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The review considers the problem of hydrogen peroxide decomposition and hydroxyl radical formation in the presence of iron in vivo and in vitro. Analysis of the literature data allows us to conclude that, under physiological conditions, transport of iron, carried out with the help of carrier proteins, minimizes the possibility of appearance of free iron ions in cytoplasm of the cell. Under pathological conditions, when the process of transferring an iron ion from a donor protein to an acceptor protein can be disrupted due to modifications of the carrier proteins, iron ions can enter cytosol. However, at pH values close to neutral, which is typical for cytosol, iron ions are converted into water-insoluble hydroxides. This makes it impossible to decompose hydrogen peroxide according to the mechanism of the classical Fenton reaction. A similar situation is observed in vitro, since buffers with pH close to neutral are used to simulate free radical oxidation. At the same time, iron hydroxides are able to catalyze decomposition of hydrogen peroxide with formation of a hydroxyl radical. Decomposition of hydrogen peroxide with iron hydroxides is called Fenton-like reaction. Studying the features of Fenton-like reaction in biological systems is the subject of future research.

α H -Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter ( D h ). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of D h of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.

β L -crystallin aggregation due to oxidative damage in the presence of H 2 O 2 and ferric chloride was studied in-vitro under conditions close to physiological. It was shown that the protein aggregation characterized by the nucleation time and the aggregation rate significantly depended on the composition of the isoosmotic buffers used, and decreased in the series HEPES buffer > Tris buffer > PBS. Ferric chloride at neutral pH was converted into water-insoluble iron hydroxide III (≡Fe III OH). According to the data of scanning electron microscopy the ≡Fe III OH particles formed in HEPES buffer, Tris buffer, and PBS practically did not differ in structure. However, the sizes of ≡Fe III OH floating particles measured by dynamic light scattering differed significantly and were 44 ± 28 nm, 93 ± 66 nm, 433 ± 316 nm (Z aver  ± SD) for HEPES buffer, Tris buffer, and PBS, respectively. It was found by the spin trap method that the ability of ≡Fe III OH to decompose H 2 O 2 with the formation of a • OH decreases in the series HEPES buffer, Tris buffer, and PBS. The authors suggest that the ability to generate • OH during the decomposition of H 2 O 2 is determined by the total surface area of ≡Fe III OH particles, which significantly depends on the composition of the buffer in which these particles are formed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.

α-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native α-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged β L -crystallin as the target protein. α-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged β L -crystallin. DSC investigation has shown that refolding of denatured UV-damaged β L -crystallin was not observed under incubation with α-crystallin. α-Crystallin and UV-damaged β L -crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged β L -crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged β L -crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75 kDa complex is indicative of α-crystallin dissociation. We suppose that α-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly β and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of α-crystallin, and thus preventing aggregation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The chronically exposure of eye lenses to ultra violet and visible light of the solar radiation is an important risk factor for development of the senile cataract diseases. Various photosensitizer molecules including riboflavin (RF) play a significant role in photo-oxidative damages of lens proteins underlying development of opacity in the lenticular tissues. In the current study, RF-mediated photo-oxidation of human αA-crystallin (αA-Cry) was assessed using SDS-PAGE analysis, dynamic light scattering and other spectroscopic assessments. The RF-photosensitized reactions led to non-disulfide covalent cross-linking, oligomerization and significant structural changes in αA-Cry. The photo-damaging of αA-Cry under solar radiation was also accompanied by the reduction in both Trp and Tyr fluorescence intensities which followed by the formation of new photosensitizer chromophores. The solvent exposed hydrophobic patches, secondary structures and chaperone-like activity of αA-Cry were significantly altered after exposure to the solar radiation in the presence of RF. Although glutathione and ascorbate were capable to partially protect the photo-induced structural damages of human αA-Cry, they also disrupted its chaperone function when co-exposed with this protein to the solar radiation. Also, the most promising data were obtained with cysteine which its availability in the lenticular tissues is a rate limiting factor for the biosynthesis of glutathione. Overall our results suggest that glutathione and ascorbate, as the major anti-oxidant compounds within lenticular tissues, demonstrate controversial effect on structure and chaperone-like activity of human αA-Cry. Elucidation of this effect may demand further experiments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

