Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

Proc. Natl. Acad. Sci. U.S.A.

Steele

1992

Self Cite

The variation of the phase-separation temperature (Td) was studied in lenses during formation of cataracts induced by a subcutaneous ihfection of sodium selenite. In normal control animals, the Tc decreased monotonically with increasing age. Approximately 2 days after administration of the selenite the T, decreased sharply to a minimum, and then at day 4 the Tc increased dramatically toward body temperature. Mature irreversible cataracts formed -6 days after injection of the selenite. (18)(19)(20)(21). The effect of WR-77913 on x-ray cataract was not entirely unexpected because the amino phosphorothioates are believed to behave as free-radical scavengers (17). The inhibition of the x-ray cataract could be explained as protection against oxidative damage to lens cells caused by free radicals as well as an effect on T, (17,22).It was not anticipated that WR-77913 would prevent cataract produced by selenite. In contrast to the x-ray cataract, T, was reported to decrease rather than increase in the selenite cataract (23). Selenite-induced opacity was associated with lenticular accumulation of calcium (24, 25) rather than free-radical formation. Oxidation of protein sulfhydryls was observed in the late stages of cataract formation (24,26,27), although oxidation of epithelial cells may be important at earlier stages of cataract formation. The differences between the x-ray and selenite mechanisms make the selenite model an interesting test for the effectiveness of WR-77913 as a PSI in vivo. In this report we show a remarkable observation: the chemical inhibition of abnormal changes in Tc, in vivo, is sufficient for the effective prevention of selenite cataract in rats. While it is possible that the properties of WR-77913 as a PSI may have nothing to do with its mode of action in preventing selenite cataract, we present direct evidence that reagents acting in vivo on a cytoplasmic phase separation have the potential to control and prevent formation of cataract. was administered by intraperitoneal injection at a dose of 820 mg/kg 15 min prior to the injection ofPBS only (control group B) or sodium selenite (group D). At various times, upl to 9 days after treatments, animals from each group were euthanized. Lenses were removed and placed in silicone oil in the temperature-controlled sample chamber (see Fig. 2). The T" was measured immediately. MATERIALS AND METHODS

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Phase-separation inhibitors and prevention of selenite cataract.

Proc. Natl. Acad. Sci. U.S.A.

Steele

1992

Self Cite

“…Under conditions favourable to normal transparency and unfavourable for protein self-assembly, the interactions between protein and solvent molecules, E ps , dominate and, with age, a change in T c favours transparent cell structure at body temperature [16,17]. Under conditions favourable for protein aggregation, the interactions between selfassembling proteins, E pp , and the interactions between solvent constituents, E ss , dominate.…”

Section: Introductionmentioning

confidence: 99%

“…The relationship between T c and the self-assembly of cytoplasmic proteins in lens was studied in diverse cataract models. The rate of change in T c was found to be a sensitive indicator of the earliest stages of protein aggregation and cataract formation in vivo [17]. Once the conditions leading to protein self-assembly were characterized empirically, it became important to identify the specific interactive sites that were responsible for the attractive forces contributing to solubility and transparency of lens cytoplasmic proteins or, in the case of cytoplasmic opacity, the self-assembly of aggregates and fibrils [18].…”

Section: Introductionmentioning

confidence: 99%

Self-assembly of protein aggregates in ageing disorders: the lens and cataract model

2013

Phil. Trans. R. Soc. B

Cataract, neurodegenerative disease, macular degeneration and pathologies of ageing are often characterized by the slow progressive destabilization of proteins and their self-assembly to amyloid-like fibrils and aggregates. During normal cell differentiation, protein self-assembly is well established as a dynamic mechanism for cytoskeletal organization. With the increased emphasis on ageing disorders, there is renewed interest in small-molecule regulators of protein self-assembly. Synthetic peptides, mini-chaperones, aptamers, ATP and pantethine reportedly regulate self-assembly mechanisms involving small stress proteins, represented by human αB-crystallin, and their targets. Small molecules are being considered for direct application as molecular therapeutics to protect against amyloid and protein aggregation disorders in ageing cells and tissues in vivo . The identification of specific interactive peptide sites for effective regulation of protein self-assembly is underway using conventional and innovative technologies. The quantification of the functional interactions between small stress proteins and their targets in vivo remains a top research priority. The quantitative parameters controlling protein–protein interactions in vivo need characterization to understand the fundamental biology of self-assembling systems in normal cells and disorders of ageing.

Phase separation and hydrogen bonding in cells of the ocular lens

1990

Biopolymers

T h e cytoplasm of the ocular lens is a concentrated protein solution that normally remains transparent. T h e conditions for cytoplasmic transparency are described in a phase diagram that has been determined by two separate experimental methods.'.' Under normal physiological conditions the cytoplasm exists as a single transparent phase. A decrease in temperature below the phase separation temperature T, produces two coexisting phases and opacity.' :' Normally, T, is well below body temperature, but during cataract formation T, increases toward body temp e r a t~r e .~-'It is well known that the value of the cytoplasmic T, is determined by the molecular interactions between proteins and solvent constituents of lens cytoplasm that are responsible for transparent cytoplasmic structure.8 " T h e nature of these interactions has been investigated by measuring the effects of changes in ionic strength, pH, or other conditions on T,. In this report, the effects of H-bonding solvents were evaluated on the T,. of lens cells.We determined d T , / d C for several solvents with different H-bonding characteristics. T h e solvents, including methanol, D 2 0 , and several glycols, were selected on the basis of ( 1 ) the strength of the H bonds formed in solution, which were readily characterized by the value of the boiling points and heats of ( 2 ) previous work that has shown t h a t MeOH, D,O, and glycols change I , and ( 3 ) finding that these particular solvents were easily diffused into the calf lens. We observed that the change in T,, d T , / d C , was directly proportional to the boiling point and heat of vaporization of the solvents. While many factors may contribute to phase behavior of lens cytoplasm, these empirical findings indicate the importance of' hydrogen bonds. ( Woburn, M A ) . The solvents were diluted with phosphate buffer so that the final buffer concentration was 0.154 M , which maintained the p H at 7.0. Calf lenses were dissected out of fresh calf eyes a n d placed in 100 m L of each test solution to soak for approximately 16 h at 4°C until the Tc equilibrated t o a constant value. It should be noted that returning the lens to buffer solution without any solvent allowed T, t o return to normal. It was interpreted t h a t this decrease and increase in T, was due t o diffusion of medium into a n d out of the lens. In the case of D20, proton NMR spectroscopy was used to measure the diffusion of D 2 0 into lens c y t~p l a s m . '~ After soaking the lens in the solvent, each lens was placed in a cuvette filled with silicone oil (Dow Corning, Midland, M I ) . The cuvette was mounted in the path of a 5 mW He-Ne LASER ( Melles-Griot, San Marcos, CA) beam. T h e beam was directed through the center of the lens onto a PIN-1OD photodiode (United Detector Technologies, Culver City, CA) , which measured the transmitted intensity of the LASER beam. T h e photocurrent produced in the photodiode was measured using a Keithley multimeter ( Keithley Instrument?, Cleveland, OH) and recorded using a Houston Instruments c...