[Oxidative stress in uveitis and its correction with superoxide dismutase antioxidative enzyme (experimental study)].

OBJECTIVE: to study the influence of experimental uveitis on those biochemical parameters of aqueous humor that reflect inflammation acuity as well as local antioxidant and local antiproteolytic activity; to study the effect of topical superoxide dismutase (SOD) on the clinical course of uveitis and ocular metabolism. MATERIAL AND METHODS: Acute uveitis was induced in rabbits by a double injection (subcutaneous and intravitreal) of normal horse serum. The following parameters of aqueous humor were measured: protein concentration, antioxidant activity, SOD activity, alpha2-macroglobulin level, total nitrates and nitrites, and leukocyte number. Clinical assessment and histopathological study were performed. RESULTS: It was found that uveitis is associated with a statistically significant increase in protein concentration, leukocyte number, SOD activity, and alpha2-macroglobulin level in aqueous humor as well as a decrease in anti-hydroxyl radical activity. SOD instillations contributed to the reduction of the listed parameters and improvement of the antioxidant activity. Clinical presentations of uveitis also became less pronounced. CONCLUSION: SOD instillations for oxidative stress correction help reduce clinical presentations of uveitis, which is confirmed by biochemical examination.

[Production of timolol containing calcium-phosphate nanoparticles and evaluation of their effect on intraocular pressure in experiment].

Methodology for production of calcium-phosphate nanoparticles is developed and its efficacy as a drug carrier system is estimated by example of timolol. Conditions for production of particles with optimal size and resistance are determined, methodology of loading of particles with timolol is developed. Physical parameters of particles (form, size, relief), kinetics of saturation with drug and its release are studied. Packaging of timolol into calcium phosphate nanoparticles was showed to enhance and prolong its hypotensive effect in experiment on healthy rabbits.

[Angiotensin-converting enzyme stability in the presence of zinc-ions different concentrations].

Stability of angiotensin-converting enzyme was studied as a dependence on the zinc-ions concentrations brining in the apo-enzyme. Our data were discussed in the terms of a set of initial permissible conformation conditions of a protein (conformation distribution). Apo-enzyme was shown to be able to the radiation activation that is disappearing in the presence of even 10(-6 )mol/l of the zinc-ions.

[Experimental rationale for local use of angiotensin-converting enzyme inhibitors for the treatment of ocular tissue ischemia on a model of postburn conjunctival ischemia].

The authors have evaluated the local renin-angiotensin system on a model of experimental postburn conjunctival ischemia from the tear activity of angiotensin-converting enzyme (ACE) and studied whether impaired microcirculation might be restored by locally applying the ACE inhibitor captopril. It has been found that in conjunctival ischemia, there is a considerable increase in the activity of ACE, the key enzyme of the renin-angiotensin system, the activity of which largely determines the microcirculation in eye tissues. Instillations of the ACE inhibitor to rabbits within 2 weeks after alkaline burn of the eye result in a reduction in ACE activity and an earlier recovery of microcirculation in the area of conjunctival ischemia. Instillations of the ACE inhibitor captopril in ocular burn facilitate the maintenance of the tear antioxidant potential at the high level, which also suggests that the ACE inhibitor has a positive effect on the course of reparative processes after ocular burn injury. The findings suggest that it is promising to locally use ACE inhibitors for the treatment of ischemic processes in the eye.

[The in vitro cross-effects of inhibitors of renin-angiotensin and fibrinolytic systems on the key enzymes of these systems].

The effects of hypotensive agents (captopril, enalaprilate, and lisinopril) on the activities of components of the fibrinolytic system (FS) and the effects of antifibrinolytic agents (6-aminohexanoic acid (6-AHA) and tranexamic acid (t-AMCHA)) on the activities of angiotensin converting enzyme (ACE) were studied in vitro. Enalaprilate did not affect the FS activity. Captopril considerably inhibited the amidase activities of urokinase (u-PA), plasminogen tissue activator (t-PA), and plasmin ([I]50 (2.0-2.6) +/- 0.1 mM), and the activation of Glu-plasminogen affected by t-PA and u-PA ([I]50 (1.50-1.80) +/- 0.06 mM), which may be due to the presence of a mercapto group in the inhibitor molecule. Lisinopril did not affect the amidase activities of FS enzymes, but stimulated Glu-plasminogen and u-PA activation and inhibited activation of t-PA-fibrin-bound Glu-plasminogen ([I]50 (12.0 +/- 0.5) mM). Presumably, these effects can be explained by the presence in lisinopril of a Lys side residue, whose binding to lysine-binding Glu-plasminogen centers resulted, on the one hand, in the transformation of its closed conformation to a semi-open one and, on the other hand, in its desorption from fibrin. Unspecific inhibition of the activity of ACE, a key enzyme of the renin-angiotensin system, in the presence of 6-AHA and t-AMCHA ([I]50 10.0 +/- 0.5 and 7.5 +/- 0.4 mM, respectively) was found. A decrease in the ACE activity along with the growth of the fibrin monomer concentration was revealed. The data demonstrate that, along with endogenous mediated interactions, relations based on the direct interactions of exogenous inhibitors of one system affecting the activities of components of another system can take place.

