[Deceleration of cataract development in rats under the action of N-acetylcarnosine and D-pantethine mixture].

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 estimation of age-related sensitivity of the retinal pigment epithelium of Japanese quail (Coturnix japonica) to the light damage].

The effect of blue light damage (445-455 nm, 4 J/cm2) to retinal pigment epithelium (RPE) subcellular structures was investigated in 4 age risk groups (9, 25, 40 and 52 weeks) of Japanese quail Coturnix japonica by light and electron microscopy. The indicator of biological aging of RPE was age-related accumulation of lipofuscin granules: 5-6-fold increase in their quantity increasing by 5-6 times in quails at 52 weeks. The main photo-induced changes observed after 24 h of the photo radiation were located in the blood-retinal barrier, such as loss of homogeneity of Bruch's membrane, disorganization of basal processes, deformations of the nuclei and mitochondria shapes. Those effects ofphotobleaching were more expressed in young birds. But for the older 52-week age birds it was not so noticeable, because their retinal pigment epithelium structures had disorders which were similar to those in younger birds after photodamage.

[Damage and functional recovery of the mouse retina after exposure to ionizing radiation and methylnitrosourea].

The eye retina consists of terminally differentiated cells that have lost their ability to proliferate. The death of these cells leads tothe loss of sight. The mice retina is characterized by relatively high resistance to radiation, which is provided by its ability to repair damage caused by environmental factors. The aim of our work was to assess the damaging effect of ionizing radiation and methylnitrosourea (MNU) on the DNA structure in the mouse retina, the functional activity of the retina, and its ability to recover in vivo. The results confirm the ability of the mature retina to structural and functional recovery. Adapting influence of low dose chemical agent increases retina resistance to cytotoxic dose of genotoxicants and prevents degeneration of photoreceptor layer of the retina. The results show the possibility of neurohormesis effect in the mice retina after exposure to ionizing radiation and chemicals.

[Experimental study of the influence of disturbing factors and chaperone-like drugs on cataractogenesis].

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.

[Galactic heavy charged particles damaging effect on biological structures].

A concept of the radiation risk of the manned interplanetary flights is proposed and substantiated. Heavy charged particles that are a component of the galactic cosmic rays (GCR) have a high damaging effect on the biological structures as great amount of energy is deposited in heavy particle tracks. The high biological effectiveness of heavy ions is observed in their action on cell genetic structures and the whole organism, including the brain structures. The hippocampus is the part of the central nervous system that is the most sensitive to radiation--first of all, to heavy charged particles. Irradiation of animals with accelerated iron ions at doses corresponding to the real fluxes of GCR heavy nuclei, to which Mars mission crews can be exposed, leads to marked behavioral function disorders in the post-irradiation period. To evaluate the radiation risk for the interplanetary flight crews, the concept of successful mission accomplishment is introduced. In these conditions, the central nervous system structures can be the critical target of GCR heavy nuclei. Their damage can modify the higher integrative functions of the brain and cause disorders in the crew members' operator performances.

[Carotenoids of the human eye in prenatal and early postnatal development].

The review deals with the role of carotenoids in the formation of the structural and functional differentiation of the macula--the area of the highest visual acuity of the human retina. The review also presents the data on detection of carotenoids (lutein) in the vitreous body of the human eye during its prenatal development and discusses their possible role in the development of the retina, particularly in relation to differentiation of the macular area. Macular dystrophy has been considered till recently as senile pathology. According to modern ophthalmologic observations, the number of cases of appearance of this pathology increases in young humans. Such a shift can be prevented by addition of carotenoids to the diet. This permits a conclusion that the permanent presence of carotenoids in the course of the whole human life is necessary for the formation and retention of structural and functional integrity of the macula.

[RPE melanosomes bind A2E fluorophore of lipofuscin granules and products of its photooxidation].

The ability of melanosomes from human, bovine and frog retinal pigment epithelium cells (RPE) to bind A2E fluorophore of RPE lipofuscin granules and products of A2E photooxidation is investigated. RPE melanosomes are found to bind A2E molecules themselves as well as the molecules formed after A2E irradiation by visible light. In our experiments single melanosome was able to bind up to 0.08 fmol A2E. Antioxidant activity of melanosomes is compared to antioxidant activity of their complexes with A2E. It is shown by luminal chemiluminescence quenching in the presence of hydrogen peroxide that in A2E/melanosomes complex the chemiluminescence quenching is not significantly reduced. Comparison of inhibitory activity of melanosomes and their complexes with A2E on UV-induced (light conditions) and Fe(2+)-ascorbate-induced (dark conditions) peroxidation of photoreceptor outer segments (POS) demonstrated that bound A2E does not affect inhibitory ability of melanosomes in both systems. Thus, binding of A2E to RPE melanosomes in concentrations from 0.01 to 0.1 fmol A2E per melanosome does not significantly alter their antioxidant properties. It is supposed that both A2E and hydrophilic products of its photooxidation could be bound by RPE melanosomes and, thus, it lost the ability to exhibit toxic properties.

