[THE MODE OF EVALUATION OF FUNCTIONAL ACTIVITY OF C3-CONVERTASE OF CLASSIC PATH OF ACTIVATION OF COMPLEMENT].

The technique of detection of stabilization of C3-convertase classical way of activation of system of complement inhuman blood serum. The technique comprises two stages and is based on applying a reaction of lysis of erythrocytes of sheep sensitized by antibodies using 0.8% human blood serum. Preliminary an incubation of two samples (experimental and control) is applied during 10 min. and then reaction of activation of complement is stopped by adding a buffer containing 10 mM of EDTA. In control sample degree of lysis of erythrocytes is established and experimental sample is additionally incubated during 30 min at 37oC and then degree of lysis is determined. The activity of C3-convertase is calculated as a difference between degree of lysis and in experimental and control samples. The difference more than 10% is considered as a pathological state conditioned by stabilization of C3-convertase of classical way of activation of system of complement. The studies were carried out concerning stabilization of C3-convertase of classical way of activation of compliment in 31 patients with abdominal obesity. It is demonstrated that in 87% of patients with abdominal obesity stabilization of C3-convertase was established.

[THE PHYSICAL CHEMICAL, BIOLOGICAL BASICS OF CELLS ABSORPTION OF UNESTERIFIED FATTY ACIDS; ALBUMIN, CAVEOLIN, CLATHRIN AND LIPID-BINDING PROTEINS OF CYTOPLASM (THE LECTURE)].

From aposition of phylogenetic theory of general pathology, obesity and metabolic syndrome are pathology of fatty cells. However, the first is a pathology of phylogenetically early visceral fatty cells of omentum. They supply with substratum of energy realization of biologic function of trophology, homeostasis, endoecology and adaptation. The visceral fatty cells of omentum have no receptors to insulin and synthesize adaptively insulin and they are not characterized by biologic reaction of proliferation. The obesity is a pathology of late in phylogenesis subcutaneous adpocytes. They are insulin-dependent and supply with substratum of energy realization of one biologic function of locomotion--movement at the expense of constriction of cross-striated miocytes. The adipocytes in terms of adaptation synthesize humoral mediator adponectin and actively implement biologic function of proliferation. Under both aphysiologic conditions increases passive by gradient of concentration, absorption by cells albumin-unbound free fatty acids in unionized form in micellae's composition. The passive aphysiologic absorption of free fatty acids by cells which under intracellular compartmentalization don't oxidize mitochondria results in synthesis, accumulation of triglycerides in cytoplasm of cells which don't implement it physiologically. The aphysiologic absorption of free fatty acids by cells, their etherification in triglyceride, in particular, in phylogenetically late ß-cells of islets and either late cardiomyocytes which fatty acids don't synthesize de novo results in development of aphysiologic processes and disorder of function. From position of biology, these cells in vivo are subjected to loss similar to apoptosis. The formation of corpuscles of apoptosis compromise biologic function of endoecology activating biologic reaction of inflammation.

[THE UNESTERIFIED FATTY ACIDS IN BLOOD PLASMA AND INTERCELLULAR MEDIUM: EFFECT OF INSULIN AND ALBUMIN (THE LECTURE)].

The high content of palmitic saturated fatty acid, palmitic triglycerides in food, the large amount of lipoproteins of very low density of the same name in blood, obvious insufficient amount of unesterified fatty acids releasing under lipolysis in blood to meet in vivo requirements in biotransforming energy of ATP are the causes of biological malfunction of homeostasis. As a rule, for every cell in vivo everything is always to be enough. The deficiency of synthesis of ATP by reason of non-optimal substratum for acquirement of ATP by mitochondria is followed by activation also phylogenetically earlier biological function of adaptation, biological reaction of stress. Thus, surplus of palmitic unesterified fatty acid after every food intake forms in vivo biological reaction of "metabolic" stress, deficiency of energy by reason of realization by mitochondria in vivo non-optimal exogenous substratum-palmitic unesterified fatty acid, deficiency of acyl- and acetyl-KoA and prognostically formation of potentially ineffective palmitic alternative of metabolism of fatty acids. The deficiency of palmitic unesterified fatty acids in biological reaction of exotrophy after every intake of food compensates biological reaction of stress, activation of releasing of palmitic unesterified fatty acids from visceral fatty cells of gland as it physiologically occurs in biological reaction of endotrophy. At that, adrenalin increases lipolysis in visceral fatty cells of gland and physiologically late insulin can't inhibit lipolysis in phylogenetically early visceral fatty cells. Increasing of content of unesterifed fatty acids in blood plasma, as it always occurs in vivo, stops absorption of glucose by cells initiating hyperglycemia, hyperinsulinemia, and syndrome of resistance to insulin. The result of such a compensation of biological reaction of exotrophy is biological reaction of endotrophy, condition of "metabolic" stress, depletion of function of ß-cells of islets with formation of diabetes mellitus type I, deficiency in vivo of insulin synthesis. The biological role of albumin - transfer of fatty acids in intercellular medium inform of unesterifed fatty acids and prevention of formation of pool of free fatty acids effecting aphysiologically.

