Causes of Decreased Phase Transition ... - Semantic Scholar
Causes of Decreased Phase Transition Temperature in Selenite Cataract Model K. P. Mitton, J. L. Hess, and G. E. Bunce
Purpose. Lenses from selenite-treated animals develop reversible "cold cataract" at a lower temperature than is required for lenses from age-matched control animals. This unexplained, stabilized phase transition is readily observed in intact lenses 36 to 48 hours after treatment and occurs in lenses before the appearance of the irreversible nuclear opacity observed 72 to 96 hours after treatment. The objective of this study was to investigate factors that may be responsible for this difference. Methods. Preweanling rats were injected with sodium selenite. Lens extracellular water volume was measured using 14C-inulin. Free amino acids were analyzed using precolumn derivatization and high performance liquid chromatography. Soluble protein was isolated from lenses of control, and treated animals and temperature-dependent changes in light scattering were measured at 490 lira. Results. Lens extracellular water was increased by the selenite treatment, with a concurrent 10% decrease in intracellular volume. Solutions of soluble protein from lenses of selenitetreated animals after postinjection hours 24 and 48 had higher critical phase transition temperatures (Tc) compared to similar proteins from control lenses. From 24 to 72 hours after injection, the free amino acid content of the lens increased 42%. Taurine levels were unchanged over the same period. The addition of 7 mM glycine and 7 mM proline to solutions of soluble protein (96 mg ml"1) decreased the phase transition temperature. Taurine (14 mM) had a similar effect. Combining taurine and the glycine plus proline solutions had an additive effect in lowering the T c . Conclusions. Increases in free amino acid concentration occur in lenses in response to the stress imposed by a systemic dose of selenite. The altered polyion content in lenses from selenite-treated animals, before nuclear cataract formation, contributes to the greater thermal stability of transparency in these lenses, thus lowering the temperature at which "cold cataract" forms. Invest Ophthalmol Vis Sci. 1995; 36:914-924.
S u b c u t a n e o u s injection of sodium selenite (Na 2 . SeO 9 ) in a single dose induces the formation of nuclear cataract in neonatal rats within 3 to 4 days. 12 The following changes 0 to 48 hours after injection precede cataract formation: increased selenium content, 1 decreased ATP content, 3 glutathione loss, 1 ' 4 increased N A D P + - N A D P H ratio, 1 elevated glycerol-3-
From the Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Btacksburg, Virginia. Supported by National Institutes of Health giant EY06123. Submitted for publication September 26, 1994; revised November 10, 1994; accepted November 11, 1994. IMtfnietary interest category: N. Repiint requests: J. L. Hess, Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State Uni sity, West Campu Drive, BUickslmrg, VA 24061-0308.
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phosphate content,3 and DNA double-strand breaks.5 From 24 to 48 hours after injection, alterations occur in lens Ca2+ homeostasis: decreased Na + -Ca 2 + exchange,6 decreased Ca2+-ATPase activity, and increased Ca2+ permeability.7 The appearance of nuclear cataract 72 to 96 hours after injection is accompanied by some recovery from glutathione and ATP loss, increases in lens Ca2+ 8fl and inorganic phosphate content,0 increased insoluble protein content,1 and elevated proteolysis in the nuclear region of the lens.10 The current hypothesis regarding the nuclear cataract is that elevated Ca2+ activates calpain II and proteolysis of /3-crystallins, causing their insolubility.11 The lenses from selenite-treated rats contain /3-crystallins missing portions of their N-termini, similar to those
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5 Copyright © Association for Research in Vision and Ophthalmology
Phase Transition in Selenite Cataract tL I ^ !~
B» ^
j *.. ,..
' V Y
+•
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'
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obtained by treatment with purified calpain in vitro.12 Mature, but clear, rat lenses contain similar amounts of insoluble y-crystallin and insoluble, fragmented /3-crystallin as are present in cataractous lenses from younger selenite-treated rats.13 This recent finding shows that this cataract does not depend solely on insoluble protein content. The developmental stage at which proteolysis occurs may also be a factor in the selenite model.13 Rat lenses exhibit a phenomenon called "cold cataract" in which phase separation occurs as a function of temperature. At 10 to 15 days of age, cold cataract appears below 32°C and is reversible upon elevation of temperature. The temperature of phase transition drops to 26°C in selenite-treated rats at 36 to 48 hours after injection14 but increases sharply above physiological temperature just before the emergence of nuclear cataract.15 Direct effects of temperature on light scattering properties of protein solutions provide information about protein-solute interactions. Solutions of bovine lens extracts or pure bovine ylV-crystallin display a temperature-dependent phase transition in vitro.16 This property of crystallin interactions may explain the formation of cold cataract in the intact lens.17 Two phases, a protein-rich phase and a protein-poor phase, coexist at and below the critical temperature (Tc) and cause light scattering.18 All y-crystallin amino acid sequences are extremely homologous. The high-Tc crystallins (yllla- and ylVa-) and low-Tc crystallins (ylland ylllb-), however, have very different maximum Tc values of approximately 35°C and 5°C, respectively.19 Lens age is an important factor for cataractogenesis in the selenite model. The rat lens is less susceptible to selenite after 16 days of age, 12 which corresponds to the end of the critical maturation period of the lens.20 During this period, the lens changes from a state of uniform hydration to a state in which the nucleus is less hydrated relative to the cortex.21 The decreased phase transition temperature in the selenite model before nuclear cataract formation suggests a transient environmental change of the lens cytosol. These studies were undertaken to examine the regional intracellular and extracellular hydration states of lenses from selenite-treated animals compared to agematched controls and also to examine how alterations in free amino acids correlated with the temperaturedependent phase transition characteristics of die intact lens. Temperature-dependent phase transition properties revealed early alterations of the soluble protein population before nuclear cataract formation. METHODS Cataract Induction Sprague-Dawley rats were housed in polypropylene shoe box cages in rooms maintained at 24°C and 80%
915 relative humidity with a 12-hour light-12-hour dark cycle. Animals were fed Agway 3000 laboratory rat chow (Agway, Syracuse, NY) and distilled water. Tenday-old animals (13-day-old animals for the phase transition temperature experiments) were injected subcutaneously with 20 mM sodium selenite (Na2SeO3), 0.9% NaCl, for a total dose of SOnmolg" 1 body weight (approximately 30 fi\ injection). Uninjected control animals from the same litter were used for all experiments. These animals served as appropriate controls because injections with 0.9% NaCl or 20 mM sodium sulfite do not cause lens pathology.1 At designated times after injection, animals were decapitated and lenses were removed immediately for extraction of free amino acids or proteins. Lenses were observed and photographed with an Olympus SZH zoom-stereo microscope equipped with a model SZH-ILLD illumination base (Olympus Optical, Tokyo, Japan). All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Amino Acid Extraction and Analysis Free amino acids were measured by precolumn derivatization and high-performance liquid chromatography.22 Pairs of lenses were removed by a posterior approach into 0.9% NaCl, blotted, and homogenized on ice in a 0.5 ml volume of 10% txichloroacetic acid using a glass Potter-Elvehjem homogenizer. Supernatant was collected after centrifugadon (10,000g X 5 minutes). Aliquots of supernatant were dried under vacuum (70 mTorr), combined 1:1 with norleucine internal standard (0.40 mM 0.1 N HC1), and again dried under vacuum (70 mTorr). The material was washed with 40% MeOH (vol/vol), 40% 1M sodium acetate, 20% triethylamine, and again dried under vacuum. Amino acids were converted to phenylthiocarbamyl amino acids by reaction with 10% phenylisothiocyanate, 70% MeOH, 10% triethylamine, and 10% H 2 O. Phenylthiocarbamyl amino acids were diluted in 0.07% Na2HPO4, pH 7.4, 5% acetonitrile, and filtered using microcentrifuge filters (Ultra-Free-MC; Millipore, Milford, MA) before chromatography. All detectable amino acids were quantified by calibradon with physiological amino acid standards (Sigma, St. Louis, MO) run with each set of analysis. The highperformance liquid chromatography system was a Waters WISP/PicoTag system using the Waters free amino acid analysis column (#10950, 3.9 X 300 mm). (Waters, Millipore)
Osmolarity Measurement Osmolality of buffers was measured using an Osmette A automatic osmometer (Precision Systems, Natick, MA) calibrated with 100 and 500 mOsm kg"1 standards .
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Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
Protein Determinations
Phase Transition Temperature Curves
Protein content was measured by using the Bradford dye-binding assay''2* using bovine serum albumin as a standard and by monitoring the A280:A260 ratio.
Phase transition was monitored at 490 nm as percent transmittance (%T) of protein samples at various temperatures. Samples were diluted with dialysis buffer to equivalent protein concentration (96 nig/ml" 1 ) with or without the inclusion of various amino acids as specified and transferred into 0.300 ml volume quartz cuvettes with teflon-plug lids to prevent evaporation. The microcuvettes were placed in the cuvette holder assembly of a Gilford Thermoset Temperature Controller installed in a Gilford 2600-UV/VIS-Spectrophotometer. The assembly included a bolt-on lid to minimize temperature gradients; integrated bidirectional, solid-state heat pumps; and an internal thermistor. The internal thermistor was calibrated for linearity from 0°C to 99°C with a National Bureau of Standards probe. Each cuvette measured 12.2 X 5.5 X 7.1 mm external dimensions. The exposed optical surface was 24 mm2, only 5% of the total surface area. The maximum surface contact with the temperature control block and the small sample volume ensured rapid thermal equilibration.
