ßA3/A1-Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis.

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell-matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated. We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for ßA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether ßA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express ßA3/A1-crystallin. The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of ßA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of ßA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that ßA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

Opacification of lenses cultured in the presence of Pb.

PURPOSE: Occupational and environmental Pb-exposure is associated with protein aggregation diseases which typically present in elderly populations (Parkinsons and cataract). Post-translational processing of crystallins, the major structural proteins of the lens, is altered with short-term Pb-exposure in Fisher 344 rats. In addition, lenses from aged rats become opaque upon long-term exposure to Pb in organ culture. To explore the route to lens opacification in the presence of Pb, cultured lenses from young rats which exhibit higher metabolic activity in lens culture and are more susceptible to experimental cataract in vivo and in vitro were exposed to Pb and evaluated for morphological and biochemical alterations. METHODS: Following culture in Pb (as lead nitrate) for four days (in the presence/absence of oxidative challenge), lenses were examined for clarity, integrity of epithelial layer, and molecular stability including crystallin post-translational modification and choline transport. Clarity of lenses cultured with/without Pb for up to 8 days was assessed to determine if Pb exposure would accelerate opacification. RESULTS: Lenses cultured in Pb for four days exhibited epithelial abnormalities including epithelial cell multilayering and nuclei abnormalities with extension of the nucleated epithelial cells past the bow region. Alterations in crystallin post-translational modifications and decreased membrane transport of choline were noted without corresponding lens opacification or altered α-crystallin chaperone activity. Lenses treated with Pb according to the same exposure protocol with subsequent challenge by hydrogen peroxide became opaque while the contralateral control lenses did not. Lenses which were cultured in the presence of Pb for longer periods with no subsequent oxidative insult exhibited lens failure at earlier time points than did the controls. CONCLUSIONS: These data indicate that Pb-exposure can accelerate the degradation of the cultured lens through induction of epithelial cell abnormalities, induce structural protein modifications before opacity, and predispose the lens to opacification with subsequent oxidant challenge.

Gene duplication and separation of functions in alphaB-crystallin from zebrafish (Danio rerio).

We previously reported that zebrafish alphaB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish alphaB-crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel alpha-crystallin transcript designated cryab2 and the resulting protein alphaB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human alphaB-crystallin and zebrafish alphaB1-crystallin, respectively. RT-PCR showed that alphaB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian alphaB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant alphaB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of alpha-lactalbumin and lysozyme. At 25 degrees C and 30 degrees C, zebrafish alphaB2 showed greater chaperone-like activity than human alphaB-crystallin, and at 35 degrees C and 40 degrees C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that alphaB2-crystallin makes up approximately 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different alphaB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish alphaB-crystallins perform divergent physiological roles. After gene duplication, zebrafish alphaB2 maintained the widespread protective role also found in mammalian alphaB-crystallin, while zebrafish alphaB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.

Pb2+ exposure alters the lens alpha A-crystallin protein profile in vivo and induces cataract formation in lens organ culture.

Epidemiological data supports lead exposure as a risk factor for cataract development. Previous studies which demonstrated oxidative imbalances in the lens following in vivo Pb(2+) exposure support the idea that lead exposure can alter the lens biochemical homeostasis which may ultimately lead to loss of lens clarity with time. alpha-Crystallin, a major lens structural protein and molecular chaperone, undergoes various post-translational modifications upon aging which may contribute to decreased chaperone function and contribute to loss of lens clarity. This study evaluated the impact of 5 weeks of oral Pb(2+) exposure (peripheral Pb(2+) level approximately 30 microg/dL) on the alphaA-crystallin protein profile of the lens from Fisher 344 rats. Decreases in relative protein spot intensity of more acidic forms of alphaA- and betaA(4)-crystallin and of truncated forms of alphaA-crystallin were noted. This data indicates that changes in post-translational processing of crystallins do occur in vivo following short courses of clinically relevant Pb(2+)-exposure. In addition, organ culture of lenses from 4.5-month-old rats in 5 microM Pb(2+) resulted in opacities, demonstrating that lead is toxic to the lens and can induce a loss of lens clarity.

