[Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms].

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.

[Influence of serum IgG antibodies to Chlamydia trachomatis on the functional activity of complement components].

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.

[Activation of pancreatic lipase by detergents. III. Oligomerization of the enzyme]. / Aktivatsiia pankreaticheskoi lipazy detergentami. III. Oligomerizatsiia fermenta.

Activation of pancreatic lipase by non-micellar solution of sodium dodecylsulphate (SDS) has been studied. By means of gel-filtration it was found that SDS forces the lipase to form an octamer. A new method of the active sites titration using alkylboronic acids is proposed. The octameric form of the lipase was shown to contain six active sites at the optimal SDS concentration. The activated form of pancreatic lipase supposedly contains six native subunits, each of them forming an active site, and two conformationally altered subunits. This model was confirmed by a probability-theoretic calculation.

[The mechanism of inhibition of the activation of the complement first component by polyanions and polycations ]. / Mekhanizm ingibirovaniia polianionami i polikationami aktivatsii pervogo komponenta komplementa.

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.

[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.

[The effect of alpha-melanocyte-stimulating hormone and its analog on the growth of human melanoma cell lines with different phenotypes]. / Vliianie al'fa-melanotsitstimuliruiushchego gormona i ego analoga na rost kletok melanomnykh linii cheloveka s razlichnym fenotipom.

Investigation of physiological effects of alpha-MSH and its analog (NLe4, D-Phe7)-alpha-MSH on human melanoma cells with different phenotypes has shown that these peptides have a growth-modulating activity. The effect of inhibition or activation of the growth of melanoma cells depended on their phenotypes. (NLe4, D-Phe7)-alpha-MSH activated 1.5-2.5-fold the growth of amelanotic BRO cells at concentrations of 10(-6)-10(-12) M, but inhibited the growth of melanin-producing MS cells under the same conditions not affecting the growth of human lung fibroblasts.

[Use of immunospecific methods for quantitative estimation of microsomal epoxide hydrolase]. / Ispol'zovanie immunospetsificheskikh metodov dlia opredeleniia kolichestva mikrosomal'noi époksidgidrolazy.

Antiserum, specific to rat liver microsomal epoxide hydrolase, was produced. Three methods for quantitative estimation of the epoxide hydrolase, base on the principle of antigen-antiserum immunoprecipitation, were used: electroimmune analysis by Laurell, radial immunodiffusion, enzyme-linked immunoassay. Distinct correlation between specific enzyme activity and its content in microsomal preparation was demonstrated using a simple and stable procedure of radial immunodiffusion. In the enzyme-linked assay conjugate of protein A with alkaline phosphate served as the secondary antibody. Sensitivity of the procedure described was about 1,000-fold higher as compared with radial immunodiffusion method.

[Inhibition of human MS and BRO melanoma cell proliferation by alpha melanocyte stimulating hormone]. / Ingibirovanie proliferatsii melanomnykh kletok cheloveka MS i BRO alpha-melanotsitstimuliruiushchim gormonom.

Effect of alpha-melanocyte stimulating hormone (MSH) on proliferating activity of MS and BRO melanoma cell strains with dissimilar phenotype was studied. MSH, apart from the effect on melanomagenesis, influenced the cell proliferation. Growth of MS cells was inhibited at 10(-8)-10(-9) M concentrations of the hormone (IC50 = 2.10(-8) M), while BRO cells were activated. The higher concentrations of MSH inhibited also BRO cells growth (IC50 = 9.10(-7) M). Ligand-receptor interaction of 125I = MSH with plasmatic membranes and cytosol fraction of the cells was studied at 4 degrees and 37 degrees.

[A drop of mink complement kills a mouse]. / Kaplia komplementa norok ubivaet mysh'.

The phenomenon of fast death of mice after parenteral administration of mink serum was explained by high activity of mink complement in particular by unusually high activity of its alternative pathway of activation. The presence of antibodies to mouse erythrocytes in mink serum was necessary precondition for their lysis under action of mink complement by classical and alternative pathways. However, removal of these antibodies resulting in cancellation of hemolysis did not effect toxicity of mink serum for nice in vivo. Partial decomplementization of mink serum zymosan completely prevented death of animals.

[Protease activity of microflora in the oral cavity of patients with periodontitis].

Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

[Determination of protease activity in blood and in microorganisms].

For determination of protease activity it is possible to use immunoglobulins. Since proteolytic products apparently do not retain substrate antigenic determinants, it is possible to use ELISA methodsfor monitoring for enzymatic process. ELISA determination of functional activity of specific IgA1-protease has been applied not only for detection of this enzyme, but also for measurement of its inhibition constants. Fixed on a micropanel IgG may be used for evaluation of total proteolytic activity. Depending on pH values, it is possible to measure activity of neutral, alkaline and acid proteases. This approach has allowed to estimate total proteolytic activity of neutral proteases of serum. Measurement of a total level of serum pepsinogene activity can have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the measured activity.

[Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors]. / Geterogennost' preparatov tirozinazy, vydelennykh iz kozhi krys, kozyi i melanomnykh opukholei cheloveka.

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.