α-Crystallin is the major eye lens protein that has been shown to support lens transparency by preventing the aggregation of lens proteins. The 3D structure of α-crystallin is largely unknown. Electron microscopy, single-particle 3D reconstruction, size exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation were used to study the structure of the native α-crystallin. Native α-crystallin has a wide distribution in size. The shape of mass distribution is temperature-dependent, but the oligomers with a sedimentation coefficient of ~22 S (750-830 kDa) strongly prevailed at all temperatures used. A 3D model of native α-crystallin with resolution of ~2 nm was created. The model is asymmetrical, has an elongated bean-like shape 13 × 19 nm with a dense core and filamentous "kernel". It does not contain a central cavity. The majority of α-crystallin particles regardless of experimental conditions are 13 × 19 nm, which corresponds to 22S sedimentation coefficient, hydrodynamic diameter 20 nm and mass of 750-830 kD. These particles are in dynamic equilibrium with particles of smaller and larger sizes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70)., (Copyright © 2013 Wiley Periodicals, Inc.)

The effect of a mixture of N-acetylcarnosine and D-pantethine (1 : 1, m/m) on UV-A induced cataract in rats was studied. It is shown that instillation of a 5% mixture into the eyes or intraperitoneal injections (25 or 150 mg/kg) inhibit the formation of cataracts, starting from 82nd day of the experiment (p < 0.03), after which the protective effect of the mixture significantly increases (p = 0.0003). UV-A irradiation significantly (p < 0.01) increased the content of water-insoluble proteins in the lens. The use of the mixture of N-Acetylcarnosine and D-pantethine prevented (p < 0.001) an increase in the content of water-insoluble proteins caused by UV-A irradiation. Gel permeation chromatography data showed that, in the control group, water insoluble proteins consist of 3 fractions (40 kDa, 100 - 200 kDa, and1000 kDa). UV-A irradiation reduced the amount of protein in fraction 1 and increases the amount of protein in the fractions 2 and 3. The use of the mixture of N-acetylcarnosine and D-pantethine reduced the effects of UV-A light. The authors attribute the effect of the N-acetylcarnosine and D-pantethine mixture to their chaperone-like properties.

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively).

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A comparative experimental study of biomicroscopic appearance of lenses in cataracts of different genesis (age-related, ultraviolet and other radiation-induced or combined) has been performed on animals (mice). It is shown that identical lens opacification can be provoked by aging (endogenous factor), as well as ultraviolet and other radiation exposure (exogenous physical factors). The only differential sign is the severity of the damage. These factors can be arranged in the following ascending order by their damaging ability: aging --> ultraviolet --> gamma rays --> gamma rays + ultraviolet. Anti-cataract effect of a chaperone-like combined drug (N-acetylcarnosin and D-pantetin) has been studied in vivo on a "prolonged" model of induced cataract in rats. The use of the combined drug (1:1 mixture of the two peptides) in the form of ocular instillations and intraperitoneal injections helped slowing the progression of the ultraviolet-induced cataract in vivo.

The effect of histidine-containing dipeptides-carnosine and N-acetylcarnosine-on preventing and treating of cataracts of various etiologic origins has been demonstrated in many studies in vivo, while the precise molecular mechanism of their action is actually obscure. Cataract has been recently attributed to conformational diseases due to the association of lens structure protein aggregation with cataract pathogenesis. In our study, effect of histidine-containing dipeptides-carnosine, N-acetylcarnosine, and anserine-on the UV induced βL-crystallin aggregation was studied in vitro. It was first demonstrated that N-acetylcarnosine and anserine (10-40 mM) considerably suppressed UV induced aggregation of βL-crystallin, while carnosine exerted no effect. Positive correlation between anti-aggregating activity of the compounds used and their hydrophobicity was obtained. It was revealed that N-acetylcarnosine and anserine inhibited the initial stages of the protein photochemical damage. A decrease in the size of protein aggregates was detected in the presence of N-acetylcarnosine and anserine. UV irradiation of βL-crystallin resulted in a significant increase in the number of protein carbonyl groups, and the dipeptides studied did not affect this process. We suppose that N-acetylcarnosine and anserine inhibit βL-crystallin aggregation via formation of a protein-dipeptide complex that prevents macromolecular conformational changes and ensuing protein aggregation.