Alterations in human vitreous humour following cataract extraction.

Cataract extraction is associated with the risk of posterior vitreous detachment, macular edema and retinal detachment possibly as a result of a disturbance to the vitreous body during surgery. While it is common for lens cortical fiber debris to leak into the vitreous humour during cataract extraction, the extent to which the vitreous humour is altered post-surgery is unknown. The current study examines the integrity of the vitreous humour of pseudophakic and phakic human donor eyes by comparing the proteome, the viscosity and the size distribution of macromolecules in different regions of the vitreous humour from human pseudophakic and phakic donor eyes. Major differences between the proteomes of anterior and posterior vitreous humour were observed in phakic and pseudophakic donor eyes. Seventeen spots identified as complete, modified or cleaved forms of alphaA-, alphaB-, betaA4-, betaB2, and gammaS-crystallins were present in the anterior vitreous humour of all pseudophakic eyes studied. Crystallins were not detected in the posterior vitreous humour of the pseudophakic eye or the vitreous humour of the phakic eye. Significant alterations in abundance and/or modification of transthyretin, alpha antitrypsin, and retinoic acid binding protein were observed in all locations of pseudophakic vitreous humour as compared to phakic samples. In addition, a significant decrease in the number and intensity of protein spots was observed for the posterior vitreous humour of pseudophakic eyes when compared to posterior vitreous humour of phakic eyes. Proteins which were affected include antioxidant proteins and enzymes such as carbonic anhydrase and trisephosphate isomerase. A reversal of the viscosity gradient, anterior to posterior, in the vitreous humour of pseudophakic eyes was observed concomitant with alterations in the distribution of 50 nm particles. These particles are likely primarily composed of hyaluronan. While varying degrees of vitreous degradation may have existed prior to surgery and may have contributed to the cataract formation, in no case did the phakic donor eyes exhibit the same alterations in the vitreous humour proteome, viscosity or particle sizes as did the pseudophakic donor eyes. The examination of phakic/pseudophakic donor eye pairs confirmed that the vitreous humour proteome and structural integrity were very similar in the matched phakic donor eye to eyes from donors with no history of cataract. Even though the number of samples for this study was limited, the observed changes support the hypothesis that alterations in the vitreous humour proteome occur in psuedophakic eyes with concurrent alterations in the structure of the vitreous humor. These modifications of the microenvironment of the retina may contribute to the development of retinal complications following cataract surgery.

A novel inflammatory eye disease induced by lymphocytes from knockout mice sensitized against the deleted ocular antigen.

Lens-associated uveitis (LAU), a severe inflammatory eye disease, is thought to be mediated by autoimmunity against lens crystallins. Previously described animal models for this disease are antibody-mediated, since no cellular response to self crystallins could be induced in experimental animals. Here, we describe a new model for LAU, in which lymphocytes from knockout mice deficient in alphaB-crystallin are sensitized against the deleted protein and induce severe ocular inflammation when adoptively transferred into wild type recipients. Similar to LAU, the experimental disease developed only following rupture of the lens capsule, produced in this study by capsulotomy; no disease was detected in recipient eyes with no capsulotomy, or in those treated with cautery, or in eyes affected by systemic treatment with sodium iodate, lipopolysaccharide or X-irradiation. The ocular changes in affected eyes included heavy cellular infiltration and proteinaceous exudate in both the anterior and posterior segments of the eye, that reached their peak on day 4 following cell transfer and subsided quite rapidly thereafter.

NMR relaxation studies of syneretic response to pressure change in bovine lenses.