Determination of Lens Extracellular Space The extracellular volume in the rat lens was quantified by equilibrating lenses individually in Hank's medium containing inulin ('''C-carboxylated, 0.5 /xCi ml"'; DuPont NEN, Boston, MA), which is permeable to the lens capsule but not to the plasma membrane.'14 Uptake by lenses from age-matched control and selenitetreated rats was determined after 24 hours at 30°C. Lenses, suspended on a glass loop, were rinsed twice by dipping in unlabeled medium and blotting with moistened filter paper. l4C-counts were measured by liquid scintillation spectrometry at 95% to 98% efficiency. A zero-time value was subtracted from all measurements because 20% of the counts were accounted for by a rapid, irreversible binding of the inulin to the capsule surface. Distribution of inulin uptake between the capsule-epithelium-cortex fraction and the nuclear portion of the lens was estimated from dissection of lenses on Parafilm "M" (American National Can, Greenwich, CT), transferring the appropriate sections of the lens to filter paper, absorbing the associated liquid, and counting the fractions. Preparation of Lens Total Soluble Protein Extract Lenses were obtained as above and frozen using liquid nitrogen for storage at -80°C to obtain sufficient tissue for the protein extract (22 lenses/extract). Pooled lenses were homogenized on ice in a glass PotterElvehjem homogenizer after the addition of 0.5 ml of buffer; 100 mM sodium phosphate, 5.0 mM ethylenediaminetetraacetic acid, 0.01% NaNH, pH 7.2 with HO, (295 mOsm kg"1). The homogenate was warmed to 15° to 17°C during the homogenization. Samples from selenite-treated rats were processed simultaneously with their controls. Insoluble cell debris and capsular material were removed after centrifuging (12,000g"X 5 minutes) at 15°C. The supernatant fraction was transferred directly into Spectra/por-1 dialysis tubing (6 to 8 kd cutoff; Spectrum, Houston, TX.) and dialyzed against three 1-1 changes of buffer, equilibrated with argon gas. Dialysis and concentration steps were performed at 21°C. After 14 hours, samples were concentrated in dialysis tubing against Sephadex G-200 (Pharmacia Biotech, Piscataway, NJ) followed by 30 minutes of dialysis against buffer.
The cuvette holder temperature, measured with a thermometer, was within 0.15°C of the thermistor readout. Argon was introduced (450 cm' minute"') into the spectrophotometer cell thermal cavity to provide a dry atmosphere for operation between 1°C to 15°C. Light transmission was monitored from high to low temperature after equilibrating samples for 1.5 minutes at a new set temperature. All solutions were transparent at 36°C and were referenced to 100% T at this temperature. The phase transition data could be fitted to the sigmoidal equation, %T = (%Tmax X k(T° -
k(T° - a)b)
with an r2 > 0.997. Differences between phase transition temperature curves were evaluated using a balanced two-way analysis of variance and Duncan's new multiple range test.25 Seven to 10 temperatures that determined the solution transmittance properties between 76%T and 90%T blocks were used in the analysis of variance and served as the basis for comparing the average Tc between group treatments.
Isolation of Lens Nuclear-Cortex Tissue For obtaining primarily nuclear and primarily cortical tissue, the greatest concern was metabolic alterations to amino acid concentrations during dissection. A technique was developed that allowed for complete freezing of the very fragile neonatal rat lens and separation of tissue. After washing the lenses in saline and blotting gently, they were frozen quickly at the surface
uid nitrogen bath. The lens was held with preforceps and carefully pierced along the postenterior axis with a 27-gauge needle and cooled liquid nitrogen bath. A small scalpel blade was o shave off the equatorial cortex first, followed anterior cortex-epithelium and then the postertex so that the lens nucleus, a sphere of 1.5 emained. Tissue shavings were transferred to oth forceps cooled in liquid nitrogen. Isolated r and cortical samples, each from 12 lenses, ored at -80°C until preparation for amino acid s. Both samples contained perinuclear material.
LTS
rance of the Lens
ure 1, we present both a posterior and a side the lens from control and selenite-treated rats. ight hours after treatment with selenite, before r cataract formation, the posterior subcapsular t is apparent, as is the greater clarity of the r region at 28°C compared to the age-matched om control animals. The temperature must be sed to 24°C for the lens from selenite-treated s to have a similar cold cataract, as seen in these from the control animals.
Hydration: Intracellular and Extracellular
1 shows the weight data for lenses from control lenite-treated animals. Although lens growth enuated in the selenite-treated animals, no dife in the ratio of dry-fresh tissue weight was ed. Significantly greater extracellular water was ed in the lenses from selenite-treated animals 2). The increased extracellular volume ocin both the capsule-epithelium-cortex and r regions of the lens (Table 2) before nuclear t formation, 48 hours after injection (age 12 and when nuclear cataract had fully formed, 96 after injection (age 14 days).
o Acid Analysis
sults of amino acid analysis of whole lenses from e-treated animals and age-matched controls are ed for 6, 12, 24, 48, 72, 96, and 192 hours after on (Fig. 2). Elevation of total amino acids ocas early as 24 hours, well before the nuclear t stage. After postinjection hour 72, the total mino acid content was 142% of controls in lenses elenite-treated rats (38.1 ± 0.7 fimo\ g~l versus 0.9/miolg- 1 ). e ratio of amino acid content of nuclear to l tissue (Fig. 3) was less than unity for control at all three time periods examined. Although
FIGURE l. Appearance of lenses from age-matched control and selenite-injected rats, 48 hours after injection. (A) Posterior view of age-matched control (right) and treated (left group lenses bathed in saline at 30°C under bright-field illumination. The control lens shows cold cataract formation in the lens nuclear region, whereas the treated lens displays only the posterior subcapsular cataract that is oriented about the posterior suture. (B) Equatorial view of control (tight) and treated (left) lenses supported on glass rods, separated by 1.75 mm, in saline at 28°C under bright-field illumination. The control lens displays a nuclear cold cataract, now more obvious than in plate (A). The corresponding volume of the lens from treated animals is clear, whereas the posterior subcapsular cataract can be seen (bottom). The typica discontinuity in light refraction, seen as a ring about the perinuclear region, is visible in the lens from the treated group. (C) Dark-field illumination of the same lenses shown in plate B enhances the light scattering areas (white) an transparent areas (black).
total amino acids were elevated at 24 hours after injection (Fig. 2), the nuclear-cortical ratio remained less than unity in lenses from selenite-treated animals (Fig. 3a). However, after entry into the nuclear cataract phase (96 hours after injection), the ratio became greater than unity in the selenite-treated group. This ratio became similar to control values at 10 days after
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Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
TABLE l.
Selenite-Dependent Decrease in Lens Growth Control
(days) 11 12 13 14 21
Weight (mg)*(n) 9.2 10.8 11.5 14.3 16.2
± ± ± ± ±
0.2 0.2 0.4 0.3 0.5
Selenite Treated
Dry/Wet Ratio 0.27 dt 0.26 1t 0.28 it 0.29 1t 0.34 it
(8) (7) (9) (7) (9)
0.01 0.01 0.01 0.01 0.01
Weight (mg) (n) 9.2 10.4 11.1 12.7 13.0
± ± ± ± ±
0.1 0.3 0.3 0.3f 0.4f
(6) (6) (10) (10) (8)
Dry/Wet Ratio 0.27 0.26 0.28 0.29 0.34
± ± ± ± ±
0.01 0.01 0.01 0.01 0.01
Selenite treatment was administered on the tenth day after birth. Lenses were weighed immediately after enucleation and after drying at 100°C to constant weight. Values are the mean ± SE for a minimum of eight lenses in each determination using lenses from at least two different litters. * Fresh tissue weight for the number of lenses designated by (n). f Weight of lenses from selenite-treated animals was statistically less than for lenses of control animals (P < 0.05 West).