[Determination of the functional activity, amount of C1 inhibitor, and autoantibodies as a tool for differential diagnosis of angioedema]. / Opredelenie funktsional'noi aktivnosti, kolichestva C1 ingibitora i autotel k nemu kak instrument differentsial'noi diagnostiki otekov.

Aetiology of angioedema (and therefore the scheme of its treatment) can be different. Angioedema may be subdivided into four categories: hereditary and acquired angioedemas, allergies and vasculitis. To establish the reason of the hereditary and acquired form of angioedema analyses of functional activity of complement components, quantities and activity of C1 inhibitor, presence (or absence) autoantibodies to C1 inhibitor allow. Sorption of the puried enzymes of activated subcomponent C1s or plasmin on micropanels allows to connect specifically in cells of plate C1 inhibitor from serum and with the help conjugate of antibodies against C1 inhibitor with a horse-radish peroxidase to determine quantity of connected functionally active C1 inhibitor. Addition of this test-system ELISA system for determination of quantitative contents of C1 inhibitor in serum, and also systems for definition IgG, IgA and IgM autoantibodies against C1 inhibitor finishes creation of a necessary set of methods of differential diagnostics.

[Isolation of growth modulating factors secreted by human malignant melanoma cells]. / Vydelenie rost-moduliruiushchikh faktorov, sekretiruemykh kletkami zlokachestvennoi melanomy cheloveka.

Novel low molecular mass growth-modulating factors-F0.5 (0.5-1.0 kDa), F1 (approximately 1 kDa) and fraction F8 (8-12 kDa)-have been isolated from a serum-free culture of human malignant pigmented melanoma mS cells using ultrafiltration, affinity and low and medium pressure gel chromatography. Factor F0.5 (10(-9)-10(-7) M) had a growth-stimulating effect on melanoma mS cells exceeding by a factor of 2 and 3 that of 5% embryonic calf serum. Factor F1 and fraction F8 inhibited melanoma cell growth when used at concentrations of 10(-9)-10(-5) M and higher than 10(-6) mg/ml, respectively. The effect of fraction F8 was concentration-dependent; that of factor F1 was more complex. The inhibiting action of fraction F8 was more pronounced when amelanotic cells of human malignant melanoma BRO were used at concentrations above 10(-8) mg/ml. Neither factors F0.5 and F1 nor fraction F8 influenced the growth of human lung fibroblast Lech 240 cells. The growth-modulating factors can participate in the autocrine regulation of malignant melanoma cell growth.

[The use of liquid exclusion chromatography for evaluating methods of treating urologic diseases]. / Ispol'zovanie zhidkostnoi ékskliuzionnoi khromatografii dlia otsenki metodov lecheniia urologicheskikh zabolevanii.

Gel chromatography was used for measuring medium-molecular levels in the blood sera of urologic patients: (1) low-pressure chromatography in Sephadex G-15 packed columns with fractionation range less than 1500 dalton or in Toyopearl HW-40 packed columns with fractionation range from 100 to 100,000 dalton; (2) high-pressure chromatography in LKB (Sweden) blue columns. Liquid exclusion chromatography was employed to assess the efficacy of blood extracorporeal detoxication of urologic patients by means of hemoperfusion, hemodiafiltration, hemodialysis, plasma perfusion, plasmapheresis. The highest correlation between patients' state of health and blood levels of medium molecules was observed in hemodiafiltration.

[Determination of proteinase activity of complement system by immunoenzyme methods]. / Opredelenie aktivnosti proteinaz sistemy komplimenta immunofermentnymi metodami.

Modern ELISA for determination of functional activity of component C2 and factors B and D, proteinases of a complement system, and component C3, substrate C3-convertases, key complex enzymes of the complement, have been developed. Essential feature of C3-convertases classical (C4bC2a) and alternative (C3bBb) pathways of the complement activation is that their substrate C3 after proteolytic cleavage is converted into C3b, carrying on the surface thioester covalent bond linking C3b with nucleophilic acceptors that results in immobilization of this proteolytic product near the activating enzyme. Cascade character of an activation of complement system allows to create artificial deficit of separate components in the experimental system and to determine (by ELISA) covalently immobilized component C3 during activation, and also to determine functional activity of any of pre-exhausted components. Use of such approach resulted in the development of the ELISA systems suitable for determination of functional activity of component C2 of classical pathway and factors B and D of the alternative pathway by testing quantity of the immobilized C3b at excess C3. The developed methods allow to investigate mechanisms of functioning of complement, inhibition of the cascade activation by endogenic and exogenous inhibitors, and also to find functional deficiency of components in serum and other biological fluids.

[The expression of P-glycoprotein in leukemia P388 cells with induced doxorubicin resistance]. / Ekspressiia P-glikoproteina v kletkakh leikoza P 388 s indutsirovannoi ustoichivost'iu k doksorubitsinu.

beta-D-galactose-containing glycoproteins were prepared from cells P-388 leukemia and from P-388 leukemia cells with induced resistance to doxorubicin. It was shown by HPLC method that plasma membranes from resistant cells contain 4-4.5% P-glycoproteins and plasma membranes from sensitive cells contain P-glycoproteins about 10 times lower.