PURPOSE: Reversible syneretic response to pressure in bovine and rhesus monkey lenses has been demonstrated previously by invasive techniques, such as differential scanning calorimetry and thermogravimetric analysis. This study is designed to investigate whether such a response could be observed by non-invasive techniques, namely by relaxographic imaging studies, in situ, in the intact, albeit excised lens. METHODS: Excised bovine lenses were incubated in media at 37 degrees C in specialized pressure chambers for 24 hrs. Three pressures, 2, 1 and 0.03 atm, were employed. The pressure chambers were placed in the cavity of an NMR magnet. Seven sections of the lens, under 2 atm pressure, from anterior outer cortex to posterior outer cortex were imaged and the T(1) (spin-lattice) and T(2 ) (spin-spin) relaxation data on each section were collected. The pressure was then released and NMR data were collected under 1 atm. Similar arrangement was followed on lenses under initial 0.03 atm pressure. T(1) and T(2) relaxations were analyzed by fitting pixel intensity to one and two term exponential expressions. RESULTS: Analysis of the time dependence of the T(2) relaxation time indicated that the response to a change in pressure is complete within 2 hours. Both T(1) and T(2) relaxation times showed minimal values in the nuclear region and maxima at the two outer cortexes. With increasing pressure both relaxation times decrease. The effect of pressure on both relaxation times was smaller in the nucleus and more enhanced at the outer cortexes. The pre-exponential terms of the fittings of both T(1) and T( 2) relaxations indicate the amount of protons participating in the relaxation. Thus they serve as a population index. The T(2) population index had a maximum in the nucleus and minima in the two cortexes. The population index of T(1) relaxation exhibited minimal value in the nucleus and maxima at the two cortexes. The pre-exponential term of T(2) relaxation increased with increasing pressure. The pre-exponential term of T(1) relaxation did not show consistent pressure dependence. CONCLUSIONS: The positional dependence of T(2) relaxation times as well as that of its population index indicated that it represents the behavior of the bound water in the lens. The positional dependence of T(1) population index suggests that this relaxation represents the total water that has a minimal value in the nucleus. Both the relaxation times as well as the population indices indicated that as pressure increases the strength of hydrogen bonding as well as the amount of bound water increases. This also means that the free water/bound water ratio decreases with increasing pressure. Thus NMR imaging and relaxation studies confirm significant syneretic response to applied hydrostatic pressure in bovine lenses.

Effect of perillic acid, a putative isoprenylation inhibitor, on the cultured rat lens.

Previous studies have demonstrated that agents affecting the cholesterol synthetic pathway can have cataractogenic effects. We have suggested that opacification of cultured lenses resulting from exposure to the cholesterol-lowering agent lovastatin is caused by inhibition of isoprenylation of small GTPases. To test that hypothesis we have investigated the effects of perillic acid, an agent reported to inhibit isoprenylation, on rat lenses in organ culture. Perillic acid caused dose and time dependent opacification of cultured lenses. While the opacities appeared grossly similar to those produced by lovastatin, they differed dramatically when analysed histologically. It also produced marked morphological changes to lens epithelial cells in culture. Analysis of small GTPases in the perillic acid treated cells failed to detect any accumulation in the water soluble fraction as would be expected if isoprenylation was inhibited. Further, studies on the isoprenylation of radiolabelled isoprenoids into proteins in cultured lenses showed no significant decrease following perillic acid exposure. It was concluded that perillic acid causes cataract in this system by a mechanism different from lovastatin and that inhibition of isoprenylation is unlikely to be a primary factor in the perillic acid cataract.

On the equilibrium between monomeric alpha-lactalbumin and the chaperoning complex of alpha-crystallin.

In chaperoning dithiothreitol-denatured alpha-lactabumin, alpha-crystallin forms a chaperoning complex. In order to study the kinetics of such chaperoning it needs to be established whether the formation of the chaperoning complex is a reversible or irreversible process. The chaperoning reaction was studied by dynamic light scattering as a function of concentration and weight ratio of alpha-lactalbumin/alpha-crystallin. HPLC and subsequent SDS-PAGE gel electrophoresis experiments established that the chaperoning complex formed contains both alpha-crystallin and alpha-lactalbumin. Upon rechromatographing the chaperoning complex, the presence of monomeric alpha-lactalbumin has been demonstrated in addition to the chaperoning complex itself. This and equilibrium dialysis experiments demonstrated conclusively the existence of an equilibrium between monomeric partially denatured alpha-lactalbumin and the chaperoning complex made of alpha-lactalbumin and alpha-crystallin.