injection. The experiment was replicated for the 96hour time period and confirmed the reversed nuclear-cortical amino acid concentration ratio. As internal comparisons from the same analysis, two nonprotein amino acids—taurine and ornithine—had a nuclear-cortical concentration ratio of less than unity throughout this period (Fig. 3b). Phase Transition Studies In Vitro Plots of light transmission as a function of temperature, for soluble protein solutions from lenses of selenite-treated and age-matched control animals, are presented in Figure 4. Solution turbidity was stable and apparent when light transmission (490 nm) decreased to 80%T. Therefore, the Tc (°C) at 80%T was compared. Statistical comparisons were based on the average Tc calculated from this most stable region of transmittance change between 76%T and 90%T (Table 3). Once light transmission fell below 75%T, solutions displayed a continuously decreasing transmittance at constant temperature. Protein solutions (without
TABLE
amino acid addition) from lenses of selenite-treated animals had a Tc approximately 2°C greater than protein solutions from lenses of age-matched controls after both 24 and 48 hours after injection (Table 3, Fig. 4). This selenite-dependent difference is inconsistent with the formation of cold cataract at lower temperatures compared to control lenses (Fig. 1). Elevated amino acid concentration stabilized the transparency of lens soluble protein solutions, seen as a shift of the Tc to lower temperatures (Fig. 5). Solutions contained glycine, proline, and taurine concentrations similar to those in lenses from selenite-treated rats. Specifically, an equimolar mixture of glycine and proline (14 mM total) or taurine (14 mM) reduced the Tc of lens protein solutions from control and selenite-treated animals (Table 4A). Taurine was more effective than the equimolar glycine-proline mixture. Further effects on Tc were examined in a solution of taurine, glycine, and proline (28 mM total) (Fig. 6). There was an additive effect on the reduction of Tc of soluble lens protein in this solution (Table 4B). The
2. Selenite-Dependent Increase in Extracellular Lens Water Lens Water (yd/lens)* Extracellular
IntraceUular Animal Age (days)
Whole Lens
Control (12) Selenite-treated (12) Control (14) Selenite-treated (14)
1A 6.5 9.5 7.6
± ± ± ±
0.2 0.3 0.3 0.7
Whole Lens 0.59 1.2 0.6 1.4
± ± ± ±
0.06 0.06f 0.05 0.19f
Cortex Epithelium 0.45 0.95 0.45 1.2
Nucleus 0.14 0.28 0.17 0.27
Selenite treatment was administered on the tenth day after birth. * Volume, estimated by equilibrating lenses in HC-inulin and from tissue weights (1 mg = 1 /til), is reported as mean value ± SE; n = 6 for all measurements. Similar overall errors occurred with the dissected tissue, although variation was greater with the estimation of the nuclear volume in the cataractous (14-day) tissue. f Extracellular volume in lenses from selenite-treated animals was significantly less compared to this volume in lenses from control animals (P < 0.05, (-test).
rf
919
Phase Transition in Selenite Cataract
40 -
•
Control
D Se
•
p
30-
20-
10-
i
1I 11
I I 11111 Hours Post Injection
FIGURE 2. Selenite treatment causes elevated amino acid content in rat lenses. Total free amino acid content, including taurine, is reported for lenses from selenite-treated animals (at designated times after injection) and for lenses from age-matched, control animals. Error bars = SE, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001.
nucleus to abnormal levels of calcium that are required for nuclear opacification. Modifications of membrane lipids after exposure of the lens to selenite in vivo could also influence the cell-to-cell associations, permeability to ions and metabolites, and the distribution of lenticular water. The critical maturation period lasts from approximately 12 to 16 days of age and is characterized by a transient sharp increase in the rate of lens dehydration, a temporary plateau in growth in the equatorial diameter and the posterior-anterior axis, a primarily intracellular water loss with constant extracellular water, a decreased penetration of extracellular space by procion yellow, and a constant increase in lens dry weight (1 to 21 days).20 During maturation, the lens epithelium displays junctions of focal adhesions on basolateral borders of adjacent cells that decrease in number with age as desmosomes appear to replace
average Tc, from 80%T to 90%T, for protein solutions at 96 mg/ml or 93 mg/ml were not significantly different.
A ) Protein amino acids
r
V_
DISCUSSION Hydration Decreased growth (Table 1) characterizes lenses from selenite-treated animals.' The dry mass concentration at various points along the lens axis is not altered in the selenite model until 6 days after injection, after the formation of nuclear cataract.26 These lenses showed no change in the wet-dry weight ratio (Table 1); therefore, hydration effects, typical of an osmotic cataract, do not occur in this model. The extracellular compartment water volume increases before nuclear cataract formation in the capsule-epithelium-cortex and nuclear regions of the lens (Table 2). The posterior liquid-filled vacuoles identified by 2 days after selenite treatment26 are consistent with the increase in extracellular water in these lenses. Posterior delivery of metabolites and selenite would be provided by the tunica vasculosa lentis at this stage of lens development.27 The posterior subcapsular cataract and the open appearance of the posterior suture (Fig. 1) indicate that this region of the lens is susceptible to local increases in the extracellular space. These changes may reflect interrupted cell maturation so that normal cell-to-cell interactions do not occur. The posterior subcapsular cataract decreases before nuclear opacification, yet an abnormal distribution of extracellular space persists (Table 2). The nature of the extracellular space during early lens development and perturbations of this space in lenses from selenite-treated rats may provide exposure of the lens
Time Post-Injection (Hours)
• D B •
B ) Taurine & Ornithine
2
0.S
Control Taurine Se Taurine Control Ornithine Se Ornithine
6
Time Post-Injection (Hours)
FIGURE 3. Distribution of free amino acid content in rat lens reported as the nucleus-cortex ratio. (A) The ratios of the total, free protein amino acid concentrations (/zmol/g fresh tissue) in the nucleus and cortex are presented. Extracts were prepared from dissected lenses of selenite-treated rats (24, 96, and 240 hours after injection) and from lenses of age-matched, control animals. The cortex fraction includes the capsule, epithelium, and cortex. (B) Ratio of taurine and ornithine content in lens fractions as noted above.
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
920 100
80-
o -» •D *
40-
10
11
12
13
Temperature (°C) them.28 The decreasing susceptibility of lenses to systemic selenite exposure beyond 18 days of age may be a result of decreasing access of selenite into the extracellular space related to the regression of the tunica vasculosa lentis.27
Temperature-dependent Phase Transition Proteins. The temperature sensitivity of the phase transition of the intact rat lens decreases with age. This age-dependent change correlates with the lower Tc for solutions of protein isolated from "older" (by 24 hours) lenses of control and selenite-treated animals (Table 3, Fig. 4). Age-dependent changes in Tc have been observed in the bovine lens,29 probably in response to an altered distribution of proteins among the chaperone
TABLE 3. Increased Apparent Tc of Solutions of Proteins Isolated From Lenses of Selenite-Treated Rats Tc, °C*
At 80%
r
Average 80% ( T to 907,i T) (n= 7)
Group (hours PI)
24
48
24
48
Controlf Selenite-treatedf
11.9 13.9
11.3 12.9
13.9 14.7
11.7 13.9
* Values of Te at 80% T are estimated from the phase transition curves in Figure 4. In addition, the average Tc is calculated for seven temperatures determining 80% T to 90% T. The average Tc for solutions of protein from lenses of selenite-treated rats is significantly higher than for solutions of protein from lenses of control rats (P < 0.05). The average Tc for the samples from lenses of 15-day-old animals (48 hours after injection) is significantly lower than the comparable sample from 14-day-old rats (24 hours after injection) (P < 0.01, Duncan's test), f Soluble lens protein concentration of 96 ± 1 mg ml"'. PI = after injection.
Control 24 hrs Se 24 hrs Control 48 hrs Se 48 hrs
FIGURE 4. Selenite treatment destabilizes phase transition properties of solutions of soluble lens proteins. Transmission (%T at 490 nm) is plotted as a function of solution temperature. Solutions, containing 96.0 ± 1 mg protein ml"1 in 300 mOsm kg"1 buffer, are prepared with soluble proteins extracted from lenses of age-matched control and selenite-treated rats 24 and 48 hours after injection.
a-crystallins and the high-Tc and low-Tc y-crystallins. Modifications to existing proteins may also cause these effects, although the relationship between "y-crystallin primary structure and Tc is not known.30'3' The phase transition properties of the soluble proteins from lenses of selenite-treated rats cannot explain the requirement for a lower temperature to cause onset of cold cataract in these lenses (Fig. 1). These protein solutions became cloudy 2°C higher than solutions of proteins isolated from control lenses (Table 4). Protein modification has not been well characterized in lenses before nuclear cataract formation. No additional disulfide bond formation occurs in soluble proteins from the lenses of selenite-treated animals.32 Although elevated selenium content is associated with the crystallins in lenses from treated animals,33 there exists no more than one Se atom per 10,000 protein molecules.1 It is unlikely that this modification could account for the observed change in phase transition properties of these protein solutions. Other uncharacterized modifications to proteins must cause the increased Tc of solutions of protein isolated from lenses of selenite-treated rats. Amino Acids. Tc was decreased in vitro using elevations of amino acid concentrations similar to those found in vivo (Table 4A, Figure 5). Glycine and proline were selected because they displayed the largest individual increases in the selenite model in vivo.3'1 The effect of taurine was also examined because it is the most abundant small molecular weight organic substance in the rat lens.35'36 All three amino acids possess uncharged side chains and an a-amino and an acidic group; therefore, all are zwitterions at physiological pH. Altered water distribution across fiber cell membranes in the lenses from selenite-treated animals (Fig. 2) would additionally increase the effective intracellular concentration of free amino acids and, thus, favor a lower T c .
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Phase Transition in Selenite Cataract
A) Control - O - Protein - • - Protein+7 mM Gly, 7 mM Pro -•*• Protein+14 mM Tau
E C
6 0-
Temperature (°C)
B) Treated FIGURE 5. Amino acids stabilize phase transition properties of solutions of soluble lens proteins. Amino acid additions—7 mM glycine and 7 mM proline or 14 mM taurine—decrease the Tc of solutions containing 96.0 ± 1 mg protein ml"'. Soluble protein is extracted from (A) age-matched control rats and (B) selenite-treated rats 48 hours after injection.
