Functional analysis of the promoter and chromosomal localization for human LEP503, a novel lens epithelium gene.

LEP503 is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503 may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503 is highly restricted to lens epithelial cells in vivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503 gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5' of the initiation methionine codon. By reporter gene transfection experiments, we found that approximately 2.5-kb of LEP503 5'-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp -401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between -401 and +22 showed that the AP-1 element at -131 and the Sp1 element at -48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503 mRNA, we found that the approximately 2.5-kb 5'-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503 promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503 gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.

Studies on lens proteins. I. Subunit structure of beta crystallins of rabbit lens cortex.

A method has been developed to isolate and characterize beta-crystallins of rabbit lens cortex. Chromatographic separation of water-soluble structure proteins of rabbit lens cortex on a Sephacryl S-200 gel column yielded four beta-crystallin peaks (beta1, beta2, beta3 and beta4), all eluting between alpha and gamma-crystallins. Their molecular weights were estimated to be 250,000, 130,000, 60,000, and 37,000 daltons, respectively. SDS-gradient gel electrophoresis of these beta-crystallins gave rise to characteristic polypeptides; beta1, two polypeptides of 30,000 and 23,000 daltons; beta2, one major polypeptide of 33,000; beta3; two polypeptides of 28,000 and 26,000; and beta4, two polypeptides of 22,500 and 11,200 daltons. From a knowledge of the molecular weights and the ratio of the polypeptides in each crystallin, their oligomeric structure was calculated to be 5:5, 4, 1:1, and 1:1. The relative abundance of these four beta-crystallins was found to be 25.6%, 7.2%, 27.2%, and 2.8% of the total water-soluble proteins of the lens cortex.

Ca++-induced cataract.

Cataracts in cultured rabbit lenses were produced by elevation of internal calcium. Experimental procedures were successful in increasing levels of total and bound Ca++, often without significant changes in sodium, potassium, or water content. Although the excess in calcium was predominantly associated with water-soluble proteins and was freely diffusible, a significant amount was bound to membranes and cytosol water-insoluble proteins. Thus, in lenses with a 10-fold increase in total Ca++, the bound Ca++ increased twofold, nearly 35% of which remained fixed to water-insoluble and membrane proteins after exhaustive (72 hr) dialysis. In contrast, over 95% of the Ca++ in water-soluble protein fractions was removed by dialysis.

Studies on lens proteins. III. Variations in polypeptides of lens beta-crystallins.

Relative amounts of two water-soluble beta-crystallin polypeptides with molecular weights of 26,000 (26K) and 28,000 daltons (28K) were measured in a number of species and also in different age groups of rats. The data indicate an age-dependent increase in the quantity of the 26K which is concomitant with a decrease in the 28K polypeptide. The ratio of these two polypeptides in the total water-soluble fraction is similar to that observed in the purified beta-crystallins. Purified beta 3-crystallin of the rabbit and its equivalent "beta L" of the bovine lens display charge heterogeneity upon DEAE chromatography, suggesting subtle qualitative changes in these crystallins. The presence of these two polypeptides in the urea-soluble fraction of lens preparations is also an indication of qualitative changes. The 26K polypeptide of beta-crystallins studied here is different from the 26K main intrinsic membrane protein in that, unlike the latter, it does not aggregate upon heating in SDS-buffer.

Lens membrane damage associated with cryoextraction.

The effects of cryoextraction on transport characteristics of rabbit lenses were evaluated by measuring Na+ and Ca++ levels, initial rates of 86Rb accumulation, and bioelectric potentials. The results demonstrate that membrane damage results from cryoextraction and is partially reversible during subsequent culture. While the decline in potential often exceeds 30 mV, maximum recovery of 80% occurs during a 1-hour incubation in TC199 at 37 C. Changes in the integrity of the membranes also are indicated by the finding that rates of 86Rb accumulation in cryoextracted lenses are approximately 44% less than the rates found in control lenses after a 20-hour culture in TC199. The degree of damage incurred by lens membranes also is reflected by a 10% increase in Na+ and 20% increase in Ca++ levels during 3 hours of culture. After extended culture periods, further increases in the concentration of Na+ (40%) and Ca++ (120%) were found to occur. Brief exposure of cryoextracted rabbit lenses to physiologic saline in a manner similar to that employed for photographic documentation of cataracts resulted in a marked decline in potential, 86Rb uptake, and an increase in Na and Ca content. In contrast, saline had little or no effect on these parameters in lenses excised by cutting the zonules. The results suggest that the observed decline in 86Rb accumulation in a limited number of cryoextracted cortico-nuclear cataracts compared with clear eye bank lenses may be attributed, in part, to damage to lens membranes by cryoextraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of X-irradiation on Na-K ATPase and cation distribution in rabbit lens.