[Experimental study of influence of different damaging factors on lens. Report 2. Features of microscopic lens changes].

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.

[Experimental study of influence of different damaging factors on lens. Report 1. Features of biomicroscopic changes].

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.

[Experimental study of influence of different damaging factors on lens. Report 3. Changes of lens protein composition].

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.

[Mechanisms of radioresistance in terminally differentiated cells of mature retina].

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.

[Comparative study of aging, UV treatment, and radiation on cataract formation].

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.

[Studies on the conformational state of the chromophore group (11-cis-retinal) in rhodopsin by computer molecular simulation methods].

Based on computer simulation methods, the molecular dynamics of the rhodopsin chromophore group (11-cis-retinal) has been analyzed. The molecular dynamics has been traced within a 3-ns time interval; thereby 3 x 10(6) discrete conformational states of opsin and rhodopsin were compared and analyzed. It was shown that, within a short time of about 0.3-0.4 ns from the start of simulation, the retinal beta-ionone ring becomes twisted around the C6-C7 bond by approximately 60 degrees compared with that of the initial configuration. The influence of retinal conformation on the positions of the maximum of the absorption band of rhodopsin at the conformational states of t=0 and t=3 ns were estimated using the ab initio methods. The results indicated that the absorption maximum for the final (3-ns) state is shifted by 10 nm toward the long wavelength region compared with the initial state. This suggests that the rhodopsin molecule with its twisted chromophore will possess a considerably lower activation energy than the rhodopsin molecule where the beta-ionone ring is in a planar orientation to the retinal polyene chain.

[Investigation of the mechanisms of crystallin aggregation induced by pulsed laser UV irradiation at 308 nm].

The results of the investigations of photoaggregation of the main eye lens proteins alpha-, beta- and gamma-crystallins and the model protein carbonic anhydrase in response to pulsed irradiation by a XeCI laser at 308 nm in the wide range of pulse energy densities (w) and pulse repetition rates (F) have been reviewed. A nonlinear dependence of aggregation efficiency on the values of w, F, and the concentration of protein solution was found. A theoretical model that qualitatively describes the experimental results was developed. The aggregation of N-amino-arm truncated beta A3-crystallin was analyzed. It was found that the loss of the N-amino-arm as a result of mutation or eye lens aging increases the probability of UV-induced beta-crystallin aggregation, thereby increasing the predisposition of eye lens to senile cataract. The influence of some short-chain peptides on the aggregation efficiency of beta-crystallin and beta-crystallin in solution with alpha-crystallin was investigated. Based on the results obtained, a combination of peptides (called "a new preparation") was found that most effectively delays the crystallin aggregation. The preparation has been probed on experimental animals. The trials showed that the preparation increases the delay in the development of UV-induced cataract in rats. The possibility of designing a drug for the prophylaxis of the development of cataract in humans based on this preparation is discussed.

[Search for chaperon-like anticataract drugs, the antiaggregants of lens crystallins. Communication. 1. Chaperon-like activity of N-acetyl carnosine dipeptide: in vitro study on a model of ultraviolet-induced aggregation of betaL-crystallin].

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.

[Like anticataract agents, the antiaggregants of lens crystallin. Communication 2. Study of the impact of chaperon-like (protective) activity of short-chain peptides on the rate of UV-induced aggregation of betaL-crystallins by eximer laser].

UV-induced aggregation of betaL-crystallin, one of the major lens proteins, was studied under its pulse radiation with XeCl laser at a wavelength of 308 nm. Unlike the in vitro tested dipeptides L-carnosine, N-acetyl carnosine, D-panthetine, and particularly their combination, the so-called new chaperon was demonstrated to slow down the rate of photoaggregatin of beta-crystallin. The new chaperon, a mixture of D-pathethine and N-acetyl carnosine was ascertained to protect a mixture of betaL- and alpha-crystallins from UV-induced aggregation to a greater extent than D-pathethine or N-acetyl carnosine used alone. An effective drug based on the new chaperon may be designed for the prevention of cataract in sight.

[Chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 4. Study of the effect of a mixture of di- and tetrapeptides on a prolonged rat model of UV-induced 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.

[Search for chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 3. Possibilities of a follow-up of caractogenesis processes on a prolonged rat model of UV-induced cataract].

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.

[Retina radioresistance: whole-body exposure of mice to gamma-irradiation induces the retina DNA damage, accumulation of p53 protein followed by DNA repair rather than the apoptosis].

Whole-body irradiation of mice with gamma-rays at 14 Gy causes DNA single and double strand breaks effectively repaired later. p53 is accumulated during the repair period. There is still some amount of DNA breaks 48-72 hours after the irradiation. Despite p53 accumulation and residual DNA lesions in the cells, mice retina demonstrated no morphological destructive changes or apoptosis signs. Retina resistance to apoptotic signals could derive from efficient repair of radiation-induced lesions in transcriptionally active regions of the genome of differentiated cells.