Taurine was more effective than an equimolar mixture of glycine and proline using extracts of the preweanling rat lens (Table 4A, Fig. 5). Taurine is also superior to glycine and glutamate in lowering Tc of soluble protein extracts from calf lens nuclear homogenates.30 The proportion of taurine to other amino acids increases substantially in the developing rat lens, from 15 mol% at day 14 to 32 mol% by day 20 (data not shown), and to about 75 mol% of the entire free amino acid pool in the adult rat lens.35'36 Taurine in the lens may function in osmoregulation.37 In the diabetic rat model of cataract, sorbitol elevation is balanced by decreased taurine concentration before initial vacuole formation38 and after vacuole formation.37 Taurine is a conditionally essential nutrient implicated in osmoregulation in the brain,39 membrane stabilization-antioxidation,40 and stimulation of cellular proliferation in retinal pigment epithelium.40 Our data support taurine's possible contribution to increased temperature stability (decreased Tc) of the lens fiber cytosol.
'rotein - • - Protein+7 mM Gly, 7 mM Pro ••*• Protein+14 mM Tau
10
13
14
Temperature (°C)
11
12
_/-
15
16
The ratio of amino acids contained in the nucleus to those in the capsule-epithelium-cortex fraction remained constant through the age of 23 days (240 hours after injection). Only in the lenses with nuclear cataract did the population of free amino acids become greater in the nucleus relative to the cortex (Fig. 3a). This effect was not seen with taurine and ornithine. These data suggest that extensive proteolysis in the lens nucleus leads not only to the accumulation of water insoluble products' 0 but also to the degradation of peptides to amino acids. Furthermore, these amino acids are not freely equilibrated throughout the lens at this stage of development. The addition of glutathione and amino acids lowers the Tc of calf lens nucleus extracts.30 Because glutathione content is decreased in lenses from selenitetreated rats,' it cannot account for lower Tc. Glutathione degradation may, in part, contribute to the elevated glycine content in the lenses from selenitetreated rats.
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Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
TABLE 4. Amino Acids Decrease Estimated T c of Lens-Soluble
Protein Solutions A. Solutions containing 96 ±1 mg protein mT' Tc, °C* Average (80% Tto 90% T) (n= 7)
At 80% T Composition
Control
Proteint Protein + 7 mM gly, 7 mM Pro Protein + 14 mM Tau
Se-treated
11.3 11.0 10.9
Control a
11.9 11.6" 11.3 C
12.9 12.8 12.5
Se-treated 13.8a 13.7" 13.5"
B. Solutions containing 93 :t 1 mg protein'' Tc, °C* Averai •ye (76% T to
Composition Protein^ Protein + 14 mM Tau Protein + 14 mM Tau + 7 mM Gly, 7 mM Pro
At 80% T
90%^ T) (n = 10)
11.3 10.9 10.7
11.4" 11.0" 10.8c
Values of Tc (at 80% T) are estimated from the phase transition curves in Figures 5 and 6. In addition, average values are calculated from (n) values of temperature that determine 76% T through 90% T. Values within a column with different superscript letters are significantly different at P < 0.05. (Duncan's test). f Soluble proteins extracted from lenses of selenite-treated rats 48 hours after injection and agematched (15-day-old) control rats. X Soluble proteins isolated from 15-day-old control rats.
SUMMARY We consider three changes in lenses from selenitetreated rats that relate to the requirement for a lower temperature to cause "cold cataract" in these lenses: intracellular volume, amino acid concentration, and changes to soluble proteins. Of these, the selenite-dependent decrease in intracellular volume and the increase in amino acid content affect greater stability of
protein associations, detected as lower Tc in temperature-dependent changes in light scattering. Changes to me lens proteins, however, result in solutions that have a higher Tc. This unexpected result reveals that lens transparency may not be driven by the nature of protein properties alone but, rather, by their interac-tion with the environment as influenced by metabolite concentrations and the properties of cellular membranes.
8 0-
tt/
uf
6 0-
4 0-
O Protein A Protein+7 mM Gly, 7 mM Pro, 14 mM Tau • Protein+14mMTau
/ 2 0-
A i
i
i
11
12
13
Temperature (°C)
i
i
FIGURE 6. Additive stabilization, caused by amino acids, of the temperature-dependent phase transition of solutions of lens soluble proteins. Amino acid additions—7 mM glycine and 7 mM proline plus 14 mM taurine or 14 mM taurine—decrease the T c of solutions containing 93.0 ± 1 mg protein ml" 1 . Protein was extracted from lenses of 15-day-old control rats, comparable to 48 hours after injection.
Phase Transition in Selenite Cataract
923
Key Wards amino acids, cataract models, lens proteins, phase transition, selenite
17.
Acknowledgments The authors thank Renu Batra for completion of the inulin experiments, Kathy Smith for technical assistance, and Dr. Kenneth Webb and Kris Lee of the Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University for amino acid analysis. References 1. Bunce GE, HessJL. Biochemical changes associated with selenite-induced cataract in the rat. Exp Eye Res. 1981;33:505-514. 2. Ostadalova I, Babicky A, Obenberger T. Cataract induced by administration of a single dose of sodium selenite to suckling rats. Experientia. 1978;34:222-223. 3. HessJL, Tarnawska E, Bunce GE. Selenite influences on adenylate pool in selenite-induced cataract in rat lens. Invest Ophthalmol Vis Sci. 1985; 26:304. 4. Wang Z, Hess JL, Bunce GE. Deferoxamine effect on selenite-induced cataract formation in rats. Invest Ophthalmol Vis Sci. 1992:33:2511-2519. 5. Huang LL, HessJL, Bunce GE. DNA damage, repair and replication in selenite-induced cataract in the rat lens. CurrEyeRes. 1990;9:1041-1050. 6. Wang Z, Hess JL, Bunce GE. Calcium efflux in rat lens: Na/Ca-exchange related to cataract induced by selenite. CurrEyeRes. 1992; 11:625-632. 7. Wang Z, Bunce GE, HessJL. Selenite and Ca2+ homeostasis in the rat lens: Effect on Ca-ATPase and passive Ca2+ transport. CurrEyeRes. 1993; 12:213-218. 8. Shearer TR, David LL. Role of calcium in selenite cataract. CurrEyeRes. 1983;2:777-784. 9. Bunce GE, Hess JL, Batra R. Lens calcium and selenite-induced cataract. CurrEyeRes.. 1984;3:315-320. 10. David LL, Shearer TR. Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest Ophthalmol Vis Sci. 1984; 25:1275-1283. 11. David LL, Wright JW, Shearer TR. Calpain II induced insolubilization of lens /3-crystallin polypeptides may induce cataract. Biochim Biophys Acta. 1992; 1139:210216. 12. David LL, Shearer TR, Shih, MJ. Sequence analysis of lens /?-crystallins suggests involvement of calpain in cataract formation./flwf Chem. 1992;268:1937-1940. 13. David LL, Azuma M, Shearer TR. Cataract and the acceleration of calpain-induced /3-crystallin insolubilization occurring during normal maturation of rat lens. Invest Ophthamol Vis Sci. 1994; 35:785-793. 14. Shearer TR, David LL, Anderson RS. Selenite decreases phase separation temperature in rat lens. Exp Eye Res. 1986;42:503-506. 15. Clark JI, Steele JE. Phase-separation inhibitors and prevention of selenite cataract. ProcNatlAcad Sci USA. 1992:89:1720-1724. 16. Siezen RJ, Benedek GB. Controlled modulation of the phase separation and opacification temperature of pu-
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
rified bovine 7lV-crystallin. CurrEyeRes. 1985;4:10771085. Delaye M, Clark JI, Benedek GB. Coexistence curves for phase separation in calf lens cytoplasm. Biochem Biophys Res Comm. 1981; 100:908-914. Thomson JA, Schurtenberger P, Thurston GM, Benedek GB. Binary liquid phase separation and critical phenomena in a protein/water solution. Proc Natl AcadSci USA. 1987;84:7079-7083. Broide ML, Berland CR, PandeJ, Ogun OO, Benedek GB. Binary-liquid phase separation of lens protein solutions. ProcNatlAcad Sci USA. 1991;88:5660-5664. Groth-Vasselli B, Farnsworth PN. A critical maturation period in neonatal-rat-lens development. Exp Eye Res. 1986:43:1057-1066. Mousa GY, Trevithick JR. Actin in the lens: Changes in actin during differentiation of lens epithelial cells in vivo. Exp Eye Res. 1979;29:71-81. Bidlingmeyer BA, Cohen SA, Tarvin TL. Rapid analysis of amino acids using pre-column derivitization. J Chrom. 1984; 336:93-104. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem. 1976:72:248-254. Thoft RA, KinoshitaJH. Metabolic studies on calcium transport in the mammalian lens. Invest Ophthalmol. 1965;4:122-128. Alder HL, Roessler ES. Introduction to Probability and Statistics. 6th ed. San Francisco: WH Freeman: 1977:330-343. Palmquist BM, Fagerholm P, Landau I. Selenium-induced cataract: A correlation of dry mass content and light scattering. Exp Eye Res. 1986;42:35-42. Latker CH, Kuwabara T. Regression of the tunica vasculosa lends in the postnatal rat. Invest Ophthalmol Vis Sci. 1981;21:689-699. Groth-Vasselli B, Farnsworth PN. Junctional alterations in lens epithelial cells during neonatal development in the rat. Lens Res. 1987;4:ll-26. Hammer P, Benedek GB. The effect of naturally occurring cellular constituents on phase separation and opacification in calf lens nuclear homogenates. Curr Eye Res. 1983; 2:809-814. Siezen RJ, Thomson JA, Kaplan ED, Benedek GB. Human lens -y-crystallins: Isolation, identification, and characterization of the expressed gene products. Proc NatlAcad Sci USA. 1987; 84:6088-6092. Hay RE, Andley UP, PetrashJM. Expression of recombinant bovine yB-, yC- and •yD-crystallins and correlation with native proteins. Exp Eye Res. 1994;58:573584. David LL, Shearer TR. State of rat lens sulfhydryl groups in selenite cataract. Toxicol Appl Pharmacol. 1984; 74:109-115. Shearer TR, Ridlington, Goeger DE, Whanger PD. Uptake of 75Se in cataractous rat lens proteins. Biol Trace Elem Res. 1984; 6:195-205. HessJL, Bunce GE, Mitton KP. Effects of selenite on
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37. 38.