The Na-K ATPase activity of rabbit lens was measured at various times after exposure to a single dose of 2000 rads of X-ray and was compared with that in contralateral control eyes. A decrease in enzyme activity in both whole lens and in isolated capsule-epithelium was first observed 3 to 4 weeks after irradiation and became increasingly marked at 7.5 weeks after X-ray. These findings are consistent with our earlier observations that active transport of cations is reduced in these lenses and support the view that loss of membrane ATPase is responsible for the impairment of the cation pump in X-irradiated lenses. Despite a significant loss of the enzyme, X-irradiated lenses were able to maintain near normal levels of total cations (Na+ + K+), thus accounting for their normal hydration. The results of the changes in lens Na+ and K+ levels revealed that between 4 and 7.5 weeks after X-ray, the gain in Na+ was compensated by an equivalent loss of K+. A breakdown of this relationship of 1:1 exchange of Na+ for K+ is accompanied by a disproportionate increase in Na+ and water.

Calcium transport in the lens.

Evidence based on the following three observations suggests the existence of a calcium transport system in the mammalian lens: calcium levels in the lens are lower than that measured in the aqueous humor; calcium efflux is temperature-dependent and is reduced by inhibitors of Ca++ transport; and there exists a calcium-acivated, magnesium-dependent ATPase. In rat, bovine, dog, and rabbit lenses, the concentration of total calcium was found to be approximately 0.2 mM, at least an order of magnitude lower than that found in the aqueous humor. To determine the nature of the mechanism responsible for maintaining these low levels, calcium fluxes were measured. During the initial rapid phase of 45Ca efflux, the rate at 4 degrees C was reduced by 85% compared with that found at 37 degrees C. Efflux was not altered in the absence of external Na+. Calcium efflux was reduced, however, by lanthanum and propranolol, inhibitors of Ca/Mg ATPase. The presence of Ca/Mg ATPase was also demonstrated in the rat, bovine, and rabbit lens and was likewise inhibited by both lanthum and propranolol.

The effects of X-irradiation on lens reducing systems.

Studies have been made of the effects of X-ray on various lens reducing systems, including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS) and of certain enzymes, including GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PG). It was found that during several weeks following X-irradiation but prior to cataract formation, there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the X-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the X-rayed lens was found to decrease significantly relative to controls 1 week prior to cataract formation, and the ratio of NADPH to NADP+ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the X-rayed lens. A corresponding decrease occurred in the activity of the HMS in X-rayed lenses as measured by culture in the presence of 1-14C-labeled glucose, G-6-PD was partially inactivated in the X-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to X-irradiation. The data indicate that X-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high-molecular-weight protein aggregates may be initiated.

Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture.

PURPOSE: A successful method to subculture human lens epithelial (HLE) cells that retain their intrinsic characteristics is of great importance. This study examines the effects of four different growth factors on proliferation and differentiation in HLE cells in early subcultures. METHODS: Specimens of HLE cells were obtained from infants. First- or second-passage cells were cultured in the presence of 10(-2) to 10(2) ng/ml acidic and basic fibroblast growth factor (aFGF, bFGF), epidermal growth factor (EGF), or insulin-like growth factor-I (IGF-I). Cell proliferation was determined from cell number, and fiber differentiation was assessed from the time of appearance, the number of lentoids formed, and the expression of gamma-crystallin. RESULTS: Cell proliferation was increased by EGF, bFGF, and IGF-I at concentrations greater than 10(-1) ng/ml; the most effective concentration was 10 ng/ml. The effect of aFGF on proliferation appeared only at a concentration of 10(2) ng/ml. EGF, bFGF, or IGF-I at 10 ng/ml affected the time of appearance and the number of lentoids formed within 5 to 7 days. In contrast, lentoids were observed after 42 days without the addition of growth factors. Lentoid formation was accompanied by the expression of gamma-crystallin. CONCLUSIONS: EGF, aFGF, bFGF, and IGF-I stimulated cell proliferation and fiber differentiation in early subcultures.

