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.

Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins and K+ were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brj 58. In Triton X-100-treated cells, the loss of K+ was complete within 2 min. The loss of K+ from the Br" 58-treated cells was complete only after 10 min and the mobilization of K+ showed sigmoid-type release kinetics. These results support the view that most of K+ and "diffusible" proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell.In the past two decades, experiments with nonionic detergents (Triton X-100, Nonidet P-40, etc.) have considerably improved our knowledge about the composition and structure of the living cell (1-9). The cellular components may be divided into detergent-resistant and detergent-soluble elements. The detergent-soluble elements are the membrane lipoids, several proteins, smaller organic molecules, and most of the inorganic ions (1,8,9). The detergent-resistant cellular components are the "detergent-resistant cytoskeleton" composed of microtubules, intermediate filaments and microfilaments, nuclear matrix, chromatin, DNA-and RNAcontaining filaments, etc. (2-7). The release kinetics of the different cellular components solubilized by detergents are not known, and only a few follow-up studies have ever been performed on the detergent-treated cells (3, 10, 11).The major goal of our experiments was to perform a careful study on the release kinetics of K+ and proteins mobilized from tissue culture cells with Triton X-100 and Brij 58 nonionic detergents. The encouragement for these studies came from earlier reports that indicated similarities in the lipo-or membranolytic effect of Triton X-100 and Brij 58 detergents but differences in protein mobilization (7, 11). As confirmed by electron microscopic observations, both detergents remove the lipoid membranes within 2-5 min; however, the release of K+ was much faster in Triton X-100-treated cells than in Brij 58-treated cells. In the case of the Brij 58, although the membranes were removed-i.e., the cells were " opened" in 2-5 min-the release of K+ took place after several minutes delay. The parallel studies of protein release in response to detergent treatment demonstrated K+ release correlated more closely with the release of proteins than with the removal of the lipoid membranes. These findings suggest that cellular structures other than the membrane are also significantly involved in the maintenance of the extra-and intracellular K+/Na+ gradients. In addition, these findings support the view that the intracellular K+ ions are not freely dissolved in the cell water (12-18) and suggest that a cocompartmentation exists within the living cel...

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IntroductionBesides routine serum markers of inflammatory diseases, the diagnostic potential of selected urinary proteins has not been fully exploited yet. Former studies revealed that urinary orosomucoid (u-ORM) might have complementary information in inflammatory disorders. Our aim was to develop and validate a fully automated method for u-ORM measurements and to evaluate its potential clinical impact on systemic inflammatory diseases.Materials and methodsA particle-enhanced immune turbidimetric assay was validated for a Cobas 8000/c502 analyzer to determine u-ORM levels. Spot urine samples from 72 healthy individuals, 28 patients with Crohn’s disease and 30 septic patients were studied.ResultsOur assay time was 10 minutes and the detection limit of u-ORM was 0.02 mg/L. The intra- and inter-assay imprecision expressed as CV was less than 5%, and the recovery ranged between 95–103%. Within 10 to 60 years of age, a preliminary reference range for urinary orosomucoid/creatinine ratio (u-ORM/u-CREAT) was found to be 0.08 (0.01–0.24) mg/mmol [median (2.5–97.5 percentiles)]. Compared to controls, a five-fold increase of u-ORM/u-CREAT values in Crohn’s disease and approximately a 240-fold increase in sepsis were observed.ConclusionsWe set up a fast, sensitive and precise turbidimetric approach for automated u-ORM determination. Our highly sensitive assay is ideal for routine u-ORM measurements and might be a potential novel laboratory test in the management of systemic inflammatory processes.