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It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.Stanovitsia vse bolee ochevidnym, chto ubikvitinirovanie vnutrikletochnykh belkov ne iavliaetsia obiazatel'nym étapom, neobkhodimym dlia ikh degradatsii v proteasomakh. Izvestno nemalo podlezhashchikh éliminatsii belkov, kotorye ne podvergaiutsia predvaritel'nomu ubikvitinirovaniiu dlia uznavaniia reguliatornymi subchastitsami 26S proteasomy, a postupaiut neposredstvenno v ee korovuiu (proteoliticheskuiu) chast' – 20S proteasomu. Neobkhodimym usloviem dlia étogo iavliaetsia nalichie v belke nestrukturirovannykh uchastkov. V dannoĭ rabote issledovali sviazyvanie riada polifunktsional'nykh belkov (glitseral'degid-3-fosfatdegidrogenaza (GAFD), al'dolaza, piruvatkinaza), a takzhe belkov, uchastvuiushchikh v protsessakh neĭrodegeneratsii (a-sinuklein, osnovnoĭ belok mielina), s immobilizovannoĭ na chipe SPR-biosensora Biacore 20S proteasomoĭ. Dlia predotvrashcheniia vozmozhnoĭ dissotsiatsii 20S proteasomu v khode immobilizatsii stabilizirovali pri pomoshchi bifunktsional'nogo sshivaiushchego reagenta – dimetilpimelimidata. Iz vsekh issledovannykh belkov tol'ko al'dolaza i piruvatkinaza vzaimodeĭstvovali s 20S proteasomoĭ (Kd sootvetstvenno 8,17´10-7 M i 5,56´10-7 M). V dopolnenie k ranee obnaruzhennomu ubikvitinirovaniiu GAFD, mass-spektrometricheskiĭ analiz preparatov étikh belkov vyiavil nalichie tak nazyvaemoĭ ubikvitinovoĭ signatury Lys-e-Gly-Gly tol'ko v preparate al'dolazy. Okislenie piruvatkinazy i al'dolazy, sposobstvuiushchee éliminatsii belkov v rezul'tate ikh neposredstvennogo vzaimodeĭstviia s 20S proteasomoĭ, privodilo k snizheniiu Kd v 2-3 raza po sravneniiu s intaktnymi belkami. Poluchennye rezul'taty svidetel'stvuiut v pol'zu togo, chto s 20S proteasomoĭ odinakovo éffektivno sviazyvaiutsia kak ubikvitinirovannye (al'dolaza), tak i neubikvitinirovannye (piruvatkinaza) belki.

The amyloid-beta peptide 1-42 formed during proteolytic processing of the amyloid precursor protein (APP) plays a key role in the development or progression of Alzheimer's disease (AD) and other pathologies associated with formation of protein aggregates in the central nervous system. Recent proteomic profiling of mouse and rat brain preparations by means of beta-amyloid peptide immobilized on Affigel-10 revealed a large group of amyloid-binding proteins (n80). Many (about 25%) of these proteins were previously identified as isatin-binding proteins. The aim of this study was to validate direct interaction between beta-amyloid peptide and highly purified intact and oxidized peroxiredoxin, M-type pyruvate kinase, alpha-enolase, and the effect of isatin on this interaction. The study performed using SPR-based Biacore 3000 and Biacore X100 biosensors has shown that all the proteins form molecular complexes with immobilized beta-amyloid peptide. The Kd values for these complexes varied from 8.36х10-8 M (peroxiredoxin) to 1.97х10-6 M (alpha-enolase). Oxidative modification of investigated proteins caused opposite effects on complexes of these peptides with beta-amyloid. The endogenous neuroprotector isatin increased dissociation of complexes formed by beta-amyloid peptide with both intact proteins (peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase) and/or oxidized proteins (peroxiredoxin, pyruvate kinase) used in this study.Beta-amiloidnyĭ peptid (1-42), obrazuiushchiĭsia v rezul'tate proteoliticheskogo protsessinga transmembrannogo belka-predshestvennika amiloida, rassmatrivaetsia v kachestve "kliuchevogo igroka" v razvitii bolezni Al'tsgeĭmera (AD) i drugikh patologicheskikh sostoianiĭ, obuslovlennykh obrazovaniem i nakopleniem belkovykh agregatov v tsentral'noĭ nervnoĭ sisteme. V khode proteomnogo profilirovaniia preparatov mozga intaktnykh krys i mysheĭ, provedennogo ranee s ispol'zovaniem v kachestve affinnogo sorbenta affigelia-10 s immobilizovannym beta-amiloidnym peptidom, bylo identifitsirovano bolee 80 individual'nykh belkov, spetsificheski sviazyvaiushchikhsia s étim ligandom. Mnogie iz nikh byli obnaruzheny ranee kak belki, sviazyvaiushchiesia s éndogennym neĭroprotektorom izatinom. Tsel'iu dannogo issledovaniia byla priamaia éksperimental'naia proverka vzaimodeĭstviia vysokoochishchennykh intaktnykh i okislennykh preparatov peroksiredoksina, piruvatkinazy myshechnogo tipa i al'fa-enolazy s immobilizovannym beta-amiloidnym peptidom i vliianie izatina na étot protsess. Po dannym biosensornogo analiza s ispol'zovaniem opticheskikh SPR-biosensorov Biacore 3000 i Biacore X100 vse issledovannye belki obrazovyvali molekuliarnye kompleksy s immobilizovannym beta-amiloidnym peptidom. Znacheniia Kd étikh kompleksov var'irovali ot 8,36kh10-8 M (u peroksiredoksina) do 1,97kh10-6 M (u al'fa-enolazy). Okislitel'naia modifikatsiia issledovannykh belkov okazyvala raznonapravlennoe vliianie na prochnost' kompleksov s beta-amiloidnym peptidom, a éndogennyĭ neĭroprotektor izatin povyshal dissotsiatsiiu kompleksov beta-amiloidnogo peptida s riadom intaktnykh (peroksiredoksin, GAFD) i/ili podvergnutykh okislitel'noĭ modifikatsii (peroksiredoksin, piruvatkinaza) model'nykh belkov.