Hypertonic stress increases NaK ATPase, taurine, and myoinositol in human lens and retinal pigment epithelial cultures.

PURPOSE: Recent evidence suggests that taurine and myoinositol may serve as organic osmolytes in a number of cells, including lens and retinal pigment epithelia, but the mechanism for their increased accumulation in response to hypertonic stress is not known. To assess whether NaK ATPase contributed to the elevated levels of taurine and myoinositol in cells exposed to hypertonic media, we measured the activity of NaK ATPase, which is known to be implicated in the transport of these substances, in human lens and retinal pigment epithelia cultured in isotonic and hypertonic media. METHODS: Primary cultures of human lens epithelial (HLE) and human retinal pigment epithelial (HRPE) cells were maintained in isotonic and hypertonic media for varying periods of time, and the activity of NaK ATPase and the levels of taurine and myoinositol were measured in cells cultured under two different conditions. The possible involvement of the transport enzyme in the accumulation of the two osmolytes was also investigated by inhibiting the enzyme with ouabain. RESULTS: When primary cultures of HLE and HRPE were exposed to hypertonic medium containing NaCl (600 mOsm) or cellobiose (500m Osm) for 72 hours, the concentration of taurine and myoinositol in HLE cells increased by 218% and 558% of control, respectively, in NaCl medium, whereas the corresponding increases in cellobiose medium were 147% and 439%. In HRPE cells, the increase in myoinositol levels in the two hypertonic media was more dramatic than that in taurine. Concomitant with the increase in the concentration of the osmolytes, there was an increase in NaK ATPase activity in both cell types. Although the accumulation of taurine in HLE cells in hypertonic media in a 6-hour culture was essentially prevented by 10(-8) mmol/l ouabain, myoinositol levels were affected to a lesser, but still significant, extent. In HRPE cells, which were cultured for 24 hrs in the presence of 10(-6) mmol/l ouabain, there was a more direct correlation between the inhibition of NaK ATPase and the decreased accumulation of taurine and myoinositol in the hypertonic media. CONCLUSION: Although the exact mechanism by which NaK ATPase activity increases in response to hypertonic stress remains to be established, the increased activity of the enzyme is related to the enhanced accumulation of the organic osmolytes, taurine, and myoinositol, in HLE and HRPE cells cultured in hypertonic medium.

The effect of hypertonicity on aldose reductase, alpha B-crystallin, and organic osmolytes in the retinal pigment epithelium.

PURPOSE: Aldose reductase (AR), an enzyme implicated in diabetic complications of ocular tissues, has been suggested to play a physiologic role in kidney and, possibly, other tissues by elevating the organic osmolytes in conditions of heightened extracellular tonicity. Hypertonicity has been shown to induce AR and alpha B-crystallin in some cells. To examine if similar mechanisms are operating in the retinal pigment epithelium (RPE), another target tissue of diabetic complications, we studied the effect of hypertonic media on the induction of AR, alpha B-crystallin, myoinositol, taurine, and other free amino acids. METHODS: Human RPE cells were cultured in normal and hypertonic media containing 150 mmol/l NaCl or 200 mmol/l cellobiose in combination with 30 mmol/l galactose from 0-8 days. Western blot analysis with antibodies were used to measure the expression of AR and alpha B-crystallin. Hybridization of northern blots using AR and alpha B-crystallin complementary DNA probes were employed for the measurement of the respective messenger RNA for these proteins. Changes in the levels of myoinositol, galactitol, taurine, and other free amino acids were determined biochemically. RESULTS: AR and alpha B-crystallin messenger RNA levels rose 16-fold and 4-fold, respectively, when human RPE cells were cultured for 3 days in media supplemented with either 150 mmol/l NaCl or 200 mmol/l cellobiose. AR and alpha B-crystallin protein levels also increased significantly, as seen by western blots. Consistent with the increased AR, galactitol accumulated to a greater extent when human RPE cells were grown in media containing 30 mmol/l galactose plus 150 mmol/l NaCl compared with cells grown in 30 mmol/l galactose alone. An 11-fold increase in cellular myoinositol and a 1.4-fold increase in taurine was observed in cells exposed to hypertonic media. CONCLUSIONS: These findings suggest that human RPE cells are responsive to hypertonic stress by elevating AR activity and use intracellular organic solutes in an interactive manner to help regulate intracellular tonicity.

Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases.

PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.

The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium.