Differential gene expression in male and female rat lenses undergoing cataract induction by transforming growth factor-beta (TGF-beta).

Epidemiologic studies in humans as well as immunohistologic studies in animals have demonstrated significant sex differences in the propensity to develop cataract. Several studies suggest that estrogen may play a protective role against cataractogenesis. Indeed, male and ovariectomized female rat lenses have a greater susceptibility to cataract induced by transforming growth factor-beta (TGF-beta) than do normal female lenses. However, in spite of the current evidence that estrogen may play a pivotal role in cataractogenesis, the molecular mechanisms behind this phenomenon are largely undetermined. Our study utilized the differential display procedure to examine gene up- and down-regulation in male, normal female and ovariectomized female rat lenses exposed to TGF-beta. Male and normal female rat lenses were cultured with or without 0.15 ng ml(-1)TGF-beta. Lenses were then harvested, and total RNA was isolated for analysis by reverse-transcriptase differential display. Differentially expressed mRNAs were subcloned, sequenced and identified through GenBank database searches. The original experiment was repeated with the addition of ovariectomized female TGF-beta(+/-) conditions, and all differential patterns of gene expression were verified using Northern blot and RT-PCR analysis. Screening of approximately 12% of the mRNA population led to the identification of 27 differentially expressed cDNAs. Notably, strong gender differences were found in expression levels of gammaB-crystallin. In addition, proteasome Z subunit was up-regulated in TGF-beta-treated male and ovariectomized female lenses, but was down-regulated in TGF-beta-treated normal female lenses. This pattern of expression is consistent with the increased susceptibility of male and ovariectomized lenses to TGF-beta-induced cataract. We conclude that differential display is a useful and expedient method for analysing changes in gene expression in the lens. Structural and functional studies of the genes identified in this study may further elucidate mechanisms underlying the TGF-beta-induced cataract formation and differential rates of cataractogenesis in males vs females. In particular, our data suggest that the role of proteasome Z subunit in TGF-beta-induced anterior subcapsular cataract warrants further investigation.

Use of a lipophilic cation to monitor electrical membrane potential in the intact rat lens.

PURPOSE: Tetraphenylphosphonium (TPP+) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP+ can be used for assessing membrane potential in intact lenses in organ culture. METHODS: Rat lenses were cultured in media containing 10 microM TPP+ and a tracer level of 3H-TPP+ for various times. 3H-TPP+ levels in whole lenses or dissected portions of lenses were determined by liquid scintillation counting. Ionophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP+ uptake. RESULTS. Incubation of lenses in low-K+ balanced salt solution and TC-199 medium, containing physiological concentrations of Na+ and K+, led to a biphasic accumulation of TPP+ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP+ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP+ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K+. The calculated membrane potential for the normal rat lens in TC-199 was -75 +/- 3 mV. Monensin (1 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM decreased TPP+ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine. CONCLUSIONS: TPP+ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP+ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.

High level of ferritin light chain mRNA in lens.

Ferritin is of particular interest with regard to cataract because (i) cataract occurs in individuals with hereditary hyperferritinemia cataract syndrome (HHCS), a condition in which ferritin light chain (L-ferritin) protein is overexpressed systemically, and (ii) ferritin is an important regulator of oxidative stress, a primary factor in the etiology of aging-related cataract. From gene array analysis two novel observations were made with respect to ferritin gene expression: first, lenses from guinea pigs and humans have disproportionately high levels of L-ferritin mRNA relative to the amounts of ferritin protein present, and second, L-ferritin message increased markedly in lenses from guinea pigs with hereditary nuclear cataract. The human lens L-ferritin sequence was identical to previous data from human liver; the guinea pig sequence was 86% identical to the human sequence at the amino acid level. Despite mRNA levels similar to those of major lens crystallins, lens ferritin was undetectable by Western blot techniques.