[The role of lipoprotein-associated enzyme paraoxonase 1 and its polymorphisms in the pathogenesis of endothelial dysfunction and somatic complications in patients with alcoholism: Review ].

A review of recent data on the role of the multifunctional enzyme, associated with high density lipoproteins - paraoxonase 1 (PON1) in maintaining healthy endothelial function by detoxifying both oxidized low density lipoproteins and homocysteine thiolactone. The additional contribution to the protection of the endothelium against damage makes organophosphatase activity of PON1 involved in the detoxification products of tobacco smoke. The reduction of antioxidant activity of PON1 promotes the differentiation of monocytes into macrophages and the development of inflammation. The reduction of thiolactonase activity of PON1 is accompanied by a decrease of methionine re-synthesis from homocysteine causing DNA- hypomethylation and alteratioin of the expression patterns of pro- and anti-atherogenic genes. Global hypomethylation of the genome is regarded as one of the three most important mechanisms of the increased risk of somatic complications of alcoholism. The accumulation of homocysteine thiolactone serving agonist of glutamate receptors and antagonist of dopamine receptors is a prerequisite to increased alcohol abuse. Clinical observations focusing on gene polymorphisms of PON indicate that three different genotypes of polymorphism PON1Q192R have unequal degrees atheroprotective properties.

LOCAL RENIN-ANGIOTENSIN SYSTEM OF SMALL INTESTINE.

The renin-angiotensin system (RAS) is the universal multiple-factor regulator of many vital processes at the organismal, tissue and cellular levels. Classical (circulating) RAS provides maintenance of arterial pressure, a water and salt balance, lipid and glucose homeostasis. Local tissue RAS are functioning independently and participating in metabolic processes and protective reactions. Local RAS of a small intestine mucosa is presented practically by a complete rangeof the components (renin, angiotensinogen, angiotensin-converting enzymes, angiotensin receptors) localized, mainly, in an intestinal epithehum, lamina propria and muscularis mucosa. Local RAS participates in regulation of the most levels of activity ofa small intestine, influencing on an intestinal motility, secretory-transport processes, adaptation and protective reactions. The experimental data presented in this review are very promising for the detection of possible complications when using drugs that alter the activity of the RAS-related unexplored effects of the interaction of these drugs with their potential targets, localized not only in the blood vessels, but also directly to the niucosa of the gastrointestinal tract. This is especially important in connection with the extensive use of drugs that modulate the activity of the RAS in the practice of the treatment of cardiovascular diseases such as hypertension, atherosclerosis, endothelial dysfunction, and others.

Estimation of Functional Activity of Ionized Calcium for Complement System Reactivity Assessment.

We proposed a new indicator for evaluation of functional activity of Ca(2+) in human blood serum based on lysis of sheep erythrocytes with 10% human blood serum in the presence of 0.55 mM ethylene glycol tetraacetic acid at 37°C for 10 min. After incubation, the degree of sheep erythrocytes lysis inhibition is estimated: inhibition of complement hemolytic activity <30% is considered as high functional Са(2+) activity, inhibition by 31-70% corresponds to normal activity, and >71% indicates low activity. The comparative studies of complement activating function of heterophilic antibodies, complement system reactivity in the presence of 0.29 M NaCl, and functional activity of ionized Ca(2+) make possible estimation of the individual's immune status.

Comparative study of the effects of free bound and carrier protein angiotensin II in experimental hypoglycemia and hyperglycemia.