An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-β-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-β-CD reveals a purely protective effect., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Comparative study of lens biomicroscopy in cataract of different etiology (senile, ultraviolet, radioactive and combined ultraviolet- radioactive exposure) is performed in experiment on animals (mice). Lens opacification pattern was showed to be similar in aging as an endogenous factor and ultraviolet (UV) and radioactive exposure as exogenous physical factors. Specificity of these factors is expressed in degree of damage only. Depending on damaging potential the factors may be arranged in a following way (in increasing manner): aging, ultraviolet irradiation, gamma-irradiation, gamma+UV-irradiation.

Microscopic lens changes are studied in mice (F1C57B1XCBA) depending on age and after ultraviolet (UV), gamma-irradiation and their combination. In all animals compared to young 3-months animals nonspecific changes due to aging were revealed: microvacuoles in cytoplasm of fiber cells, flattening of epithelial cells and nuclei fragmentation, swelling and confluence of cortical layer cells in anterior and posterior subcapsular zones. In epithelial cells the following changes were noted: nuclei polymorphism, cavities in a cell layer formation of multilayered structures of fibroblast-like cells and cell desquamation. Degree of nuclei vacuolization was significantly lower in groups with gamma- and gamma+UV-irradiation but higher in a group of UV exposure. The number of cells per unit area was significantly decreased in a group of gamma-irradiation after 7 and 10 months of exposure. Aging and UV exposure caused decrease of nuclei area, whereas affect of gamma-irradiation depended on proliferative capacity of cells: size of central cells increased and of peripheral cells reduced. Specific changes were not revealed for any of the factors.

Using differential electrophoresis protein composition of lens major proteins in hybrid mice F1 (C57B1XCBA) with cataracts of different etiology (senile, ultraviolet, radioactive and combined ultraviolet-radioactive exposure) was studied Changes that may be specific for cataract caused by aging, ultraviolet and/or gamma-irradiation were not revealed in water-soluble and water-insoluble protein fractions.

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.

Thermal denaturation and aggregation of UV-irradiated β(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of β(L)-crystallin at 10 °С resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type β(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated β(L)-crystallin was identical to that for wild type β(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured β(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of β(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated β(L)-crystallin at 37 °С using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The structure of the oligomeric protein α-crystallin from bovine eye lens was investigated by small-angle neutron scattering (SANS) with contrast variation. Based on the SANS curves, the match point for α-crystallin (43% D2O) and its average scattering length density at this point (2.4·10(10) cm(-2)) were evaluated. The radius of gyration and the distance distribution functions for α-crystallin were calculated. On the basis of these calculations, it was concluded that α-crystallin is characterized by homogeneous distribution of scattering density in the domains inaccessible for water penetration, and all polypeptide subunits in α-crystallin oligomers undergo equal deuteration. The latter indicates that all α-crystallin subunits are equally accessible for water and presumably for some other low molecular weight substances. These conclusions on the α-crystallin structure (homogeneous distribution of scattering density and equal accessibility of all subunits for low molecular weight substances) should be taken into account when elaborating α-crystallin quaternary structure models.