The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-crystallin performs such a function in the ocular lens. To gain insight into the mechanism of the anti-aggregation activity of alpha-crystallin, we performed dynamic light scattering (DLS) measurements investigating its interaction with partially denatured alpha-lactalbumin over a 24 hr period. Analyses were conducted as a function of the concentration of alpha-lactalbumin as well as the bovine alpha-crystallin/alpha-lactalbumin ratio. Additional studies of the systems were performed by HPLC and SDS gel electrophoresis. The particle distribution patterns derived from the DLS data indicated that the chaperoned complex (lactalbumin plus crystallin) is a loose fluffy globular entity. After the complex becomes saturated with lactalbumin, it appears to release the partially denatured lactalbumin which may aggregate into high molecular weight moieties. These eventually may precipitate out of solution. On longer standing, 24hr and over, the chaperoned complex as well as the lactalbumin aggregates become more compact. The chaperoned complex (alpha-crystallin plus alpha-lactalbumin) is in dynamic equilibrium both with the monomeric and the aggregated alpha-lactalbumin population.

Thiamine deficiency in vivo produces fiber cell degeneration in mouse lenses.

Thiamine (Vitamin B1) is a co-factor for enzymes key in bridging aerobic and anaerobic metabolism. One such enzyme, transketolase, catalyzes two of three reactions for entry into the pentose-phosphate pathway, a major source of chemical reducing power. Thus, thiamine deprivation (TD) is considered a classic model of systemic oxidative stress and is linked with degenerative diseases. TD in mice and rats produces neurodegeneration with Alzheimer's disease characteristics. Age-related disease of the lens, commonly cataract, is also linked with thiamine and oxidative stress. To test the effects of TD on mice, we used a previously defined protocol involving a thiamine free diet and a thiamine antagonist. After 12 days, lens fiber cell degeneration was observed primarily along the lens posterior beneath the intact capsule. These regions exhibited a localized increased expression of Alzheimer precursor protein, Abeta peptides, and presenilin 1. These data indicate that TD in mice produces fiber cell degeneration and suggest common mechanisms for TD-induced lens fiber and neuronal cell degeneration.

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Pressure-induced syneretic response in rhesus monkey lenses.

PURPOSE: To investigate the effect of pressure on the freezable and nonfreezable water content of the lens. METHODS: Excised rhesus monkey lenses in tissue culture media were subjected to three different hydrostatic pressures (2 atm, 1 atm, and 0.03 atm) for 24 hours. Then while still under the experimental pressure, the vessels were cooled in dry ice-acetone until the lenses were frozen. While the lenses were kept frozen, nuclear and cortical samples were dissected, enclosed in a sample pan, and weighed. Differential scanning calorimetry (DSC) measurements were performed between -30 degrees C and 30 degrees C. Total water content of each lens sample was obtained by thermogravimetric analysis at 105 degrees C. The nonfreezable water content was obtained by subtracting the freezable water content calculated from the DSC data from the total water content. RESULTS: The total water content of the lenses did not change significantly as a function of pressure applied. This was true both for cortical and for nuclear sections. The freezable water content increased as the pressure decreased both in cortex and nucleus. Similarly, the freezable water/nonfreezable water ratio also decreased with increasing pressure. CONCLUSIONS: External hydrostatic pressure would generate an influx of water into the lens. To alleviate this diluting tendency and to prevent turbidity as a result of dilution, the lens must effect an osmotic pressure change equivalent to the applied pressure. Change in the osmotic pressure is caused by changing the activity of the water (i.e., converting free water to bound water). This is a reversible and energetically the least expensive response. The release of bound water from the hydration layers of macromolecules and its conversion to free water in condensed systems are known as syneresis. In the lens decreasing pressures induce syneresis as demonstrated by the increase in freezable water content and the freezable water/nonfreezable water ratio. Such a response may be operative also in accommodating lenses.

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.

Oxidative stress induces differential gene expression in a human lens epithelial cell line.