Experimental hypoglycemia and hyperglycemia eliminated the differences in the regulatory functions of free angiotensin II and its complexes with carrier proteins (transport protein BSA and neurospecific protein S100b) in rats. Under these conditions, free and protein-bound angiotensin II primarily suppressed operant drinking behavior and reduced the hypertensive and tachyarrhythmic effects in comparison with control rats. These changes were most pronounced during acute hyperglycemia. We hypothesized that complexes of angiotensin II with functionally different proteins are differentially and simultaneously involved in not only compensation of behavioral and hemodynamic disturbances during acute and/or chronic hypoglycemia and hyperglycemia, but also their transformation into pathological processes mediated by the so-called metabolic memory mechanisms.

[Estimation of atherogenic immune complexes containing modified lipoproteins in complement fixation tests].

A method for determining atherogenicity of the immune complexes containing multiple modified low-density lipoprotein (mmLDL) in complement fixation test has been found. In the proposed method, the precipitate immune complexes containing mmLDL (IC mmLDL) was prepared from human serum by treating it with buffer (8.3% of th PEG 3350 and 3.3% PVP 12600 th in the ratio 1: 1.2) for 10 min at 23 °C. IC mmLDL aggregates were separated by centrifugation at 3100g for 10 min at 23 °C. The precipitate IC mmLDL was dissolved in buffer without PEG and PVP, cholesterol content and the degree of binding of guinea pig complement were measured. Atherogenicity of the IC mmLDL was registered as the ratio of the degree of complement binding to cholesterol in the the immune complexes.

[A method of rapid determination of cholesterol in immune complexes].

A method of rapid determination of cholesterol in immune complexes (CIC). In the proposed method precipitate immune complexes containing multiple modified low density lipoproteins from human serum are prepared by treatment with a buffer containing 8.3 PEC 3350, and 3.3% PVP 12600, in a ratio of 1:1.2, incubated for 10 min at room temperature. The precipitate containing the CIC is separated by centrifugation at 3100 g for 5 min at 23 degrees C, dissolved in a buffer without PEG and PVP, the cholesterol is determined using an enzymatic kit and at a level of CIC more than 8.3 mg/dl ascertain higher level. The method improves the accuracy of the quantitative determination of CIC, conduct extensive screening tests to detect atherosclerosis as the pre-clinical stage and monitor the effectiveness of the therapy.

[Rapid assessment of the complement system reactivity].

The authors developed a new indicator of humoral immunity, which characterizes the complement system reactivity (CSR). Complement system reactivity has been investigated in haemolytic assay using sheep red blood cells, autologous heterophile antibodies and complement in a "load" as 0.29 M NaCl in the system. There has been made a study of blood sera of 10 healthy donors and 20 patients who undergo screening for chronic infections in clinical-diagnostic centers. Inhibition of complement--0.29 M NaCl at a level of 30-70% in healthy donors is taken as an indicator that characterizes normal complement system reactivity of a human serum. Investigations have revealed in 11 patients (55%) of the 20 examined people high complement system reactivity (lysis degree more than 70%), in 6 patients (30%)--normal complement system reactivity, and in 3 patients (15%)--low complement system reactivity (lysis degree less than 30%). Thus, the study of the complement system reactivity in a "load", in the presence of 0.29 M NaCl in the test sample, detects functional changes, both reinforcing and reducing complement activity.

Free and protein-bound angiotensin II(1-7)in the regulation of drinking behavior and hemodynamics in rats.

We compared physiological activity of synthetic analogues of endogenous protein-peptide compounds, complexes of angiotensin II(1-7)with functionally different proteins (transport protein, serum albumin; and neurospecific Ca(2+)-binding protein, S100b). Physiological activity of angiotensin II(1-7)was shown to depend on the type of a carrier protein. Our results suggest that complexes of angiotensins with BSA and S100b are strong factors for the integration of central and peripheral functions at the homeostatic and behavioral level.

[Mechanisms of the liver anti-endotoxin defence].

Current knowledge of immunocellular and lipoprotein mechanisms of the liver-induced anti-endotoxin tolerance has been summarized. The role of T regulatory cells, different macrophage phenotypes, high density lipoproteins, oxidized low density lipoproteins and their receptors as the key players in mechanism of tolerance to endotoxin has been discussed.

[A simple method for quantification of modified low-density lipoproteins].

