[Bradykinin and angiotensin-converting enzyme in serum of patients with diabetic retinopathy and the prognosis of diabetic macular edema development (pilot study)].

BACKGROUND: Diabetic macular edema (DME) is a microvascular complication of diabetic retinopathy. One of the key roles in the pathogenesis of DME may belong to the components of rennin-angiotensin and kallikrein-kinin systems: bradykinin (Bk) and angiotensin-converting enzyme (ACE). PURPOSE: To determine the Bk and ACE concentration and ACE activity in serum of patients with proliferative diabetic retinopathy (PDR) and to estimate the significance of these parameters for the early diagnostic and prognosis of DMO. MATERIALS AND METHODS: Serum was collected from the 2 groups of patients with II type diabetes. Group I (n=9) had DME, group II (n=27) had PDR without DME. Control group (n=14) consisted of adult volonteers without diabetes and ophthalmic diseases. Concentration of Bk and ACE was measured using ELISA kits, ACE activity was determined enzymatically with specific fluorogenic substrate. RESULTS: Concentration of Bk in serum of patients without DME did not differ from one in controls (12,00 (9,70; 12,40) pg/ml) while all patients with DME had Bk level of 14,69 (13,68; 16,78) pg/ml that was significantly higher (p<0,01). In patients without DME ACE concentration (88,60 (77,30; 97,45) ng/ml) and ACE activity (6,8 (5,1;7,1) nmol/min·ml) were higher than normal (p<0,01) while in the case of DME concentration of ACE increased (77,36 (70,24; 86,29 ng/ml, p<0,01) and activity remained normal. The Bk/ACE concentrations ratio decreased in patients without DME and increased in those having DME. CONCLUSION: Patients with DME have increased Bk concentration along with nearly normal ACE concentration that indicate predominance of Bk synthesis over its degradation that may lead to the DME development. The Bk/ACE ratio decrease in patients with uncomplicated PDR and increase significantly in ones with DME. It means that determination of Bk in serum of patients with PDR may be used for the prediction of DME development. The Bk/ACE concentrations ratio may be even more informative.

[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.

[Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme].

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

[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.

Role of two chloride-binding sites in functioning of testicular angiotensin-converting enzyme.

Modeling the structure of the C-domain of bovine angiotensin-converting enzyme revealed two putative chloride-binding sites. The kinetic parameters, K(m) and k(cat), of hydrolysis of the substrate Cbz-Phe-His-Leu catalyzed by the testicular (C-domain) enzyme were determined over a wide range of chloride concentrations. Chloride anions were found to be enzyme activators at relatively low concentrations, but they inhibit enzymatic activity at high concentrations. A general scheme for the effect of chloride anions on activity of the C-domain of bovine angiotensin-converting enzyme accounting for binding the "activating" and "inhibiting" anions is suggested.

[Radioenzymatic investigation of membrane and soluble forms of angiotensin-converting enzyme in acetate-phosphate buffer]. / Radioénzimologicheskoe izuchenie membrannoi i rastvorimoi form angiotenzinprevrashchaiushchego fermenta v atsetatno-fosfatnom bufere.

The dose response of soluble and membrane forms of angiotensin-converting enzyme to gamma-irradiation is investigated at different pH values of the medium and at different concentrations of acetate-phosphate buffer. Membrane form of the enzyme is more stable shows principally other conformational equilibrium than the soluble form. "Splitted" activation peaks on the curves of the enzyme dose response are observed.

[Structure-functional features of homologous domains of angiotensin-converting enzyme]. / Strukturno-funktsional'nye osobennosti gomologichnykh domenov angiotenzinprevrashchaiushchego fermenta.

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each of them containing an active site. Differences in substrate specificities and affinity to inhibitors of the active sites of the two domains of bovine ACE are described. The ACE domains demonstrate different thermostability, and the reasons for this difference are analyzed. A structural model of the ACE domains is suggested, which allows us to reveal the structural subdomain important for the protein stability and localize the hydrophobic and the carbohydrate-binding sites.

[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.

A hydrophobic site on the surface of the angiotensin-converting enzyme molecule.

Using the hydrophobic fluorescent dye 8-anilino-1-naphthalenesulfonic acid (8-ANS), a hydrophobic site on the surface of the protein globule of angiotensin-converting enzyme (ACE) from bovine lung was found. The dissociation constant of the ACE-8-ANS complex was estimated as 1.5 +/- 0.2 microM. This hydrophobic site is far from the ACE catalytic sites because the binding of the hydrophobic dye does not influence ACE activity. Shielding of the ACE hydrophobic site due to the complex formation with 8-ANS or Triton X-100 resulted in pronounced stabilization of the enzyme against the action of water radiolysis products during gamma-irradiation of dilute solutions of ACE.

[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 (?).

Fluorescence polarization studies of different forms of angiotensin-converting enzyme.

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied using fluorescence polarization. The dissociation constants Kd of the enzyme-inhibitor complexes in 50 mM Hepes-buffer (pH 7.5) containing 150 mM NaCl and 1 microM ZnCl2 at 37 degrees C were (2.3 +/- 0.4).10(-8), (2.1 +/- 0.3).10(-8), and (2.1 +/- 0.2).10(-8) M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 +/- 0.2).10(-3) M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 +/- 0.01) sec(-1) and (0.026 +/- 0.001) sec(-1) that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 +/- 0.001) and (0.041 +/- 0.001) sec(-1) for the N-domain and for testicular ACE, respectively.

[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.

Characterization of bovine atrial angiotensin-converting enzyme.

Bovine atrial angiotensin-converting enzyme (ACE) was purified to electrophoretic homogeneity. The purification procedure included ion-exchange chromatography on DEAE-Toyopearl 650M, affinity chromatography on lisinopril-agarose and gel filtration on Sephadex G-100. The bovine atrial ACE exhibited similar sensitivities to inhibition by lisinopril and captopril as lung ACE (the Ki values for the atrial and lung enzymes differed insignificantly). However, the kinetic parameters of hydrolysis of some synthetic tripeptide substrates (FA-Phe-Gly-Gly, FA-Phe-Phe-Arg, Cbz-Phe-His-Leu, Hip-His-Leu) catalyzed by bovine atrial and lung ACE varied to a greater extent. The enzymes were also characterized by some differences in activation by chloride, nitrate, and sulfate anions. These data support the hypothesis of tissue specificity of ACEs.

Unusual behavior of membrane somatic angiotensin-converting enzyme in a reversed micelle system.

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.