of DTT released major heat stable 27,000 and 24,000 dalton peptides. 7. The data support the concept that fiber cell membranes contain high levels of extrinsic peptides and intrinsic peptides whose stability depends strongly on oxidized crosslinks, probably disulfide bonds.

Effects of H2O2 on the dogfish (Mustelus canis) ocular lens.

The influence of in vitro exposure of dogfish (Mustelus canis) lenses to H2O2 was studied. Concentrations of H2O2 greater than 10(-4)M caused opacities in the outer cortex of the lens within a few hr. Evidence of protein aggregation within the lens and in extracted lens protein solutions was found. Dithiothreitol inhibits aggregation. Little if any influence of H2O2 was observed on 86Rb exchange of the dogfish lens.

Comparative study of lens proteins of gray squirrel and human.

1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.