PURPOSE: To identify differentially expressed genes in a human lens epithelial cell line exposed to oxidative stress. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) differential display was used to evaluate differential gene expression in a human lens epithelial cell line (SRA 01-04) when cells were exposed for 3 hours to a single bolus of 200 microM hydrogen peroxide. Differentially expressed genes were identified through DNA sequencing and a nucleotide database search. Differential expression was confirmed by northern blot and RT-PCR analyses. RESULTS: Using 18 primer sets, 28 RT-PCR products were differentially expressed between control and hydrogen peroxide-treated cells. In stressed cells, mitochondrial transcripts nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 and cytochrome b were downregulated 4-fold. Of the cytoplasmic mRNAs, glutamine cyclotransferase decreased 10-fold, whereas cytokine-inducible nuclear protein, alternative splicing factor 2, and beta-hydroxyisobutyryl-coenzyme A hydrolase increased 2-, 4-, and 10-fold, respectively. Analysis of mitochondrial transcripts in a 24-hour time course showed that NADH dehydrogenase subunit 4 mRNA decreased by 2-fold as early as 1 hour after oxidative stress, whereas the rate of decrease was slower for cytochrome b, cytochrome oxidase III, and 16S rRNA. CONCLUSIONS: Oxidative stress induced specific expressed gene changes in hydrogen peroxide-treated lens cells, including genes involved in cellular respiration and mRNA and peptide processing. These early changes may reflect pathways involved in the defense, pathology, or both of the lens epithelium, which is exposed to oxidative stress throughout life.

The effects of digitalis-like compounds on rat lenses.

PURPOSE: Fundamental to the maintenance of ionic concentration gradients and transparency of the lens is the activity of Na+,K+-adenosine triphosphatase (ATPase) in the epithelial layer. Recent studies have identified endogenous digitalis-like compounds (DLCs) and 19-norbufalin and its peptide derivatives in human cataractous lenses. These compounds inhibit the activity of Na+,K+-ATPase and have been suggested to be involved in cataract formation. The present experiments were designed to test this hypothesis by determining the ability of digitalis and DLCs to induce changes in protein composition and leakage from rat lenses in organ culture. METHODS: DLCs were determined in rat lenses using three independent assays: interaction with ouabain antibodies, interaction with bufalin antibodies, and inhibition of [3H]-ouabain binding to red blood cells. Rat lenses were incubated in modified TC-199 medium in 5% CO2 atmosphere at 37 degrees C for the time of the experiment. The onset of cataractogenesis was assessed by measuring protein leakage from lenses and by crystallin composition in the lens and media. RESULTS: DLCs were present in rat lens with concentrations 7 to 30 times higher in the capsular-epithelial layer than in the lens fibers regions. Ouabain, bufalin, digoxin, and DLC induced dose- and time-dependent leakage of protein from rat lenses. Lenses incubated with these compounds showed alterations in crystallin content consistent with changes that initiate opacity. All the compounds caused a multilayering of epithelial cells in the region surrounding the mitotic area and, at the same time, cell death in the central anterior region. CONCLUSIONS: Digitalis and endogenous DLCs are cataractogenic factors. These results, together with the demonstration of DLCs in the normal lens and their increased levels in human cataractous lenses, strongly suggest their involvement in the molecular mechanisms responsible for cataract formation.

Betaine-homocysteine methyltransferase is a developmentally regulated enzyme crystallin in rhesus monkey lens.

We describe herein the characterization of a major 45-kDa protein from the soluble betaH-crystallin fraction of rhesus monkey (Macaca mulatta) lens. Based on partial peptide sequence, immunoreactivity, and enzymatic activity, this protein has been identified as betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5), an enzyme that catalyzes the methylation of homocysteine using either betaine or thetins as methyl donors. This protein was found to be expressed abundantly in the nuclear region of the monkey lens, reaching approximately 10% of the total nuclear protein, but was barely detectable in the epithelium and cortex regions of the lens. Because the nucleus represents the early embryonic and fetal stages of lens development, we infer that BHMT expression in the lens of the eye is developmentally regulated. By virtue of its high abundance, BHMT can be considered an enzyme crystallin (psi-crystallin). This is the first enzyme crystallin to be found in primate lenses.