Four randomized groups of male mice F1 (C57Black/CBA) were investigated: a) UV-irraidated (UV-A, 15 min daily during 9 months, dose 5.6 + 1.2 W/m2), b) gamma-rays irradiated (2 Gy), single, c) influence for combination of UV- and gamma-rays treatment, d) aging. The lens opacities were measured on 7th and 10th month. The expert method based on six grade scale was used for cataract measure. On 7th month the median of lens opacities were Aging group = 0; UV-irradiated group = 2.5; gamma-irradiated group = 4.75; gamma- and UV-irradiated group = 6.0. The difference between all groups was significant (p < 0.004, Kruskall-Wallis ANOVA test). The Conover post hock test has shown the significant difference for all comparison pairs (p < 0.002) with the exception of UV-irradiated group, gamma-irradiated group, gamma- and UV-irradiated group. On 10th month the lens opacities were strongly increased: Aging group = 2.5; UV-irradiated group = 5.0; gamma-irradiated group = 6.5; gamma- and UV-irradiated group = 7.5 (median). The difference between groups was significant (p < 0.0001, Kruskall-Wallis ANOVA test). The Conover post hock test has shown the significant difference for all comparison pairs (p <0.003) with the exception ofthe UV-irradiated group and gamma-irradiated group. The formation of the specific lens opacities for any group was not found. Morphology and protein composition have been investigated on 10th month. The results of morphological changes study show destructive and degenerative impairments of capsule, epithelium cells and lens fibers. However, no specific changes related to some particular actions have been found. In addition, there were no specific changes of protein composition of both water-soluble and water-insoluble fractions estimated with Differential Gel Electrophoresis technique. Obtained data mean that aging, UV-treatment, and gamma-radiation causes the similar changes of lens. It was supposed that UV-treatment or/and gamma-radiation act as an aging factor on the lens.

The kinetics of dithiothreitol (DTT)-induced aggregation of alpha-lactalbumin from bovine milk has been studied using dynamic light-scattering technique. Analysis of the distribution of the particles formed in the solution of alpha-lactalbumin after the addition of DTT by size showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with the hydrodynamic radius (R(h)) of 80-100nm. Further growth of the protein aggregates proceeds as a result of sticking of the start aggregates. Suppression of alpha-lactalbumin aggregation by alpha-crystallin is mainly due to the increase in the duration of the lag period on the kinetic curves of aggregation. It is assumed that the initially formed complexes of unfolded alpha-lactalbumin with alpha-crystallin were transformed to the primary clusters prone to aggregation as a result of the redistribution of the denatured protein molecules on the surface of the alpha-crystallin particles., (2009 Elsevier B.V. All rights reserved.)

Effects of alpha-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of alpha-crystallin or GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster-cluster aggregation to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower the unity. In contrast to alpha-crystallin, GroEL does not interfere with formation of the start aggregates which include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that the products of dissociation of GAPDH and alpha-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones at elevated temperatures.

The damaging effect of UV radiation (lambda > 260 nm) on bovine alpha-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an alpha-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, alpha-crystallin is able to prevent aggregation of destabilized proteins (especially of gamma- and beta-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of alpha-crystallin).

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster-cluster aggregation. Suppression of mAAT aggregation by alpha-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster-cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.

Effect of alpha-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of alpha-crystallin. According to the data of differential scanning calorimetry, alpha-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 degree C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of alpha-crystallin proceeds in the regime of reaction-limited cluster-cluster aggregation.

Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.

Aggregation ofcrystallins, the lens proteins, is one of the basic stages of cataract formation. Among the protein aggregation models used to study the molecular mechanisms of the initial stages of lenticular opacity, UV-induced aggregation of betaL-crystallin is most close to the in vivo conditions. The carnosine derivative N-acetyl carnosine has been shown to be effective in inhibiting the UV-induced aggregation of betaL-crystallin. Examination of the accumulation kinetics of carbonyl groups in betaL-crystallin under UV irradiation has indicated that neither carnosine nor N-acetyl carnosine fails to affect this parameter--an indicator of oxidative protein damage. By taking into account also the fact that N-acetyl carnosine is not an antioxidant, it can be believed that the molecular mechanism of action of this compound on UV-induced aggregation of betaL is unassociated with its antioxidative properties. The authors hypothesize that the molecular chaperon-like properties similar to those of alpha-crystallin underlie the mechanism of action of the acetyl derivative carnosine. The prospects for searching anticataract agents of a new chaperon-like class are discussed.

