Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Proteases and protease inhibitory activities in normal mammalian lenses and human cataractous lenses

Trypsin inhibition (reduction in benzoyl arginine p-nitroanilide hydrolysis), elastase inhibition (reduction in succinyl trialanyl p-nitroanilide hydrolysis), and chymotrypsin inhibition (reduction in acetyl tyrosine ethyl ester hydrolysis) by neutral extracts of mammalian lenses were estimated. The activities were found to be markedly elevated in human cortical cataract lenses compared to normal adult lenses (antielastase 7.21 +/- 3.90 units (mean +/- SD) in cataract compared to 1.46 +/- 0.57 in normals; antitryptic, 0.54 +/- 0.38 and 0.12 +/- 0.04; antichymotryptic, 1.03 +/- 0.61 and 0.297 +/- 0.055). Antielastase activity was distinctly higher in adult normal human lenses compared to infant lenses (0.159 +/- 0.068). Elastase- and trypsin-like activities were detected at low levels in all mammalian lenses. Chymotrypsin-like activity could not be observed in the lenses. The cataractous lenses had lower trypsin- and elastase-like activities compared to normal human lenses (elastase 1.20 +/- 0.643 in normal compared to 0.062 +/- 0.035 in cataract; trypsin, 0.367 +/- 0.154 and 0.069 +/- 0.038). The role of protease: inhibitor complexes in the expression of the individual activities and their role in cataractogenesis are discussed.     

CRE-decoy oligonucleotide-inhibition of gene expression and tumor growth

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF , both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.     

Cholesterol and lipid peroxidation in 3 beta-(2-diethylaminoethoxy)- androst-5-en-17-oneHCl (U18666A) induced cataractogenesis in rats.

Decrease in cholesterol was observed in precataractous, cataractous, advance nuclear cataractous and non-cataractous lenses when 3 beta-(2-diethylaminoethoxy)-androst-5-en-17- oneHCl (U18666A) was injected, sc, to rats. Significant increase in lipid peroxidation was observed before the onset of any apparent lenticular opacity in U18666A treated rats. The results suggest that decrease in cholesterol is capable of altering the structural integrity of lens fibers. However, 12.5% decrease in cholesterol and 5% increase in lipid peroxidation observed in non-cataractous lenses indicated that these changes are not sufficient for any apparent opacification.     

Determination of 19 elements in human eye lenses

Neutron activation analysis was used to determine the concentrations of 19 elements in normal and senile human cataractous lenses. It was found that the concentrations of Ca, Na, Cl, Eu, Sb, and Fe were significantly higher, and those of K, Rb, Cs, Cr, Mn, Co, Sc, and Ce were significantly lower in senile mature cataractous lenses than those in normal human eye lenses. No changes were found for the concentrations of Se, Zn, Mg, S, and Th in the two groups. Positive correlations between Na, Cl, and Ca and K, Rb, and Cs were found, whereas a significantly negative correlation between na, Ca, Cl and K, Rb, Cs were found. The roles of these elements in the evolution of cataract are discussed.     

Cataract produced by tyrosinase and tyrosine systems in rabbit lens in vitro

1. Cataracts were developed by incubating rabbit lenses for 22hr. at 37 degrees in a culture medium containing tyrosine and tyrosinase (EC 1.10.3.1). 2. A 45% diminution in the content of GSH and significant inhibition of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) activity were observed in the cataractous lenses compared with controls. 3. GSSG accumulated in both cataractous and control lenses. Significant amounts of GSSG were transported outward from the cataractous lenses and small amounts from control lenses. 4. Transport of GSSG from rabbit lens incubated in a diffusate of plasma from a naphthalene-fed rabbit was also observed. 5. GSSG was found in the aqueous humour obtained between 2 and 24hr. after feeding of naphthalene to rabbits; subsequently the GSSG in the aqueous humour decreased to almost undetectable amounts in 48hr.; in controls, GSSG was not detectable. 6. A possible mechanism of formation of experimental and senile cataract is briefly discussed.     

Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.     

Lens methionine sulfoxide reductase

Investigation of human and bovine lenses has demonstrated the presence of a methionine sulfoxide (Met(O)) peptide reductase activity. The reductase can use either dithiothreitol or thioredoxin but not glutathione as a reducing agent. The enzyme is present primarily in the water soluble fraction. The highest specific activity is in the outer epithelial layer with decreasing activity in the inner layers of the tissue. The known high level of methionine sulfoxide residues in cataractous lens protein is not due to a decreased level of Met (O)-peptide reductase itself since a comparison of normal and cataractous human lenses showed no statistically significant decrease in reductase activity in the cataract population. However, it is not known whether the reducing system for Met (O)-peptide reductase (probably the thioredoxin system) is deficient in cataractous lenses.     

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.     