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5 rat lens amino acid metabolism. ARVO Abstracts. Invest Ophihamol Vis Sti. 1994;35:2208. Kinoshita JH, Barber GW, Merola LO, Tung B. Changes in the levels of free amino acids and myoinositol in the galactose exposed lens. Invest Ophthalmol. 1969;8:625-632. Reddy DVN. Distribution of free amino acids and related compounds in ocular fluids, lens and the plasma of various mammalian species. Invest Ophthalmol. 1967; 6:478-483. Malone JI, Lovvitt S, Cook WR. Non-osmotic diabetic cataracts. Pediatr Res 1990;27:293-296. Mitton KP, Dzialoszynski T, Sanford SE, TrevithickJR.
Multiple antioxidant deficit in precataractous streptozotocin diabetic rat lens: A result of osmotic compensation? ARVO Abstracts. Invest Ophthalmol Vis Sti. 1994; 35:1570 39. ThurstonJH, HauhartRE, Dirgo JA. Taurine: A role in osmotic regulation of mammalian brain and possible clinical significance. Life Sti. 1980; 26:1561-1568. 40. Pasantes-Morales H, Wright CE, Gaull GE. Taurine protection of lymphoblastoid cells from iron-ascorbate induced damage. Biochem Pharmacol. 1985; 34:2205-2207. 41. Gabrielian K, Wang H, Ogden TE, Ryan SJ. In vitro stimulation of retinal pigment epithelium proliferation by taurine. Curr Eye Res. 1992; 11:481-487.
Purpose. Lenses from selenite-treated animals develop reversible "cold cataract" at a lower temperature than is required for lenses from age-matched control animals. This unexplained, stabilized phase transition is readily observed in intact lenses 36 to 48 hours after treatment and occurs in lenses before the appearance of the irreversible nuclear opacity observed 72 to 96 hours after treatment. The objective of this study was to investigate factors that may be responsible for this difference. Methods. Preweanling rats were injected with sodium selenite. Lens extracellular water volume was measured using 14C-inulin. Free amino acids were analyzed using precolumn derivatization and high performance liquid chromatography. Soluble protein was isolated from lenses of control, and treated animals and temperature-dependent changes in light scattering were measured at 490 lira. Results. Lens extracellular water was increased by the selenite treatment, with a concurrent 10% decrease in intracellular volume. Solutions of soluble protein from lenses of selenitetreated animals after postinjection hours 24 and 48 had higher critical phase transition temperatures (Tc) compared to similar proteins from control lenses. From 24 to 72 hours after injection, the free amino acid content of the lens increased 42%. Taurine levels were unchanged over the same period. The addition of 7 mM glycine and 7 mM proline to solutions of soluble protein (96 mg ml"1) decreased the phase transition temperature. Taurine (14 mM) had a similar effect. Combining taurine and the glycine plus proline solutions had an additive effect in lowering the T c . Conclusions. Increases in free amino acid concentration occur in lenses in response to the stress imposed by a systemic dose of selenite. The altered polyion content in lenses from selenite-treated animals, before nuclear cataract formation, contributes to the greater thermal stability of transparency in these lenses, thus lowering the temperature at which "cold cataract" forms. Invest Ophthalmol Vis Sci. 1995; 36:914-924.
S u b c u t a n e o u s injection of sodium selenite (Na 2 . SeO 9 ) in a single dose induces the formation of nuclear cataract in neonatal rats within 3 to 4 days. 12 The following changes 0 to 48 hours after injection precede cataract formation: increased selenium content, 1 decreased ATP content, 3 glutathione loss, 1 ' 4 increased N A D P + - N A D P H ratio, 1 elevated glycerol-3-
From the Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Btacksburg, Virginia. Supported by National Institutes of Health giant EY06123. Submitted for publication September 26, 1994; revised November 10, 1994; accepted November 11, 1994. IMtfnietary interest category: N. Repiint requests: J. L. Hess, Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State Uni sity, West Campu Drive, BUickslmrg, VA 24061-0308.
914
phosphate content,3 and DNA double-strand breaks.5 From 24 to 48 hours after injection, alterations occur in lens Ca2+ homeostasis: decreased Na + -Ca 2 + exchange,6 decreased Ca2+-ATPase activity, and increased Ca2+ permeability.7 The appearance of nuclear cataract 72 to 96 hours after injection is accompanied by some recovery from glutathione and ATP loss, increases in lens Ca2+ 8fl and inorganic phosphate content,0 increased insoluble protein content,1 and elevated proteolysis in the nuclear region of the lens.10 The current hypothesis regarding the nuclear cataract is that elevated Ca2+ activates calpain II and proteolysis of /3-crystallins, causing their insolubility.11 The lenses from selenite-treated rats contain /3-crystallins missing portions of their N-termini, similar to those
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5 Copyright © Association for Research in Vision and Ophthalmology
Phase Transition in Selenite Cataract tL I ^ !~
B» ^
j *.. ,..
' V Y
+•
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obtained by treatment with purified calpain in vitro.12 Mature, but clear, rat lenses contain similar amounts of insoluble y-crystallin and insoluble, fragmented /3-crystallin as are present in cataractous lenses from younger selenite-treated rats.13 This recent finding shows that this cataract does not depend solely on insoluble protein content. The developmental stage at which proteolysis occurs may also be a factor in the selenite model.13 Rat lenses exhibit a phenomenon called "cold cataract" in which phase separation occurs as a function of temperature. At 10 to 15 days of age, cold cataract appears below 32°C and is reversible upon elevation of temperature. The temperature of phase transition drops to 26°C in selenite-treated rats at 36 to 48 hours after injection14 but increases sharply above physiological temperature just before the emergence of nuclear cataract.15 Direct effects of temperature on light scattering properties of protein solutions provide information about protein-solute interactions. Solutions of bovine lens extracts or pure bovine ylV-crystallin display a temperature-dependent phase transition in vitro.16 This property of crystallin interactions may explain the formation of cold cataract in the intact lens.17 Two phases, a protein-rich phase and a protein-poor phase, coexist at and below the critical temperature (Tc) and cause light scattering.18 All y-crystallin amino acid sequences are extremely homologous. The high-Tc crystallins (yllla- and ylVa-) and low-Tc crystallins (ylland ylllb-), however, have very different maximum Tc values of approximately 35°C and 5°C, respectively.19 Lens age is an important factor for cataractogenesis in the selenite model. The rat lens is less susceptible to selenite after 16 days of age, 12 which corresponds to the end of the critical maturation period of the lens.20 During this period, the lens changes from a state of uniform hydration to a state in which the nucleus is less hydrated relative to the cortex.21 The decreased phase transition temperature in the selenite model before nuclear cataract formation suggests a transient environmental change of the lens cytosol. These studies were undertaken to examine the regional intracellular and extracellular hydration states of lenses from selenite-treated animals compared to agematched controls and also to examine how alterations in free amino acids correlated with the temperaturedependent phase transition characteristics of die intact lens. Temperature-dependent phase transition properties revealed early alterations of the soluble protein population before nuclear cataract formation. METHODS Cataract Induction Sprague-Dawley rats were housed in polypropylene shoe box cages in rooms maintained at 24°C and 80%
915 relative humidity with a 12-hour light-12-hour dark cycle. Animals were fed Agway 3000 laboratory rat chow (Agway, Syracuse, NY) and distilled water. Tenday-old animals (13-day-old animals for the phase transition temperature experiments) were injected subcutaneously with 20 mM sodium selenite (Na2SeO3), 0.9% NaCl, for a total dose of SOnmolg" 1 body weight (approximately 30 fi\ injection). Uninjected control animals from the same litter were used for all experiments. These animals served as appropriate controls because injections with 0.9% NaCl or 20 mM sodium sulfite do not cause lens pathology.1 At designated times after injection, animals were decapitated and lenses were removed immediately for extraction of free amino acids or proteins. Lenses were observed and photographed with an Olympus SZH zoom-stereo microscope equipped with a model SZH-ILLD illumination base (Olympus Optical, Tokyo, Japan). All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Amino Acid Extraction and Analysis Free amino acids were measured by precolumn derivatization and high-performance liquid chromatography.22 Pairs of lenses were removed by a posterior approach into 0.9% NaCl, blotted, and homogenized on ice in a 0.5 ml volume of 10% txichloroacetic acid using a glass Potter-Elvehjem homogenizer. Supernatant was collected after centrifugadon (10,000g X 5 minutes). Aliquots of supernatant were dried under vacuum (70 mTorr), combined 1:1 with norleucine internal standard (0.40 mM 0.1 N HC1), and again dried under vacuum (70 mTorr). The material was washed with 40% MeOH (vol/vol), 40% 1M sodium acetate, 20% triethylamine, and again dried under vacuum. Amino acids were converted to phenylthiocarbamyl amino acids by reaction with 10% phenylisothiocyanate, 70% MeOH, 10% triethylamine, and 10% H 2 O. Phenylthiocarbamyl amino acids were diluted in 0.07% Na2HPO4, pH 7.4, 5% acetonitrile, and filtered using microcentrifuge filters (Ultra-Free-MC; Millipore, Milford, MA) before chromatography. All detectable amino acids were quantified by calibradon with physiological amino acid standards (Sigma, St. Louis, MO) run with each set of analysis. The highperformance liquid chromatography system was a Waters WISP/PicoTag system using the Waters free amino acid analysis column (#10950, 3.9 X 300 mm). (Waters, Millipore)
Osmolarity Measurement Osmolality of buffers was measured using an Osmette A automatic osmometer (Precision Systems, Natick, MA) calibrated with 100 and 500 mOsm kg"1 standards .