To study the mechanisms of action of new-generation anticataract drugs, it is necessary to have an accessible and adequate model of age-related cataract. A model of UV-induced cataract is pathogenetically closest to that of age-related cataract. A prolonged rat model of UV-induced cataract developing within 10 months is proposed; the clinical features of UV-induced cataract have been established at different stages of its development. A moderate homogeneous cloud-like lenticular opacity was observed at the end of the experiment; a less pronounced homogeneous opacity was seen in the anterior and posterior cortical layers. Cataract development was assessed by the appraisal method using the developed rat lenticular transparency scale, as well as by microdensitometry of biomicroscopic lenticular optical sections. Within the proposed model, the pathomorphological lenticular changes are largely similar to the histological pattern of age-related cataract.

There is a potential of therapeutic action on certain stages of caractogenesis, in particular on the aggregation of water-soluble proteins of cytoplasmic lens fiber cells, giving rise to insoluble protein complexes. The effect of a combined preparation (N-acetyl carnosine and D-patethine), acting by the chaperon-like mechanism, was studied in vivo on a prolonged rat model of UV-induced cataract. The use of the combined preparation consisting of a mixture of peptides of N-acetyl carnosine and D-patethine in a ratio of 1:1 as ocular instillations and intraperitoneal injections could slow down the development of UV-induced cataract in vivo. Pathomorphological studies suggest that the combined preparation has a protective effect on lens tissue when the rat model of UV-induced cataract is employed.

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed.

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.

Studies of molecular mechanisms of chaperone-like activity of alpha-crystallin became an active field of research over last years. However, fine interactions between alpha-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between alpha- and betaL-crystallins was studied with thermal denaturation of betaL-crystallin at 60 degrees C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography and electrophoresis. A mixed solution of alpha- and betaL-crystallins in concentrations about 10 mg/ml incubated at 60 degrees C was found to contain their soluble complexes with mean radius of gyration approximately 14 nm, mean molecular weight approximately 4000 kDA and maximal size approximately 40 nm. In pure betaL-crystallin solution, complexes were not observed at 60 degrees C. In SAXS studies, transitions in the alpha-crystallin quaternary structure at 60 degrees C were shown to occur and result in a double increase of the molecular weight. It suggests that during the temperature-induced denaturation of betaL-crystallin it binds with modified alpha-crystallin or, alternatively, alpha-betaL-crystallin complexation and alpha-crystallin modifications are concurrent. Estimates of the alpha-betaL-crystallin dimensions and relative contents of alpha- and betaL-crystallins in the complex suggest that several alpha-crystallin molecules are involved in complex formation.

The supramolecular structure of crystallins in intact ocular lenses of carp, frog and rat as well as in the interior (nuclear) and outer (cortical) parts of these lenses was studied by the small-angle X-ray scattering method. The results show that the supramolecular structure of crystallins substantially varies both in lenses of different vertebrate species and in various parts of the same lens. In carp lens and in the cortical part of rat lens, crystallins have an ordered supramolecular structure, as indicated by a small-angle X-ray diffraction maximum in the region of Bragg distances 15-20 nm, whereas in frog lens and in the nuclear part of rat lens, the supramolecular structure of these proteins is disordered. The power-law X-ray scattering by rat lens nucleus may be evidence of fractal structures in the lens. A comparison of these results with literary data indicates that there is no obvious correlation between the type of supramolecular structure of crystallins and their polypeptide composition in lenses of different vertebrate species. The results suggest that the supramolecular ordering (short-range order) of crystallins is not a necessary condition for lens transparency.