正常与患白内障大鼠晶状体中脲溶性蛋白质性质的研究

本文研究了正常及三种类型白内障大鼠晶状体中脲溶性蛋白质的含量及性质的变化,发现在每种类型白内障晶状体中,水溶性蛋白质均减少,水不溶性蛋白质则都相对增加。经SephadexG-200柱层析及SDS聚丙烯酰胺凝胶电泳发现,晶状体中脲溶性蛋白质主要是由二硫键交联而成的高分子聚合物。经巯基乙醇还原后,绝大部分高分子聚合物可分解成低分子量蛋白质,其分子量与水溶性的γ晶体蛋白相同。这提示晶状体中脲溶性蛋白质的主要成分很可能是以二硫键交联而成的γ晶体蛋白聚合物。此结果与本实验室所得白内障晶状体水溶性蛋白质的变化相吻合。     

Mechanisms underlying increases in rat soleus Na+-K+-ATPase activity by induced contractions.

Acute regulation of the Na(+)-K(+)-ATPase activity in rat soleus muscle was investigated in response to 15 and 90 min of electrically induced contractile activity (500-ms trains at 30 Hz every 1.5 s). Kinetic measurements of Na(+)-K(+)-ATPase activity, assessed by the 3-O-methylfluorescein K(+)-stimulated phosphatase assay (3-O-MFP), were performed on crude homogenates (Hom) and on tissue separated into two membrane fractions, the sarcolemmal/particulate (SLP) and endosomal (En), in both stimulated (Stim) and contralateral control (Con) muscles. Maximal 3-O-MFP activity (V(max), nmol.mg protein(-1).h(-1)) was elevated (P < 0.05) in Stim by 40% and by 53% in Hom and by 37 and 40% in SLP at 15 and 90 min, respectively. The 38% increase (P < 0.05) in the alpha(2)-isoform subunit distribution in SLP at 15 min, as assessed by quantitative immunoblotting, persisted at 90 min, whereas for En a 42% decrease (P < 0.05) was observed only at 15 min. For the alpha(1)-subunit at 15 min, a 27% decrease (P < 0.05) was observed in En, whereas the 13% increase observed in SLP was not significant (P = 0.08). At 90 min, alpha(1) was increased (P < 0.05) by 14% in SLP and by 29% in En. No changes were observed in beta(1)-subunit distribution in En and SLP regardless of time of stimulation. Immunoprecipitation with antiphosphotyrosine antibody and quantitative immunoblotting with alpha(1)- and alpha(2)-antibodies indicated increases (P < 0.05) in tyrosine phosphorylation of 51% in alpha(2) at 15 min only. These results suggest that the increases in V(max) during contractile activity are mediated both by increased phosphorylation and by translocation of the enzyme to the plasma membrane.     

Elevated tropomyosin expression is associated with epithelial–mesenchymal transition of lens epithelial cells

Injury to lens epithelial cells (LECs) leads to epithelial–mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α‐smooth muscle actin (α‐SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up‐regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non‐cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α‐SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm‐based inhibitors for postponing PCO and cataractogenesis.     

Peroxide-metabolizing systems of the crystalline lens

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the surrounding medium was evaluated. Using a chemiluminescence method in a system of luminol-horseradish peroxidase and a photometric technique, the temperature-dependent kinetics of H₂O₂ decomposition by lenses were measured. The ability of opaque human lenses to catalyze the decomposition of 10^?4 M H₂O₂ was significantly decreased. However, this was reserved by the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H₂O₂ 10^?4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activities of glutathione reductase, glutathione peroxidase (catalyzing reduction of organic hydroperoxides including hydroperoxides of lipids), superoxide dismutase, but no signs of depletion in activities of catalase or glutathione peroxidase (utilizing H₂O₂). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses compared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens.     

Lipid peroxidation in cataract of the human

Lipid peroxidation was investigated as one of the possible mechanisms of cataractogenesis in the human. Malondialdehyde (MDA), a major breakdown product of lipid peroxides, was significantly higher in cataractous lenses as compared to that in normal lenses. 2-Thiobarbituric acid-reactive material, isolated from cortical cataracts and purified by Sephadex G-10 column chromatography, was identified as MDA. In cataractous lenses the enzymic defenses against reactive species of O2 were impaired as evidenced by the significant decrease in activities of superoxide dismutase, catalase and glutathione peroxidase. Hydrogen peroxide in aqueous humor and vitreous humor of human eyes associated with cataract was increased 2-3 fold. It is possible that carbonyl groups of MDA could interact with primary amino groups of proteins and phospholipids of lenticular plasmalemmae by a cross-linking reaction forming Schiff-base conjugates and these mechanisms might be involved in the pathogenesis of cataract.     