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Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
Protein Determinations
Phase Transition Temperature Curves
Protein content was measured by using the Bradford dye-binding assay''2* using bovine serum albumin as a standard and by monitoring the A280:A260 ratio.
Phase transition was monitored at 490 nm as percent transmittance (%T) of protein samples at various temperatures. Samples were diluted with dialysis buffer to equivalent protein concentration (96 nig/ml" 1 ) with or without the inclusion of various amino acids as specified and transferred into 0.300 ml volume quartz cuvettes with teflon-plug lids to prevent evaporation. The microcuvettes were placed in the cuvette holder assembly of a Gilford Thermoset Temperature Controller installed in a Gilford 2600-UV/VIS-Spectrophotometer. The assembly included a bolt-on lid to minimize temperature gradients; integrated bidirectional, solid-state heat pumps; and an internal thermistor. The internal thermistor was calibrated for linearity from 0°C to 99°C with a National Bureau of Standards probe. Each cuvette measured 12.2 X 5.5 X 7.1 mm external dimensions. The exposed optical surface was 24 mm2, only 5% of the total surface area. The maximum surface contact with the temperature control block and the small sample volume ensured rapid thermal equilibration.
Determination of Lens Extracellular Space The extracellular volume in the rat lens was quantified by equilibrating lenses individually in Hank's medium containing inulin ('''C-carboxylated, 0.5 /xCi ml"'; DuPont NEN, Boston, MA), which is permeable to the lens capsule but not to the plasma membrane.'14 Uptake by lenses from age-matched control and selenitetreated rats was determined after 24 hours at 30°C. Lenses, suspended on a glass loop, were rinsed twice by dipping in unlabeled medium and blotting with moistened filter paper. l4C-counts were measured by liquid scintillation spectrometry at 95% to 98% efficiency. A zero-time value was subtracted from all measurements because 20% of the counts were accounted for by a rapid, irreversible binding of the inulin to the capsule surface. Distribution of inulin uptake between the capsule-epithelium-cortex fraction and the nuclear portion of the lens was estimated from dissection of lenses on Parafilm "M" (American National Can, Greenwich, CT), transferring the appropriate sections of the lens to filter paper, absorbing the associated liquid, and counting the fractions. Preparation of Lens Total Soluble Protein Extract Lenses were obtained as above and frozen using liquid nitrogen for storage at -80°C to obtain sufficient tissue for the protein extract (22 lenses/extract). Pooled lenses were homogenized on ice in a glass PotterElvehjem homogenizer after the addition of 0.5 ml of buffer; 100 mM sodium phosphate, 5.0 mM ethylenediaminetetraacetic acid, 0.01% NaNH, pH 7.2 with HO, (295 mOsm kg"1). The homogenate was warmed to 15° to 17°C during the homogenization. Samples from selenite-treated rats were processed simultaneously with their controls. Insoluble cell debris and capsular material were removed after centrifuging (12,000g"X 5 minutes) at 15°C. The supernatant fraction was transferred directly into Spectra/por-1 dialysis tubing (6 to 8 kd cutoff; Spectrum, Houston, TX.) and dialyzed against three 1-1 changes of buffer, equilibrated with argon gas. Dialysis and concentration steps were performed at 21°C. After 14 hours, samples were concentrated in dialysis tubing against Sephadex G-200 (Pharmacia Biotech, Piscataway, NJ) followed by 30 minutes of dialysis against buffer.
The cuvette holder temperature, measured with a thermometer, was within 0.15°C of the thermistor readout. Argon was introduced (450 cm' minute"') into the spectrophotometer cell thermal cavity to provide a dry atmosphere for operation between 1°C to 15°C. Light transmission was monitored from high to low temperature after equilibrating samples for 1.5 minutes at a new set temperature. All solutions were transparent at 36°C and were referenced to 100% T at this temperature. The phase transition data could be fitted to the sigmoidal equation, %T = (%Tmax X k(T° -
k(T° - a)b)
with an r2 > 0.997. Differences between phase transition temperature curves were evaluated using a balanced two-way analysis of variance and Duncan's new multiple range test.25 Seven to 10 temperatures that determined the solution transmittance properties between 76%T and 90%T blocks were used in the analysis of variance and served as the basis for comparing the average Tc between group treatments.
Isolation of Lens Nuclear-Cortex Tissue For obtaining primarily nuclear and primarily cortical tissue, the greatest concern was metabolic alterations to amino acid concentrations during dissection. A technique was developed that allowed for complete freezing of the very fragile neonatal rat lens and separation of tissue. After washing the lenses in saline and blotting gently, they were frozen quickly at the surface
uid nitrogen bath. The lens was held with preforceps and carefully pierced along the postenterior axis with a 27-gauge needle and cooled liquid nitrogen bath. A small scalpel blade was o shave off the equatorial cortex first, followed anterior cortex-epithelium and then the postertex so that the lens nucleus, a sphere of 1.5 emained. Tissue shavings were transferred to oth forceps cooled in liquid nitrogen. Isolated r and cortical samples, each from 12 lenses, ored at -80°C until preparation for amino acid s. Both samples contained perinuclear material.
LTS
rance of the Lens
ure 1, we present both a posterior and a side the lens from control and selenite-treated rats. ight hours after treatment with selenite, before r cataract formation, the posterior subcapsular t is apparent, as is the greater clarity of the r region at 28°C compared to the age-matched om control animals. The temperature must be sed to 24°C for the lens from selenite-treated s to have a similar cold cataract, as seen in these from the control animals.
Hydration: Intracellular and Extracellular
1 shows the weight data for lenses from control lenite-treated animals. Although lens growth enuated in the selenite-treated animals, no dife in the ratio of dry-fresh tissue weight was ed. Significantly greater extracellular water was ed in the lenses from selenite-treated animals 2). The increased extracellular volume ocin both the capsule-epithelium-cortex and r regions of the lens (Table 2) before nuclear t formation, 48 hours after injection (age 12 and when nuclear cataract had fully formed, 96 after injection (age 14 days).
o Acid Analysis
sults of amino acid analysis of whole lenses from e-treated animals and age-matched controls are ed for 6, 12, 24, 48, 72, 96, and 192 hours after on (Fig. 2). Elevation of total amino acids ocas early as 24 hours, well before the nuclear t stage. After postinjection hour 72, the total mino acid content was 142% of controls in lenses elenite-treated rats (38.1 ± 0.7 fimo\ g~l versus 0.9/miolg- 1 ). e ratio of amino acid content of nuclear to l tissue (Fig. 3) was less than unity for control at all three time periods examined. Although
FIGURE l. Appearance of lenses from age-matched control and selenite-injected rats, 48 hours after injection. (A) Posterior view of age-matched control (right) and treated (left group lenses bathed in saline at 30°C under bright-field illumination. The control lens shows cold cataract formation in the lens nuclear region, whereas the treated lens displays only the posterior subcapsular cataract that is oriented about the posterior suture. (B) Equatorial view of control (tight) and treated (left) lenses supported on glass rods, separated by 1.75 mm, in saline at 28°C under bright-field illumination. The control lens displays a nuclear cold cataract, now more obvious than in plate (A). The corresponding volume of the lens from treated animals is clear, whereas the posterior subcapsular cataract can be seen (bottom). The typica discontinuity in light refraction, seen as a ring about the perinuclear region, is visible in the lens from the treated group. (C) Dark-field illumination of the same lenses shown in plate B enhances the light scattering areas (white) an transparent areas (black).
total amino acids were elevated at 24 hours after injection (Fig. 2), the nuclear-cortical ratio remained less than unity in lenses from selenite-treated animals (Fig. 3a). However, after entry into the nuclear cataract phase (96 hours after injection), the ratio became greater than unity in the selenite-treated group. This ratio became similar to control values at 10 days after
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Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
TABLE l.