A simple method for quantification of modified low-density lipoproteins (mLDL) in the blood serum containing 8.9% polyvinylpyrrolidone solution 12600 +/- 2700 has been developed. The results show that 10 min incubation of serum in a buffer containing 8.9% PVP leads to complete aggregation mLDL. Atherogenicity of aggregated mLDLs is experimentally confirmed by two independent tests (mLDLs bind and activate the complement system of a guinea pig (pro-inflammatory effect) and cause platelet hyperaggregation (thrombogenic effect)). The proposed method is simple and involves only two steps: mixing the serum with a solution of PVP and recording the turbidity. The method allows to register the presence of mLDL directly in serum without its prior fractionation.

[Modified method for determining circulating immune complexes in the complement fixation test with polyethylene glycol precipitate].

The authors have modified a method for determining circulating immune complexes in the complement fixation test. It is shown that with the 7% concentration of polyethylene glycol (PEG)-6000, there is a more complete precipitation of low- and medium-molecular weight immune complexes. The time and temperature of serum incubation were optimized when PEG-6000 was obtained. The use of the soluble circulating immune complexes (sCIC) prepared from human serum as a standard for the construction of a standard plot could substantially enhance the sensitivity of the method (0.325 microg/ ml). The content of circulating immune complexes was studied in donors and in patients with connective tissue dysplasia (CTD) by the improved procedure. In the group of donors, the mean level of sCIC was 0.62 +/- 0.24 mg/ml. In the CTD group, that was 2.32 +/- 0.93 mg/ml; the serum concentrations of sCIC ranging from 1.1 to 5.0 mg/ml. In the donors and patients, the detection rate of serum sCIC was 100%. The proposed method may be clinically used to measure the human serum levels of sCIC.

[Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes]. / Izotipirovanie komponenta C4 komplementa cheloveka s ispol'zovaniiem razlichii v funktsional'noi aktivnosti isotipov C4A i C4B.

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.

[Inhibition of binding of activated compliment component C4b with its target]. / Ingibirovanie sviazyvaniia aktivirovannogo C4b-komponenta komplementa s mishen'iu.

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of the Neisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption to its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.

[Major complement inhibiting factors from the venom of the Central Asian cobra Naja naja oxiana]. / Osnovnye faktory iada sredneaziatskoi kobry Naja naja oxiana, ingibiruiushchie komplement.

The properties of two anticomplementic factors isolated by CM-Sepharose chromatography from the basic non-adsorbed on DEAE-Sepharose fraction of the Central Asian cobra Naja naja oxiana venom, were studied. Of these three factors (CFB-I, CFB-II and CFB-III) the latter had been characterized earlier. CFB-I was shown to be a protein with an N-terminal Asp and a molecular mass of about 39 kDa (data from gel chromatography); its content in the venom is 3.6 mg/g of dry venom. The protein inhibits mainly the classical pathway of the complement activation, being bound to component C4 (Ki = 9 nM). CFB-I seems to be analogous to the CI inhibitor from the venom of the Naja haje cobra. An analysis of the N-terminal sequence of CFB-II showed it to be identical to the earlier characterized cytotoxin I. CFB-I inhibits the formation of C3 convertase with Ki = 2.2-2.8 microM by way of binding to C4b and thus interfering with the component C2 sorption.

[Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent]. / Stupenchataia dissotsiatsiia subkomponentov C1, pervogo komponenta komplementa cheloveka, pri aktivatsii na affinnom sorbent.

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.

[A factor from the venom of the Central Asiatic cobra Naja naja oxiana, which inactivates component C4 of human complement]. / Faktor iada sredneaziatskoi kobry Naja naja oxiana inaktiviruiushchii komponent C4 komplementa cheloveka.

An acid glycoprotein (mol. m. 60 kDa) containing 6 sialic acid residues and N-terminal Thr was isolated from the venom of the central asian cobra Naja naja oxiana. The protein has an anticomplementary activity selectively inactivating of the C4 component of the human complement. This factor (CFA-Ib) binds C4 with Ki = 0.27 +/- 0.13 microM and then irreversible inactivates it with a rate constant k = 0.75 +/- 0.25 min-1. Membrane bound C4b restores its ability of CFA-Ib binding. This binding hinders component C2 sorption on C4b and C3 convertase formation.