PURPOSE: High levels of ascorbic acid are known to be present in the aqueous humor of many diurnal species, whereas nocturnal animals have low concentrations of the compound. The purpose of this study was to test the hypothesis that the high concentration of aqueous ascorbate in diurnal animals protects the lens against ultraviolet (UV)-induced damage to the eye. This study compares the effect of UV-B-induced DNA strand breaks on the lens epithelia of guinea pigs and rats after depletion or elevation of aqueous humor ascorbate, respectively. METHODS: Eyes of guinea pigs and rats were exposed to UV-B radiation (0.25-0.75 J/cm2 on the cornea) for 10 minutes, and DNA strand breaks in lens epithelium were measured by single-cell gel electrophoresis. Ascorbic acid concentration in the aqueous humor, lens, and lens-capsule epithelium were assayed by spectrophotometric and electrochemical methods. For depletion of aqueous humor and lens ascorbate in guinea pigs, the animals were maintained on an ascorbate-deficient diet. Aqueous ascorbic acid was elevated in the rat by intraperitoneal injections of sodium ascorbate (1 g/kg). RESULTS: The ascorbate concentration in the aqueous humor of the normal rat was approximately 3% that of the guinea pig, whereas the concentration of the compound in the lens of the normal rat was 10% that of the guinea pig. Guinea pigs fed an ascorbate-deficient diet showed a dramatic drop of more than 80% in aqueous humor ascorbate in the first week, whereas lens ascorbate decreased by approximately 25% during this time period. After a single intraperitoneal injection of sodium ascorbate in the rat, aqueous humor ascorbic acid increased nearly 30 times that in the control, whereas lens ascorbate increased by approximately 30%. The extent of DNA damage in the lens epithelium of a normal rat exposed to UV-B was significantly greater than that occurring in lenses of normal guinea pigs after exposure to the same dose of radiation. Lenses from ascorbate-deficient guinea pigs showed 50% more DNA damage than those from normal guinea pigs after UV exposure, whereas the lenses in ascorbate-injected rats exhibited significant protection against UV-induced DNA strand breaks. CONCLUSIONS: High levels of ascorbic acid in the aqueous humor had a protective effect against UV-induced DNA damage to lens epithelium. The results were consistent with the hypothesis that high ascorbic acid in diurnal animals protects the lens against the cataractogenic effect of UV radiation in sunlight.

The role of glutathione metabolism in the detoxification of H2O2 in rabbit lens.

Aqueous humor is known to contain a significant level of H2O2, but the mechanisms by which ocular tissues protect against oxidative damage are not well understood. With the use of C-1-, C-2-, and C-6-labeled glucose, the contribution of glutathione (GSH) metabolism and the hexose monophosphate shunt (HMS) to the detoxification of peroxide in the lens has been evaluated. It was observed that H2O2 in the culture medium disappeared rapidly (0.5 mumol H2O2/lens/hr) upon incubation of a rabbit lens at 37 degrees C. At 0 degrees to 3 degrees C, however, the rate of disappearance of H2O2 was only one fifth of that observed at the higher temperature. In the absence of a lens or after pretreatment of the lens with methyl mercuric hydroxide, the rate of disappearance of peroxide from the medium was reduced to nearly zero. When a nearly constant level of H2O2 (0.05 to 0.07 mM) was maintained in the medium by means of a peristaltic pump, the amount of CO2 liberated by the HMS at 37 degrees C was found to be three times that liberated from lenses cultured in the absence of peroxide. No change was noted in the level of GSH in the H2O2-treated lenses at 37 degrees C. A significant decrease in GSH was observed, however, at 0 degrees to 3 degrees C, suggesting nonenzymatic oxidation of the tripeptide at the lower temperature. The results indicate that GSH metabolism and the HMS pathway contribute significantly to the detoxification of H2O2 in the lens.

Changes in the distribution of lens calcium during development of x-ray cataract.

The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks postx-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase.

Enhancement of differentiation of human lens epithelium in tissue culture by changes in cell-substrate adhesion.