Selenite-Dependent Decrease in Lens Growth Control
(days) 11 12 13 14 21
Weight (mg)*(n) 9.2 10.8 11.5 14.3 16.2
± ± ± ± ±
0.2 0.2 0.4 0.3 0.5
Selenite Treated
Dry/Wet Ratio 0.27 dt 0.26 1t 0.28 it 0.29 1t 0.34 it
(8) (7) (9) (7) (9)
0.01 0.01 0.01 0.01 0.01
Weight (mg) (n) 9.2 10.4 11.1 12.7 13.0
± ± ± ± ±
0.1 0.3 0.3 0.3f 0.4f
(6) (6) (10) (10) (8)
Dry/Wet Ratio 0.27 0.26 0.28 0.29 0.34
± ± ± ± ±
0.01 0.01 0.01 0.01 0.01
Selenite treatment was administered on the tenth day after birth. Lenses were weighed immediately after enucleation and after drying at 100°C to constant weight. Values are the mean ± SE for a minimum of eight lenses in each determination using lenses from at least two different litters. * Fresh tissue weight for the number of lenses designated by (n). f Weight of lenses from selenite-treated animals was statistically less than for lenses of control animals (P < 0.05 West).
injection. The experiment was replicated for the 96hour time period and confirmed the reversed nuclear-cortical amino acid concentration ratio. As internal comparisons from the same analysis, two nonprotein amino acids—taurine and ornithine—had a nuclear-cortical concentration ratio of less than unity throughout this period (Fig. 3b). Phase Transition Studies In Vitro Plots of light transmission as a function of temperature, for soluble protein solutions from lenses of selenite-treated and age-matched control animals, are presented in Figure 4. Solution turbidity was stable and apparent when light transmission (490 nm) decreased to 80%T. Therefore, the Tc (°C) at 80%T was compared. Statistical comparisons were based on the average Tc calculated from this most stable region of transmittance change between 76%T and 90%T (Table 3). Once light transmission fell below 75%T, solutions displayed a continuously decreasing transmittance at constant temperature. Protein solutions (without
TABLE
amino acid addition) from lenses of selenite-treated animals had a Tc approximately 2°C greater than protein solutions from lenses of age-matched controls after both 24 and 48 hours after injection (Table 3, Fig. 4). This selenite-dependent difference is inconsistent with the formation of cold cataract at lower temperatures compared to control lenses (Fig. 1). Elevated amino acid concentration stabilized the transparency of lens soluble protein solutions, seen as a shift of the Tc to lower temperatures (Fig. 5). Solutions contained glycine, proline, and taurine concentrations similar to those in lenses from selenite-treated rats. Specifically, an equimolar mixture of glycine and proline (14 mM total) or taurine (14 mM) reduced the Tc of lens protein solutions from control and selenite-treated animals (Table 4A). Taurine was more effective than the equimolar glycine-proline mixture. Further effects on Tc were examined in a solution of taurine, glycine, and proline (28 mM total) (Fig. 6). There was an additive effect on the reduction of Tc of soluble lens protein in this solution (Table 4B). The
2. Selenite-Dependent Increase in Extracellular Lens Water Lens Water (yd/lens)* Extracellular
IntraceUular Animal Age (days)
Whole Lens
Control (12) Selenite-treated (12) Control (14) Selenite-treated (14)
1A 6.5 9.5 7.6
± ± ± ±
0.2 0.3 0.3 0.7
Whole Lens 0.59 1.2 0.6 1.4
± ± ± ±
0.06 0.06f 0.05 0.19f
Cortex Epithelium 0.45 0.95 0.45 1.2
Nucleus 0.14 0.28 0.17 0.27
Selenite treatment was administered on the tenth day after birth. * Volume, estimated by equilibrating lenses in HC-inulin and from tissue weights (1 mg = 1 /til), is reported as mean value ± SE; n = 6 for all measurements. Similar overall errors occurred with the dissected tissue, although variation was greater with the estimation of the nuclear volume in the cataractous (14-day) tissue. f Extracellular volume in lenses from selenite-treated animals was significantly less compared to this volume in lenses from control animals (P < 0.05, (-test).
rf
919
Phase Transition in Selenite Cataract
40 -
•
Control
D Se
•
p
30-
20-
10-
i
1I 11
I I 11111 Hours Post Injection
FIGURE 2. Selenite treatment causes elevated amino acid content in rat lenses. Total free amino acid content, including taurine, is reported for lenses from selenite-treated animals (at designated times after injection) and for lenses from age-matched, control animals. Error bars = SE, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001.
nucleus to abnormal levels of calcium that are required for nuclear opacification. Modifications of membrane lipids after exposure of the lens to selenite in vivo could also influence the cell-to-cell associations, permeability to ions and metabolites, and the distribution of lenticular water. The critical maturation period lasts from approximately 12 to 16 days of age and is characterized by a transient sharp increase in the rate of lens dehydration, a temporary plateau in growth in the equatorial diameter and the posterior-anterior axis, a primarily intracellular water loss with constant extracellular water, a decreased penetration of extracellular space by procion yellow, and a constant increase in lens dry weight (1 to 21 days).20 During maturation, the lens epithelium displays junctions of focal adhesions on basolateral borders of adjacent cells that decrease in number with age as desmosomes appear to replace
average Tc, from 80%T to 90%T, for protein solutions at 96 mg/ml or 93 mg/ml were not significantly different.
A ) Protein amino acids
r
V_
DISCUSSION Hydration Decreased growth (Table 1) characterizes lenses from selenite-treated animals.' The dry mass concentration at various points along the lens axis is not altered in the selenite model until 6 days after injection, after the formation of nuclear cataract.26 These lenses showed no change in the wet-dry weight ratio (Table 1); therefore, hydration effects, typical of an osmotic cataract, do not occur in this model. The extracellular compartment water volume increases before nuclear cataract formation in the capsule-epithelium-cortex and nuclear regions of the lens (Table 2). The posterior liquid-filled vacuoles identified by 2 days after selenite treatment26 are consistent with the increase in extracellular water in these lenses. Posterior delivery of metabolites and selenite would be provided by the tunica vasculosa lentis at this stage of lens development.27 The posterior subcapsular cataract and the open appearance of the posterior suture (Fig. 1) indicate that this region of the lens is susceptible to local increases in the extracellular space. These changes may reflect interrupted cell maturation so that normal cell-to-cell interactions do not occur. The posterior subcapsular cataract decreases before nuclear opacification, yet an abnormal distribution of extracellular space persists (Table 2). The nature of the extracellular space during early lens development and perturbations of this space in lenses from selenite-treated rats may provide exposure of the lens
Time Post-Injection (Hours)
• D B •
B ) Taurine & Ornithine
2
0.S
Control Taurine Se Taurine Control Ornithine Se Ornithine
6
Time Post-Injection (Hours)
FIGURE 3. Distribution of free amino acid content in rat lens reported as the nucleus-cortex ratio. (A) The ratios of the total, free protein amino acid concentrations (/zmol/g fresh tissue) in the nucleus and cortex are presented. Extracts were prepared from dissected lenses of selenite-treated rats (24, 96, and 240 hours after injection) and from lenses of age-matched, control animals. The cortex fraction includes the capsule, epithelium, and cortex. (B) Ratio of taurine and ornithine content in lens fractions as noted above.
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
920 100
80-
o -» •D *
40-
10
11
12
13
Temperature (°C) them.28 The decreasing susceptibility of lenses to systemic selenite exposure beyond 18 days of age may be a result of decreasing access of selenite into the extracellular space related to the regression of the tunica vasculosa lentis.27
Temperature-dependent Phase Transition Proteins. The temperature sensitivity of the phase transition of the intact rat lens decreases with age. This age-dependent change correlates with the lower Tc for solutions of protein isolated from "older" (by 24 hours) lenses of control and selenite-treated animals (Table 3, Fig. 4). Age-dependent changes in Tc have been observed in the bovine lens,29 probably in response to an altered distribution of proteins among the chaperone
TABLE 3. Increased Apparent Tc of Solutions of Proteins Isolated From Lenses of Selenite-Treated Rats Tc, °C*
At 80%
r
Average 80% ( T to 907,i T) (n= 7)
Group (hours PI)
24
48
24
48
Controlf Selenite-treatedf
11.9 13.9
11.3 12.9
13.9 14.7
11.7 13.9
* Values of Te at 80% T are estimated from the phase transition curves in Figure 4. In addition, the average Tc is calculated for seven temperatures determining 80% T to 90% T. The average Tc for solutions of protein from lenses of selenite-treated rats is significantly higher than for solutions of protein from lenses of control rats (P < 0.05). The average Tc for the samples from lenses of 15-day-old animals (48 hours after injection) is significantly lower than the comparable sample from 14-day-old rats (24 hours after injection) (P < 0.01, Duncan's test), f Soluble lens protein concentration of 96 ± 1 mg ml"'. PI = after injection.
