[Biological role of adipokines and their association with morbid conditions].

Adipose tissue is the source of adipokines (leptine, adiponectine, resistine, interleukin-1, 6, 7, 8, 15, visfatine, PPAR-γ, tumor necrosis factor-α, vaspine, chemerine, progranuline, endocannabinoids, lipocaline-2, apleine, omentine, nesfatine-1) - biological active molecules of adipose tissue that have local and systematic effect on body. Changing of their level in the body is associated with insulin resistance, endothelium dysfunction, arterial hypertension, bronchial asthma and obesity progressing. Adipokines are heterogenous group of molecules, one part of them is produced directly by abdominal adipose tissue; another part of them is produced in other tissues but they indirectly affect development and functioning of adipose tissue. The study of adipokines lets us observe the pathogenesis of obesity, associated cardiovascular diseases and diabetes mellitus type 2 with another way. It will give us an opportunity to compose scientific base for recognizing, preventing and treatment of such diseases. It is necessary to realize that obesity with diabetes mellitus type 2 and atherosclerosis are inflammatory diseases, that's why we need to study pro- and anti-inflammatory factors of adipokines. The production of majority of inflammatory mediators is increased in case of obesity and contributes progressing of obesity and metabolic diseases. It is actual to observe adipokines as biomarkers of pathological processes, in future it will be available to prevent pathological processes, to establish prophylaxis of disease and to support positive treatment.

[The complexes of copper, manganese and chromium with enzymatic hydrolysate of pig spleen: research in vitro].

This report describes the preparation and the results of physical and chemical analysis of complexes of enzymatic hydrolysate of pig spleen (EHPS) with manganese, copper and chromium. The complexes were prepared using schemes including the reaction of complexation of inorganic cations with EHPS-peptides structures and application of membrane technology. The process of microfiltration of the resulting mixtures was carried out in tangential flow and low molecular weight fractions were collected. Solutions of copper and manganese complexes with EHPS were subjected to nanofiltration to remove inorganic ions from the reaction mixture. The obtained preparations were lyophilic dried and the molecular weight distribution of the protein fractions in Cu-EHPS, Mn-EHPS and Cr-EHPS complexes was analyzed by exclusion medium pressure liquid chromatography. The percentage relation of fractions with specific molecular weight range was calculated by applying the weighted integration of chromatograms. The determination of copper, manganese and chromium levels in the complexes was performed by atomic absorption method. The content of microelements in the preparations is for copper 16.5 ± 0.3 mg/g, for manganese--24.9 ± 0.5 mg/g and for chromium--2.5 ± 0.2 mg/g.

[Modeling role of pyruvate in the processes of protein-protein interaction].

Using the ABO antibody-antigen model the influence of natural metabolite pyruvate on the antibody interaction with of erythrocyte antigens, defining their group specificity has been investigated. Before agglutination reaction erythrocytes of A(II)-AB(IV) blood groups, monoclonal anti-A and anti-B antibodies were incubated with sodium pyruvate. Visualization of agglutinates was performed by means of flow cytometry and laser scanning confocal microscopy. Computer-aided prediction of the spectrum of biological activity of pyruvate by a PASS program proposed major regulatory pathways, in which pyruvate may be involved. It has been demonstrated that pyruvate can regulate the intensity of antigen-antibody interaction. These results suggest the possibility of using small molecules, for example pyruvate, as molecular probes and prospects of the use of erythrocytes with antigenic determinants of the ABO system expressed on their membranes for studies of protein-protein interactions due to convenient visualization and possibility of quantitative evaluation of this process.

[A possible influence of nutrient factors in the mechanism development of the neurodegenerative diseases--alzheimerrs disease and sprongiform encelphalopathy with people].

The article presents a review of the literature data, as well as the author's experimental observations concerning the role of nutrient components in the development of neurodegenerative diseases of amyloidal nature. In particular, the role of antioxidants in the prevention of the accumulation of oxidized proteins blocking the chaperone system is discussed. A separate section is devoted to the formation of the large amorphous aggregates on the association of caseins with the precursors of amyloidal structures (amyloidal beta peptidel-42 and recombinant prions). A possible role of nutrient factors in the development of the neurodegenerative diseases is discussed.

Production of different antibodies after simultaneous immunization of animals with two antigens.

We propose a method of simultaneous immunization with two different antigens for isolation of two types of antibodies from the same antiserum. Bacterial proteins (Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and Escherichia coli GroEL chaperonin) served as the antigens. Affinity purification of antibodies was carried out using two columns: with covalently immobilized glyceraldehyde-3-phosphate dehydrogenase or GroEL chaperonin. During stage I, the antiserum was applied onto the column with immobilized glyceraldehyde-3-phosphate dehydrogenase, after which antibodies to glyceraldehyde-3-phosphate dehydrogenase were eluted. During the next stage, the antiserum without antibodies to glyceraldehyde-3-phosphate dehydrogenase was passed through the column with immobilized GroEL and antibodies to chaperonin were isolated. Antibodies to glyceraldehyde-3-phosphate dehydrogenase and to GroEL had high titers and exhibited no cross-reaction.

[The non-functioning chaperonin GroEL stimulates protein aggregation].

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to decrease in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The sizes of individual proteins (GroEL, GAPDH, and the recombinant prion), as well as aggregates formed after their mixing, were determined by the dynamic light scattering method. It was shown that at 25 degrees C the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the aggregate size to 70-80 nm. On the blocking of one of the chaperonin centers by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.

Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo- and holoforms of the enzyme.

Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which were selected using the immunosorbent with unfolded enzyme form, do not affect the activity of native holo- and apo-GAPDH, but prevent the reactivation of the denatured GAPDH, binding the denatured forms of the enzyme.

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase.

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD(+), and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.

[Isolation of renin from human kidneys]. / Vydelenie renina iz pochek cheloveka.

Isolation and partial purification of renin from human kidney was carried out as follows: defatting of the homogenized kidney; extraction with methyl cellosolve; chromatography on DEAE cellulose; affinity chromatography using immobilized pepstain. Activity of the preparation obtained was 0.1 GU/mg of protein. The preparation of human renin might be used for measurement of renin activity in blood of patients.

[Isolation and purification of angiotensinogen from swine and sheep plasma]. / Vydelenie i ochistka angiotenzinogena iz plazmy krovi svinei i ovets.

Angiotensinogen (a substrate of renin) was isolated from pig and sheep blood plasma. Content of angiotensinogen was estimated in the preparations obtained by an indirect biological method, which was based on estimation of an increase in blood pressure in rats, caused by angiotensin, liberated from angiotensinogen during incubation with renin. The preparation, obtained by ammonium sulphate fractionation of pig blood plasma, contained 0.4% of angiotensinogen. Angiotensinogen was also isolated from sheep blood plasma using ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration through Sephadex G-100; this preparation contained 10-13% of angiotensinogen.