[Mental rationale for local use of angiotensin-converting enzyme in the treatment of inflammatory processes in the eye].

A rabbit model of deep alkaline-induced corneal burn was used to study the involvement of the local renin-angiotensin system of the eye in the development of an inflammatory process and wound healing. Corneal burn injury was shown to cause a significant increase in the activity of angiotensin-converting enzyme (ACE) in the tear and internal ocular tissue structures, promoting their microcirculatory disorders and inflammation development. The local use of ACE inhibitors as instillations substantially reduces an inflammatory reaction and the incidence of deep and extensive corneal ulcers. The study performed provides experimental rationale for the local use of ACE inhibitors for the treatment of inflammatory processes in the eye.

[Activity of angiotensin-converting enzyme in the blood and tear of patients with diabetic retinopathy].

There have recently been data on the important role of the renin-angiotensin system (RAS) in the pathogenesis of diabetes mellitus and its complications; however, the relationship of systemic and local RAS to the development of diabetic retinopathy (DR) remains little studied. This study determined the activity of angiotensin-converting enzyme (ACE), the key enzyme of RAS in the blood and tear of patients with DR. There was an increase in the blood activity of ACE and a decrease in its lacrimal activity as compared with their normal levels. A relationship was found between the activity of ACE in the blood and tear, on one hand, and the stage of DR, the type of diabetes mellitus, its severity and compensation, and the presence of nephropathy in a patient, on the other.

[Diagnostic implications of detecting antibodies to angiotensin-converting enzyme and its substrates]. / Diagnosticheskaia vozmozhnost' opredeleniia antitel k angiotenzinprevrashchaiushchemu fermentu i ego substratam.

The results of the analysis of diagnostic significance of examination of natural antibodies (Nab) to agiotensin-converting enzyme (ACE) and its substrates in the serum of hypertensive patients indicate that concentration of Nab to ACE differ from this mean concentration in donors. An elevated level of Nab to ACE may be considered as a compensatory reaction to increased content of the enzyme in vascular endothelium and blood flow. The same patients were examined for antibodies to peptide angiotensin II (AT-II). Enzyme immunoassay has shown that a significantly elevated level of antibodies to AT-II was only in 5 examinees. The same patients had also high Nab to ACE. The study of a group of ischemic heart disease patients with adverse effects attributed to bronchial affection treated with enalapril and diroton (ACE inhibitors) demonstrates that deterioration of cough and external respiration function is not related to exacerbation of existing chronic pulmonary inflammation. None of the patients had elevated body temperature or inflammatory changes in the blood, other signs of inflammation. Enzyme immunoassay also proved that the initial level of Nab equaled mean value for donors or was insignificantly lower. Blood serum patients with side effects contained a significantly (p < 0.02) elevated quantities of Nab to bradikinin vs initial values. Thus, the proposed method of solid phase enzyme immunoassay quantifies Nab to ACE and its substrates in the patients.

[The role of the components of the renin-angiotensin system in the ocular tissues in norm and pathology]. / Rol' komponentov renin-angiotenzinovoi sistemy v tkaniakh glaza v norme i patologii.

Different components of the renin-angiotensin system (RAS), whose content and activity are predetermined by local factors, are generated in the ocular tissue structures. The local eye RAS plays an important role in pathogenesis of different eye diseases and in the local manifestations of general pathological processes. Therefore, a study of the eye RAS components in norm and in disease contributes to understanding the pathogenesis of eye diseases and opens up new possibilities for an adequate treatment. The RAS components in the lacrimal fluid can be an important characteristics of such diseases like keratitis, diabetic retinopathy etc.

[Change of natural antibody levels in patients with cardiovascular diseases by the use of anti-atherogenic diets with processed soy product foods]. / Izmenenie urovnia estestvennykh antitel u bol'nykh s serdechno-sosudistymi zabolevaniiami pod vliianiem antiaterogennykh diet s vkliucheniem produktov pererabotki soi.

It was investigated the influence of a diet supplemented with soy oil and soy protein on dynamic of clinic manifestation and immune status patients with IND and HBP. The results of investigations indicated that a (?).

[Autoantibodies to vasoactive peptides and angiotensin converting enzyme in patients with systemic diseases of the connective tissue]. / Autoantitela k vazoaktivnym peptidam in angiotenzinprevrashchaiushchemu fermentu u bol'nykh s sistemnymi zabolevaniiami soedinitel'noi tkani.