Control 24 hrs Se 24 hrs Control 48 hrs Se 48 hrs
FIGURE 4. Selenite treatment destabilizes phase transition properties of solutions of soluble lens proteins. Transmission (%T at 490 nm) is plotted as a function of solution temperature. Solutions, containing 96.0 ± 1 mg protein ml"1 in 300 mOsm kg"1 buffer, are prepared with soluble proteins extracted from lenses of age-matched control and selenite-treated rats 24 and 48 hours after injection.
a-crystallins and the high-Tc and low-Tc y-crystallins. Modifications to existing proteins may also cause these effects, although the relationship between "y-crystallin primary structure and Tc is not known.30'3' The phase transition properties of the soluble proteins from lenses of selenite-treated rats cannot explain the requirement for a lower temperature to cause onset of cold cataract in these lenses (Fig. 1). These protein solutions became cloudy 2°C higher than solutions of proteins isolated from control lenses (Table 4). Protein modification has not been well characterized in lenses before nuclear cataract formation. No additional disulfide bond formation occurs in soluble proteins from the lenses of selenite-treated animals.32 Although elevated selenium content is associated with the crystallins in lenses from treated animals,33 there exists no more than one Se atom per 10,000 protein molecules.1 It is unlikely that this modification could account for the observed change in phase transition properties of these protein solutions. Other uncharacterized modifications to proteins must cause the increased Tc of solutions of protein isolated from lenses of selenite-treated rats. Amino Acids. Tc was decreased in vitro using elevations of amino acid concentrations similar to those found in vivo (Table 4A, Figure 5). Glycine and proline were selected because they displayed the largest individual increases in the selenite model in vivo.3'1 The effect of taurine was also examined because it is the most abundant small molecular weight organic substance in the rat lens.35'36 All three amino acids possess uncharged side chains and an a-amino and an acidic group; therefore, all are zwitterions at physiological pH. Altered water distribution across fiber cell membranes in the lenses from selenite-treated animals (Fig. 2) would additionally increase the effective intracellular concentration of free amino acids and, thus, favor a lower T c .
921
Phase Transition in Selenite Cataract
A) Control - O - Protein - • - Protein+7 mM Gly, 7 mM Pro -•*• Protein+14 mM Tau
E C
6 0-
Temperature (°C)
B) Treated FIGURE 5. Amino acids stabilize phase transition properties of solutions of soluble lens proteins. Amino acid additions—7 mM glycine and 7 mM proline or 14 mM taurine—decrease the Tc of solutions containing 96.0 ± 1 mg protein ml"'. Soluble protein is extracted from (A) age-matched control rats and (B) selenite-treated rats 48 hours after injection.
Taurine was more effective than an equimolar mixture of glycine and proline using extracts of the preweanling rat lens (Table 4A, Fig. 5). Taurine is also superior to glycine and glutamate in lowering Tc of soluble protein extracts from calf lens nuclear homogenates.30 The proportion of taurine to other amino acids increases substantially in the developing rat lens, from 15 mol% at day 14 to 32 mol% by day 20 (data not shown), and to about 75 mol% of the entire free amino acid pool in the adult rat lens.35'36 Taurine in the lens may function in osmoregulation.37 In the diabetic rat model of cataract, sorbitol elevation is balanced by decreased taurine concentration before initial vacuole formation38 and after vacuole formation.37 Taurine is a conditionally essential nutrient implicated in osmoregulation in the brain,39 membrane stabilization-antioxidation,40 and stimulation of cellular proliferation in retinal pigment epithelium.40 Our data support taurine's possible contribution to increased temperature stability (decreased Tc) of the lens fiber cytosol.
'rotein - • - Protein+7 mM Gly, 7 mM Pro ••*• Protein+14 mM Tau
10
13
14
Temperature (°C)
11
12
_/-
15
16
The ratio of amino acids contained in the nucleus to those in the capsule-epithelium-cortex fraction remained constant through the age of 23 days (240 hours after injection). Only in the lenses with nuclear cataract did the population of free amino acids become greater in the nucleus relative to the cortex (Fig. 3a). This effect was not seen with taurine and ornithine. These data suggest that extensive proteolysis in the lens nucleus leads not only to the accumulation of water insoluble products' 0 but also to the degradation of peptides to amino acids. Furthermore, these amino acids are not freely equilibrated throughout the lens at this stage of development. The addition of glutathione and amino acids lowers the Tc of calf lens nucleus extracts.30 Because glutathione content is decreased in lenses from selenitetreated rats,' it cannot account for lower Tc. Glutathione degradation may, in part, contribute to the elevated glycine content in the lenses from selenitetreated rats.
922
Investigative Ophthalmology & Visual Science, April 1995, Vol. 36, No. 5
TABLE 4. Amino Acids Decrease Estimated T c of Lens-Soluble
Protein Solutions A. Solutions containing 96 ±1 mg protein mT' Tc, °C* Average (80% Tto 90% T) (n= 7)
At 80% T Composition
Control
Proteint Protein + 7 mM gly, 7 mM Pro Protein + 14 mM Tau
Se-treated
11.3 11.0 10.9
Control a
11.9 11.6" 11.3 C
12.9 12.8 12.5
Se-treated 13.8a 13.7" 13.5"
B. Solutions containing 93 :t 1 mg protein'' Tc, °C* Averai •ye (76% T to
Composition Protein^ Protein + 14 mM Tau Protein + 14 mM Tau + 7 mM Gly, 7 mM Pro
At 80% T
90%^ T) (n = 10)
11.3 10.9 10.7
11.4" 11.0" 10.8c
Values of Tc (at 80% T) are estimated from the phase transition curves in Figures 5 and 6. In addition, average values are calculated from (n) values of temperature that determine 76% T through 90% T. Values within a column with different superscript letters are significantly different at P < 0.05. (Duncan's test). f Soluble proteins extracted from lenses of selenite-treated rats 48 hours after injection and agematched (15-day-old) control rats. X Soluble proteins isolated from 15-day-old control rats.
SUMMARY We consider three changes in lenses from selenitetreated rats that relate to the requirement for a lower temperature to cause "cold cataract" in these lenses: intracellular volume, amino acid concentration, and changes to soluble proteins. Of these, the selenite-dependent decrease in intracellular volume and the increase in amino acid content affect greater stability of
protein associations, detected as lower Tc in temperature-dependent changes in light scattering. Changes to me lens proteins, however, result in solutions that have a higher Tc. This unexpected result reveals that lens transparency may not be driven by the nature of protein properties alone but, rather, by their interac-tion with the environment as influenced by metabolite concentrations and the properties of cellular membranes.
8 0-
tt/
uf
6 0-
4 0-
O Protein A Protein+7 mM Gly, 7 mM Pro, 14 mM Tau • Protein+14mMTau
/ 2 0-
A i
i
i
11
12
13
Temperature (°C)
i
i
FIGURE 6. Additive stabilization, caused by amino acids, of the temperature-dependent phase transition of solutions of lens soluble proteins. Amino acid additions—7 mM glycine and 7 mM proline plus 14 mM taurine or 14 mM taurine—decrease the T c of solutions containing 93.0 ± 1 mg protein ml" 1 . Protein was extracted from lenses of 15-day-old control rats, comparable to 48 hours after injection.
Phase Transition in Selenite Cataract
923
Key Wards amino acids, cataract models, lens proteins, phase transition, selenite
17.
Acknowledgments The authors thank Renu Batra for completion of the inulin experiments, Kathy Smith for technical assistance, and Dr. Kenneth Webb and Kris Lee of the Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University for amino acid analysis. References 1. Bunce GE, HessJL. Biochemical changes associated with selenite-induced cataract in the rat. Exp Eye Res. 1981;33:505-514. 2. Ostadalova I, Babicky A, Obenberger T. Cataract induced by administration of a single dose of sodium selenite to suckling rats. Experientia. 1978;34:222-223. 3. HessJL, Tarnawska E, Bunce GE. Selenite influences on adenylate pool in selenite-induced cataract in rat lens. Invest Ophthalmol Vis Sci. 1985; 26:304. 4. Wang Z, Hess JL, Bunce GE. Deferoxamine effect on selenite-induced cataract formation in rats. Invest Ophthalmol Vis Sci. 1992:33:2511-2519. 5. Huang LL, HessJL, Bunce GE. DNA damage, repair and replication in selenite-induced cataract in the rat lens. CurrEyeRes. 1990;9:1041-1050. 6. Wang Z, Hess JL, Bunce GE. Calcium efflux in rat lens: Na/Ca-exchange related to cataract induced by selenite. CurrEyeRes. 1992; 11:625-632. 7. Wang Z, Bunce GE, HessJL. Selenite and Ca2+ homeostasis in the rat lens: Effect on Ca-ATPase and passive Ca2+ transport. CurrEyeRes. 1993; 12:213-218. 8. Shearer TR, David LL. Role of calcium in selenite cataract. CurrEyeRes. 1983;2:777-784. 9. Bunce GE, Hess JL, Batra R. Lens calcium and selenite-induced cataract. CurrEyeRes.. 1984;3:315-320. 10. David LL, Shearer TR. Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest Ophthalmol Vis Sci. 1984; 25:1275-1283. 11. David LL, Wright JW, Shearer TR. Calpain II induced insolubilization of lens /3-crystallin polypeptides may induce cataract. Biochim Biophys Acta. 1992; 1139:210216. 12. David LL, Shearer TR, Shih, MJ. Sequence analysis of lens /?-crystallins suggests involvement of calpain in cataract formation./flwf Chem. 1992;268:1937-1940. 13. David LL, Azuma M, Shearer TR. Cataract and the acceleration of calpain-induced /3-crystallin insolubilization occurring during normal maturation of rat lens. Invest Ophthamol Vis Sci. 1994; 35:785-793. 14. Shearer TR, David LL, Anderson RS. Selenite decreases phase separation temperature in rat lens. Exp Eye Res. 1986;42:503-506. 15. Clark JI, Steele JE. Phase-separation inhibitors and prevention of selenite cataract. ProcNatlAcad Sci USA. 1992:89:1720-1724. 16. Siezen RJ, Benedek GB. Controlled modulation of the phase separation and opacification temperature of pu-
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