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[gamma]S-crystallin ([gamma]S) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine [gamma]S solutions, we have observed liquid-liquid phase separation (LLPS) at -8[degrees]C and revealed that, in the binary [gamma]S-water system, this phase transition would occur at -28[degrees]C. We have measured both the effect of PEG concentration on the LLPS temperature and protein/PEG partitioning between the two liquid coexisting phases. We use our measurements of protein/PEG partitioning to determine the nature and the magnitude of the [gamma]S-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for [gamma]S. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary [gamma]S-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between [gamma]S molecules in the binary [gamma]S-water system are attractive. We compare the magnitude of the attraction found for [gamma]S with the results obtained for the other [gamma]-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein-water systems than known previously. protein | cataract | polyethylene glycol | phase transition | partitioning

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ER[DELTA]3, a dominant-negative form of ER[alpha] that inhibits ER[alpha] function. In the ER[DELTA]3 transgenic mice, cortical cataracts spontaneously form in ER[DELTA]3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ER[DELTA]3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ER[DELTA]3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ER[DELTA]3 repressor. Evidence of DES-induced cataracts in the ER[DELTA]3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ER[DELTA]3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.

Women have a higher incidence of cataracts, and epidemiologic data suggest that the increased risk may be caused by a lack of estrogen in postmenopausal years. We have examined the effects of estrogen on methylnitrosourea (MNU)-induced cataractogenesis in Sprague-Dawley rats. Animals were ovariectomized, injected with MNU, and treated with estradiol or estrone by a continuous-release, subcutaneous Silastic implant, or they received an empty Silastic implant (no hormone). In the no-hormone group, rats developed opaque lenses approximately 6 months after MNU treatment. By 8 months, 74% (14/19) of the no-hormone rats had evident opacity in one or both eyes by simple gross inspection; 58% (22/38) of the eyes in this group were opaque. Estradiol or estrone treatment reduced the incidence of cataractous eyes to 12% or 25%, respectively. Lenses were examined under a dissecting microscope for light transmission. The lenses of the group treated with no hormone had light transmission of 26% [+ or -] 9.2%, whereas lenses from the estradiol-treated animals had light transmission of 72% [+ or -] 5.8%. Histological examination revealed that the anterior cortices of the opaque lenses were disrupted and showed the hallmark signs of age-related cataracts; in addition, some eyes that appeared clear by macroscopic examination showed the early histologic signs of cataractogenesis. It was demonstrated with reverse transcription-PCR that lens cells express both [Alpha] and [Beta] types of estrogen receptor, suggesting that the protective effects of the hormones may be a direct, receptor-mediated phenomenon. Thus, the MNU-treated, ovariectomized rat serves as a model for age-related cataractogenesis, and observation of a clear protective effect of estrogens in this system supports the implications of epidemiologic data.