AIM: To estimate the level of natural autoantibodies (NAAb) to angiotensin-converting enzyme (ACE) and endogenic mediators affecting vascular tone (bradykinin--BK, angiotensin II--AII, vasopressin--VP) as well as the activity of serum ACE in patients with systemic diseases of the connective tissue. MATERIAL AND METHODS: Levels of NAAb were measured by enzyme immunoassay in sera from 30 patients with SLE, 19 patients with rheumatoid arthritis (RA) and 36 patients with scleroderma systematica (SS). Serum from donors served control. IgM NAAb to ACE were measured by a new technique. Serum ACE activity was determined by the initial velocity of hydrolysis reaction using spectrofluometry. RESULTS: IgM NAAb were detected in the sera of both patients and donors. SS patients had the level of NAAb to ACE in diffuse form significantly higher than in limited (p < 0.05). In SLE and SS patients ACE activity was significantly lower (p < 0.05) than in healthy subjects and RA patients. Levels of NAAb to BK was significantly elevated (p < 0.01) in patients with SLE and RA vs donors while to AII in SS patients it was lowered (p < 0.001). Patients with diffuse SS had NAAb to BK higher than patients with SS limited form (p < 0.01). In SLE the lowest levels of NAAb to all the mediators studied were observed in patients with nephritis, for NAAb to VP the differences were significant (p < 0.05). In patients with urinary syndrome concentration of NAAb to BK was significantly higher (p < 0.01), differences between their levels in patients with nephritis and urinary syndrome were also significant (p < 0.05). CONCLUSION: Further studies are needed for specification of physiological or pathological role of NAAb to endogenic mediators.

[Experimental validation of licopin-containing drug tomatol use in combined therapy of patients with diabetic retinopathy]. / Eksperimental'noe obosnovanie ispol'zovaniia likopinsoderzhashchego preparata "tomatol" v kompleksnom lechenii diabeticheskoi retinopatii.

Effects of tomatol on metabolic changes in the blood and lacrimal fluid and ocular capillaries of rabbits with alloxane diabetes were studied. Tomatol is a drug containing licopin carotenoid characterized by a high biological activity. The intensity of lipid peroxidation was notably decreased, antioxidant activity of the blood increased, lipid metabolism parameters in the blood and some parameters of proteinase inhibitory balance in the blood and lacrimal fluid normalized in diabetic rabbits treated with tomatol in comparison with untreated rabbits. Changes in perilimbic capillaries were less expressed in treated vs. untreated animals. Hence, tomatol is recommended for combined therapy of patients with diabetes as a drug normalizing metabolism and probably decelerating the development of diabetic retinopathy.

[Detection of natural anti-angiotensin converting enzyme antibodies in human serum using immunoenzyme technique ]. / Opredelenie estestvennykh antitel k angiotenzinprevrashchaiushchemu frmentu v syvorotke krovi cheloveka immunofermentnym metodom.

Solid-phase enzyme immunoassay (EIA) was developed for detecting natural antibodies to angiotensine-converting enzyme (ACE). Optimal conditions for detecting natural anti-ACE by EIA in the sera of donors and patients with disorders of arterial pressure are selected. The findings indicate that the level of natural anti-ACE is normally constant, while in the patients it is increased in 50% cases.

[Radiation activation of angiotensin-converting enzyme during low-dose irradiation]. / Radiatsionnaia aktivatsiia angiotenzin-prevrashchaiushchego fermenta pri malykh dozakh oblucheniia.

Radiation activation of angiotensin-converting enzyme (respect to Cbz-Phe-His-Leu as substrate) was obtained at the gamma (137Cs, t(irr) = 10s-2h, D approximately 3 Gy)- and X (plasma foces source, t(irr) = 10(-9)s, Cu-filter, D approximately 2 x 10(-5) Gy)-irradiation. The inactivation of the horseradish peroxidase at the same X-irradiation dose (2 x 10(-5) Gy) took place. Based on the experimental data and on the mathematical model proposed by us we made a conclusion that the special points exist on the dose response curves. Besides, the deduction that an activation is a common process at radiation changes of the different enzymes follows from our model. The activation of tobacco peroxidase (in presence of Ca(2+)-cations and without them) and of recombinant horseradish peroxidase (in presence of H2O2) by gamma-irradiation was really observed (respect to guaiacol as substrate).

[DNA-methylase activity in human cardiac muscle. Association of DNA methylase activity with actin protein fractions]. / DNK-metilaznaia aktivnost' v serdechnoi myshtse cheloveka. Assotsiatsiia DNK-metilaznoi aktivnosti s fraktsiei aktinovykh belkov.