The effects of antioxidants (3-hydroxypyridines, 5-hydroxypyrimidines, hindered phenols) on platelet aggregation were studied. All the compounds under study possessed low anti-aggregation activity against indometacin-sensitive aggregation (activation with arachidonic acid, 50 M). Half-maximal inhibition of aggregation was achieved at a concentration similar to that of the compounds used (10(-3) M in cases of indomethacin-insensitive aggregation, platelet activation by thrombine 1.5 mu/ml and Ca2+-ionophore A23187 1.5 g/ml). 4-methyl-2.6-ditretbutyl phenol (BHT) in the concentration range of 10(-5)-4 X 10(-5) M inhibited and in the concentration range of 4 X 10(-5)-10(-4) M activated indomethacin-sensitive aggregation. The latter effect was not observed in the absence of Ca2+ ions in the incubation medium. It is concluded that the effects of the antioxidants studied on platelet aggregation were due to their non-specific action on platelet membranes.

The effects of 3-hydroxypyridine (3-HP) derivatives on platelet aggregation and platelet phosphodiesterase (PDE) of cyclic nucleotides (cAMP-dependent) were studied. It was shown that some derivatives of 3-HP inhibit platelet aggregation (the most pronounced effect was exerted by 2-benzyl-3- oxypyridine ). Several derivatives o 3-HP given in a concentration 10(-3) M were discovered to inhibit PDE by 40 to 75%. No correlation was found between the efficacy of 3-HP as antiaggregation agents and PDE inhibitors.

It has been shown that illumination of rod outer segment suspension in the presence of photosensitizers (methylene blue lambda greater than or equal to 620 nm; retinal 370 less than or equal to lambda less than or equal to 390 nm) results in chemical modification of the lipid and protein components of the photo-receptor membranes. This modification can be registered by accumulation of lipid peroxidation (LPO) products as well as oligomerization of rhodopsin and a decrease of rhodopsin thermal stability. These effects are prevented by 'O2-quenchers and free radical scavengers. It has been found that the electric activity (ERG) of isolated frog retina is inhibited due to photosensitized generation of 'O2 which can be overcome by preliminary addition of 'O2-quenchers and free radical scavengers to the incubation medium. The LPO products are accumulated in the retinae of rats exposed to high intensity light in vivo. It is concluded that 'O2 and LPO are involved in light-induced damage of the retina.

The effect of six 3-oxypiridine derivatives (at a concentration of 10(-3) and 5 X 10(-3) M) on cyclic nucleotide level in human platelets and platelet aggregation was studied. Five 3-oxypiridine derivatives were shown to depress platelet aggregation, four of them causing the increase in cAMP platelet level. The correlation between antiaggregation activity of 3-oxypiridine derivatives and their ability to rise cyclic nucleotide level in human platelets is discussed.

The effects of free radical scavengers of different chemical structure (hydroxypyridines, hindered phenols, hydroxyphenothiazines) on processes of 3H-hydroxytryptamine uptake and release by rat brain synaptosomes were studied. All free radical scavengers used inhibited 3T-hydroxytryptamine uptake and stimulated 3H-hydroxytryptamine release, with the efficacy being reduced in the following order: 7-hydroxyaminazine greater than butylated hydroxytoluene greater than paginol-2-methyl-6-ethyl-3-hydroxypyridine greater than alpha-tocopherol. The effects of free radical scavengers were eliminated in the presence of EGTA or after cooling to 4 degrees C.

The effect of synthetic (BHT, 2,2,5,7,8-pentamethyl-6-hydroxychroman) and natural (alpha-tocopherol) antioxidants on Ca++-transporting systems was compared in platelets, brain synaptosomes, and skeletal muscle sarcoplasmic reticulum. It was shown that synthetic antioxidants, in contrast to alpha-tocopherol, induced Ca++-release manifested in platelet aggregation, stimulation of 5-hydroxytryptamine release by synaptosomes, synaptosome depolarization and inhibition of Ca++-transport and Ca++-ATPase activity in the sarcoplasmic reticulum. The disturbances of Ca++-homeostasis induced by synthetic antioxidants are considered as molecular mechanisms of complications encountered upon their application.