亚硒酸钠诱发大鼠白内障晶状体前列腺素生物合成的研究

 用亚硒酸钠诱发大鼠产生白内障后,将晶状体微粒体与外源性花生四烯酸共同孵育,用放射免疫方法测定白内障晶状体前列腺素E_2(PGE_2)及前列腺素F_2α(PG-F_2α)的生物合成情况,并与正常晶状体进行了比较,结果表明大鼠晶状体具有酶促合成PGs的能力。正常晶状体及白内障晶状体合成PGE_2的能力分别为687.75±113.97及1095.00±79.39pg/100mg晶状体湿重/15分钟,PGE_2α则分别为51.45±36.72及158.83±115.94pg/100mg晶状体湿重/15分钟(平均数±S.D.)。这说明大鼠白内障晶状体合成PGs的能力明显增高,与正常晶状体相比有显著性差异(PGE_2P<0.001,PGF_2αP<0.02)。在前2次注射亚硒酸钠后,大鼠白内障晶状体PGs的合成能力逐渐高于正常晶状体,并随注射亚硒酸钠的次数增加和白内障晶状体混浊程度加重,PGs在晶状体内的含量增加。     

Inversion and isomerization of Asp-58 residue in human alphaA-crystallin from normal aged lenses and cataractous lenses.

We have previously shown that L-Asp-151 in alphaA-crystallin from the human lens is converted to the biologically uncommon D-isomer. This process was not simple racemization, but stereoinversion, accompanied by isomerization to form the beta-Asp residue, such that L-beta-Asp, D-alpha-Asp and D-beta-Asp were formed. The present study shows that Asp-58 of human alphaA-crystallin is also converted to the D-isomer to a high degree to form the same isomers with age. The D/L ratio of beta-Asp-58 in aged normal lens increased to more than 3.0, showing stereoinversion by the 60 year range, then decreased to 1.0 in the 80 year range, while the isomerization of Asp-58 increased in the 80 year range. We also measured inversion and isomerization of the same residue from cataractous and normal human lenses of the 60 year range. The D/L ratio of Asp-58 from cataractous lenses was significantly lower than that from normal lenses, while the isomerization at Asp-58 in cataractous alphaA-crystallin was significantly higher than that of normal alphaA-crystallin. These results indicate that isomerization to the beta isomer of Asp-58 in cataractous alphaA-crystallin increased more than inversion to the D-isomer, suggesting that there are changes in the native structure of alphaA-crystallin in the human cataractous lens.     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses

Cataractous lenses have been found to have a distribution of the intracellular ionic environment, the concentrations of potassium and magnesium decreasing and the concentrations of sodium and calcium increasing relative to the cytosol of most cells. This arises as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. These changes have been found to be initiated as a result of normal ageing of the human lens. In this study, total Ca2+, K+, Na+ and Mg2+ contents have been determined in human normal and cataractous lenses using atomic absorption and flame emission spectroscopy. The normal human lens Ca2+ is between 0.15 and 0.5 miromol g(-1) fresh lens weight; in senile cataracts the value increased up to 9.31 micromol g(-1) ( p < 0.0001). The normal levels of Na+, Mg2+ and K+ are 20, 5.5 and 60 micromol g(-1) respectively; these changed to 136.10, 3.60 and 9.33 micro mol g(-1), respectively in cataractous senile human lenses ( p < 0.002, p < 0.002 and p < 0.01). The remarkable differences in these elements may play some role in cataractogenesis.     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.     

Lipid composition of lens plasma membrane fractions enriched in fiber junctions

Little is known about the lipid environment of lens fiber junctions, the plasma membrane structure proposed to be responsible for passage of low molecular weight metabolites between adjacent lens fiber cells. Plasma membranes of the ocular lens are especially rich in fiber junctions. The resistance of junctional domains to disruption by detergent or alkali treatment provides the opportunity to isolate a lens plasma membrane fraction enriched in fiber junctions. When examined by electron microscopy, the fiber junction fraction prepared from bovine lenses was enriched with junctional structures by about twofold when compared to total plasma membrane. We compared the protein, phospholipid, and cholesterol concentration of total plasma membrane with fiber junctional membrane from rat and cow lens and from aged normal cataractous human lenses. The principal finding was that junctional membrane contained 20-40% more total lipid than that of the total plasma membrane. This was due to a proportionate increase in the relative content (mg/mg protein) of both phospholipid and cholesterol. Exclusive of one exception (nucleus of bovine lens), the cholesterol/phospholipid molar ratios of the two fractions were similar. In the bovine nucleus, the cholesterol/phospholipid molar ratio was substantially higher in the fiber junctional-enriched membrane fraction than in the total plasma membrane, suggesting a special association of cholesterol with bovine nuclear fiber junctions. The relative lipid compositions of the plasma membrane and fiber junction-enriched fractions from human normal and cataractous lenses were similar, suggesting that human senile cataractogenesis involves changes in the lens plasma membrane more subtle than would be reflected by gross changes in the membrane lipid composition.