Distinct energy requirement for nuclear import and export of importin beta in living cells

Carbohydrates with reactive aldehyde and ketone groups can undergo Maillard reactions with proteins to form advanced glycation end products. Oxalate monoalkylamide was identified as one of the advanced glycation end products formed from the Maillard reaction of ascorbate with proteins. In these experiments, we have analyzed human lens proteins immunochemically for the presence of oxalate monoalkylamide. Oxalate monoalkylamide was absent in most of the very young lenses but was present in old and cataractous lenses. The highest levels were found in senile brunescent lenses. Incubation experiments using bovine lens proteins revealed that oxalate monoalkylamide could form from the ascorbate degradation products, 2,3-diketogulonate and L-threose. These data provide the first evidence for oxalate monoalkylamide in vivo and suggest that ascorbate degradation and its binding to proteins are enhanced during lens aging and cataract formation.     

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.     

Shortwave light filtration effect on spectral sensitivity of two shrimp populations of <Emphasis Type="Italic">M. relicta</Emphasis> (Mysida)

Microspectrophotometric measurements of screening granules in Mysis relicta eyes showed that most of the granules have xanthommatin spectra (7n_max 455 nm) with selective absorption of blue light. We calculated spectral sensitivity of M.relicta eyes using screening granules absorption spectra and visual pigment absorption spectra. According to our computations the calculated spectral sensitivity curve appears to be in a good correspondence with the real spectral sensitivity.     

The effect of eye colour pigments on the action spectrum of Drosophila

In vivo absorption spectra for Drosophila melanogaster eye colour pigment classes (drosopterins and ommatins) were constructed by subtracting the whole eye electroretinographic (ERG) spectral sensitivities of cn and bw respectively from the sensitivities of white-eyed strains. In situ microspectrophotometric (MSP) absorption spectra were also obtained. Both the ERG and MSP drosopterin spectra show a visible peak at 500 nm compared to the 480 nm peak of in vitro drosopterins. For the ommatins, the ERG absorption spectrum peaks at 450 nm while the MSP spectrum peaks at 400 and 525 nm. The ERG spectrum is similar to the in vitro absorption spectrum of xanthommatin while the MSP spectrum is similar to the in vitro absorption spectrum of reduced xanthommatin. The ERG absorption spectra for the drosopterins and the ommatins yield an accurate prediction of the effect of the combined pigments in wild-type eyes. Newly emerged and 7 day post-emergence bw flies show quantitatively similar pigment absorption effects while the drosopterins depress the sensitivity of newly emerged cn flies to a greater extent than that of cn flies 7 days after emergence.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins: comparison with pentosidine

Methylglyoxal (MG), an alpha-dicarbonyl compound, can be produced in vivo by several metabolic pathways and the Maillard reaction. It reacts rapidly with proteins to form advanced glycation end products or AGEs. We previously isolated and characterized a blue fluorescent product of the reaction between MG and arginine, which we named argpyrimidine. We found that argpyrimidine was stable to acid hydrolysis, which allowed us to hydrolyze tissue proteins with 6 N HCl and quantify argpyrimidine by high-performance liquid chromatography. Here we report argpyrimidine concentrations in human lens and serum proteins as determined by HPLC. We have also measured pentosidine, a fluorescent AGE derived from pentose sugars, and compared the concentrations of pentosidine and argpyrimidine. We found two- to threefold higher argpyrimidine concentrations in diabetic serum proteins than in nondiabetic controls (9.3 +/- 6.7 vs 4.4 +/- 3.4 pmol/mg). We found a significant correlation (P = 0.0001) between serum protein argpyrimidine and glycosylated hemoglobin. Argpyrimidine concentrations were approximately seven times greater in brunescent cataractous lenses than in aged noncataractous lenses. Pentosidine concentrations in serum and lens proteins were much lower than argpyrimidine concentrations; in general, argpyrimidine levels were 10--25 times higher than pentosidine. Results from our study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein-arginine modification by MG in aging and diabetes.     

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.     

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。     

晶状体生化的研究——亚硒酸钠诱发大鼠白内障晶状体中巯基、二硫键及维生素C的含量

我们测定了正常及亚硒酸钠诱发的白内障大鼠晶状体中非蛋白质巯基、蛋白质巯基、蛋白质结合巯基和维生素C的含量,发现随着白内障的进展非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基在核混浊时增加,而在整个晶状体混浊时下降到与正常对照组相近,在白内障形成过程中二硫交联的蛋白质含量明显增加,而维生素C含量似乎无明显变化。     

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.     

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.     

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Further studies on elastase and trypsin inhibitory activities in mammalian lenses

Elastase and trypsin inhibitory capacities increased significantly on heat treatment of the lens extract for 15 min at 60 degrees C in human infant (mean increase 290 and 335%), human adult (130 and 245%), ovine (90 and 140%), and bovine (70 and 90%) lenses. No increase was observed in human cataractous lenses. Preincubation with target enzymes in the absence of substrate abolished the antitryptic activity in lenses whereas antielastase activity was more resistant. No decrease in antielastase activity in human adult and cataractous lenses was observed on 15-min preincubation whereas about 50% of activity was abolished in human infant lenses. The differences were attributed to the changes in the levels of endogenous proteinases and proenzymes during cataractogenesis and aging.     

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.     

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

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.     

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.