The column isoelectrofocusing activity of the nuclear extracts of the human cardiac muscle has revealed at pH 3.5-8.2 5 peaks of DNA-methylase. When one of these peaks (II) was analyzed by the two-dimensional gel electrophoresis 6 proteins (10, 25, 35, 43, 67 and 120 kDa) were separated. 43 kDa protein had electrophoretic properties similar to actins and was able to methylate cytosine in the DNA molecules. The comparative computer analysis of the primary structure of human actins and several bacterial DNA-methylases has shown the homology of the extensive fragments of these molecules.

[Angiotensin-converting enzyme in an OAT-octane reversed micelle system: interaction with the matrix]. / Angiotenzinprevrashchaiushchii ferment v sisteme obrashchennykh mitsell AOT v oktane: vzaimodeistvie s matritsei.

Regulation of catalytic activity and intramolecular structure of the bovine lung angiotensin-converting enzyme was studied using reversed micelles in a sodium docusate-water-octane system, which model the enzyme's environment in vivo. The catalytic parameters of monomeric and dimeric forms of the enzyme in the reversed micellar system were evaluated. The catalytic activity of the angiotensin-converting enzyme extracted from bovine lung with Triton X-100 did not depend on the detergent concentration at a constant level of hydration. An artificially hydrophobized form of the angiotensin-converting enzyme was obtained by modifying the enzyme with stearic acid chloride. The modification leads to the dependence of the catalytic activity on the surfactant concentration, which provides evidence that the enzyme interacts with the micellar matrix. The modified enzyme showed a significant increase in catalytic activity in the reversed micellar system.

[Regulation of catalytic activity and supramolecular structure of angiotensin-converting enzyme in reversed micelles of aerosol OT in octane]. / Reguliatsiia kataliticheskoi aktivnosti i nadmolekuliarnoi struktury angiotenzin-prevrashchaiushchego fermenta v obrashchennykh mitsellakh aérozolia OT v oktane.

Regulation of the catalytic activity and the supramolecular structure of the angiotensin-converting enzyme isolated from bovine lungs has been studied in a system of reversed micelles of aerosol OT (AOT) in octane. The curve for the dependence of the enzyme catalytic activity on the degree of the surfactant hydration (micellar size) has two maxima at the hydration degrees of [H2O]/[AOT] 27 and 31. Data from velocity sedimentation suggest that depending on the hydration degree, the angiotensin-converting enzyme occurs in the system of reversed micelles in both monomeric and dimeric forms, the latter being catalytically active. In contrast with aqueous media, in the reversed micelle system the angiotensin-converting enzyme does not require chloride anions for its catalytic activity. In the system of reversed micelles of AOT in octane the holoenzyme is stable, while the apoenzyme rapidly and irreversibly loses its activity. Under these conditions the apoenzyme shows an ability to incorporate the Zn2+ ions into the enzyme active center; however, only in the presence of a substrate or an inhibitor.

[Human thyroid gland DNA methylase in nodular and diffuse goiter]. / DNK-metilazy shchitovidnoi zhelezy cheloveka pri uzlovom i diffuznom zobe.

The heterogeneity and some properties of DNA-methylases isolated from nonmalignant human thyroid formations--nodular and diffuse goiters--have been studied. Isoelectrofocusing of methylase preparations produced 6-7 distinct activity peaks distinguished by pI, activity towards Ca2+ and Mg2+, sensitivity towards dithiothreitol and capacity to methylase cytosine into mono-, di- and tripyrimidine blocks in vitro. The degree of DNA methylation in vivo depended on the origin of of the goiter. Analysis of several methylase fractions by two-dimensional electrophoresis performed according to O'Farrell revealed the presence of major polypeptides having similar molecular masses (approximately 36 kDa). Besides the major component, all the preparations under study contained 6-10 characteristic minor components. The data obtained are suggestive of a structural and functional heterogeneity of human methylases.

[A new method of isolation and purification of SsoII restriction endonuclease]. / Novye sposob vydeleniia i ochistki éndonukleazy restriktsii SsoII.

A new method for isolation and purification of restriction endonuclease SsoII which results in a homogeneous preparation suitable for all types of fine physico-chemical assays has been elaborated. The procedure includes four chromatographic steps: fractionation on butyl-Toyopearl, combined chromatography on SP-Toyopearl and phosphocellulose PII, and chromatography on DEAE-Toyopearl and on QAE-Toyopearl. The use of fast flow sorbents (Toyopearl) makes it possible to reduce the time needed for the separation of proteins and to optimize the fractionation conditions, thus avoiding the dialysis between the chromatographic steps which significantly decreased the enzyme activity yields in previous purification schemes. The isolation of restriction endonuclease SsoII by the new method usually takes four days.