Differentiation of human lens epithelial (HLE) cells cultured in vitro was drastically accelerated when the cells were cultured on cell-substrate adhesion-free surfaces. Spontaneous differentiation of the lens epithelial cells in monolayer cultures could be recognized with the appearance of lentoid bodies after 40-50 days if maintained without further passage. Although dissociated HLE cells reconstituted into monolayers consistently on the haptotactic substrates (either gold-coated biopore membrane or regular plastic dishes), the cells from the same batches exclusively formed cell aggregates when cultured on either biopore membrane or agarose-coated plastic dishes (nonhaptotactic). The cells on nonadhesion substrate first aggregate, then synchronously develop into lentoids by the 10th day of culture. The differentiation of HLE cells into lentoid structures with lens-fiber characteristics was documented by both ultrastructural and biochemical markers, such as loss of cytoplasmic organelles, formation of gap junctions, and the expression of gamma-crystallin and MP26. The system, in which differentiation of epithelial cells can be induced predictably in a short period of time, provides an excellent model for the study of differentiation and gene expression in human lens cells cultured in vitro.

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level.

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein synthesis in x-irradiated rabbit lens.

The present study deals with the incorporation of 35S methionine into lens crystallins as a function of time after x-irradiation. Crystallin synthesis is first affected approximately 4 weeks following x-irradiation. This coincides with the time period at which the ratio of the two cations in the lens is affected, as shown in earlier studies. A greater decrease in 35S-methionine incorporation into crystallins is observed between 5-7 weeks following x-irradiation in good agreement with a cation imbalance at these time intervals. These studies also revealed for the first time that the change in cation distribution can affect not only crystallin synthesis, but also the synthesis of certain polypeptides of lens membranes. No alteration in protein synthesis could be detected in lens epithelium even after 7 weeks following irradiation. In addition to the effect of Na+ and K+ levels on protein synthesis, an impaired transport of amino acids into the x-rayed lens was also found to be a factor in the observed reduction in synthesis of the crystallin, cytoskeletal and membrane proteins of the fiber cells. It is concluded that Na+/K+ ratio as well as the availability of amino acids in the lens are important factors in protein synthesis of x-ray cataracts.

Membrane glycoproteins of Philly mouse lens.

The Philly mouse is a new model for genetic cataracts, in which there is an apparent defect in lens membrane permeability. This abnormality results in electrolyte imbalance and lens hydration, typical of an osmotic cataract. Since membrane glycoproteins are believed to be involved in transport processes, we have studied the changes in these polypeptides in the Philly mouse lens during cataract development and compared them with those in the control Swiss-Webster mouse. The membrane glycoproteins were labeled by treatment with galactose oxidase and tritiated borohydride. Radioactivity was found to be incorporated into six glycoproteins of approximate molecular weights of 128 k, 103 k, 82 k, 71 k, 35 k, and 22 k daltons. The 35 k polypeptide is the major glycoprotein in the mouse lens membrane and shows increased incorporation of the tritium label with progression of the cataract. In contrast to the murine lens, the 35 k peptide could not be detected in rabbit lens membranes. Other changes observed in glycoproteins of the Philly mouse lens during cataract development were a loss of the 103 k and 71 k polypeptides and a corresponding increase in the 66 k polypeptide. These changes in glycoproteins may be related to the permeability changes and cataract development in the Philly mouse lens.

Synthesis of lens capsule in long-term culture of human lens epithelial cells.

PURPOSE: This study examined the extent to which human lens epithelial (HLE) cells in tissue culture retain the potential for differentiation, expression of lens-specific marker proteins, and the synthesis of lens capsule, the major characteristics of lens epithelium in vivo. METHODS: Primary cultures of HLE cells were maintained for up to 450 days. Transmission and immunoelectron microscopy were used to study the thickness of the synthesized capsule and the formation of type IV collagen and laminin, two major protein components of the basement membrane of lens capsule in vivo. RESULTS: In a long-term HLE culture system, without subcloning, lens fiber differentiation and capsular synthesis were maintained over a period of 450 days. In these cultures, the cell sheet showed three distinct zones: (1) a central zone with tight monolayer; (2) a mild peripheral zone with irregularly aggregated multilayer; and (3) a peripheral zone with loose monolayer. The basement membrane-like material was synthesized in the central zone and lentoids, which serve as a model for fiber differentiation, developed primarily in the mid peripheral zone. No capsular material or lentoids were observed in the peripheral zone. The capsule-like material was 2 to 2.5 microns thick and showed the presence of type IV collagen and laminin, as detected by antibody reaction. CONCLUSION: This study demonstrates for the first time that HLE cells in long-term cultures synthesize a continuous sheet of capsule-like material. The findings also suggest that reformation of a tight cell-to-cell relationship or generation of high cell density similar to that found in vivo may be an important factor for the synthesis of lens capsule.