Amyloid-β peptide (1-42) (Aβ1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aβ1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aβ1-42. It is reasonable to expect that dissociation of the Aβ1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aβ1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aβ1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aβ-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35). Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aβ1-42 and Aβ25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aβ peptides except Aβ1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aβ1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aβ12-28 and Aβ25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aβ studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aβ1-42, and its aggressive truncated peptides (Aβ25-35).Beta-amiloidnyĭ peptid (1-42) (Aβ1-42) iavliaetsia odnim iz “kliuchevykh igrokov”, vovlechennykh v razvitie bolezni Al'tsgeĭmera (AD) i drugikh patologicheskikh sostoianiĭ, obuslovlennykh obrazovaniem i nakopleniem belkovykh agregatov v tsentral'noĭ nervnoĭ sisteme. Ego sviazyvanie s vazhnymi vnutrikletochnymi misheniami s posleduiushcheĭ ikh inaktivatsieĭ, po-vidimomu, mozhet predstavliat' soboĭ odno iz rannikh sobytiĭ, predshestvuiushchikh formirovaniiu vnekletochnoĭ patogennoĭ oligomerizatsii/agregatsii Aβ1-42. Mozhno ozhidat', chto dissotsiatsiia kompleksov Aβ1-42 s belkami-misheniami pod deĭstviem ingibitorov kompleksoobrazovaniia i posleduiushchaia degradatsiia Aβ1-42 budet ne tol'ko zashchishchat' kriticheski vazhnye vnutrikletochnye misheni, no takzhe prepiatstvovat' nakopleniiu Aβ1-42 vnutri kletok. Tsel'iu dannoĭ raboty bylo issledovanie vliianiia neĭroprotektora izatina (100 mkM) na vzaimodeĭstvie izvestnykh Ab-sviazyvaiushchikh belkov – glitseral'degid-3-fosfatdegidrogenazy (GAFD) i piruvatkinazy – s Aβ1-42 i ego fragmentami (Aβ1-28, Aβ12-28, Aβ25-35). Immobilizovannye na chipe opticheskogo biosensora Biacore Aβ1-42 i ego fragmenty (Aβ1-28, Aβ12-28, Aβ25-35) vzaimodeĭstvovali s GAFD i piruvatkinazoĭ. Naibolee nizkie i primerno ravnye znacheniia Kd byli polucheny dlia kompleksov étikh fermentov s Aβ1-42 i Aβ25-35. V prisutstvii 100 mkM izatina sushchestvennoe uvelichenie Kd v 5 i bolee raz otmecheno dlia kompleksov GAFD so vsemi issledovannymi Ab peptidami za iskliucheniem Aβ1-28. V otlichie ot GAFD, izatin sposobstvoval dissotsiatsii kompleksov piruvatkinazy tol'ko s polnorazmernym Aβ1-42 (vyzyvaia uvelichenie Kd bolee chem v tridtsat' raz) i v men'sheĭ stepeni s Aβ25-35 i Aβ1-28 (uvelichenie Kd primerno na odin poriadok). Pri étom znacheniia Kd dlia kompleksov piruvatkinazy i GAFD so vsemi issledovannymi Ab peptidami v prisutstvii 100 mkM izatina nakhodilis' v bolee uzkom diapazone (10-7 M – 10-6 M), chem v otsutstvie étogo neĭroprotektora (10-8 M – 10-6 M). Poluchennye dannye svidetel'stvuiut o sushchestvovanii printsipial'noĭ vozmozhnosti (farmakologicheskoĭ) zashchity vazhnykh vnutrikletochnykh misheneĭ ot deĭstviia kak Aβ1-42, tak i ego agressivnykh ukorochennykh peptidov (Aβ25-35).

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (K d = 2.4·10–6 M) and pyruvate kinase (K d = 2.8·10–5 M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10–8 and 3.2·10–9 M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity-based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC-MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR-based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein-protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

A comprehensive study of the state of urban soils revealed the altered structural and functional organization of microbiocenosis versus that of the soils of intact forest ecosystems. The indicator microbial and biochemical parameters of the state of the soils under urban technological pressure were identified. The findings may be used to evaluate the natural environment, to make an urban environmental monitoring.

We tested the hypothesis that serum heart fatty-acid binding protein (FABP), an early marker of myocardial necrosis, is related to prognosis of patients hospitalized because of worsening heart failure (HF).Sixty nine patients (64% men, age 66.6 +/- 11.0 years) with NYHA class II, III, IV HF (1, 18, and 50 patients, respectively) at hospital admission were followed for 6-12 (mean 11.6 +/- 1.3) months. Forty seven patients (68.1%) had history of myocardial infarction (MI), 56 (81.2%) - hypertension, 15 (21.7%) -- diabetes, and 17 (24.6%) had echocardiographical signs of aortic stenosis. Median left ventricular ejection fraction was 28%. Serum FABP, cardiac troponin I (Tn I) and N-aminoterminal pro brain natriuretic peptide (NT proBNP) were measured within 3 days after admission ( " admission " levels) and 2 weeks later (minimal hospital stay). Manufacturer recommended upper limits of norm (ULN) were 4.0 ng/ml for FABP, 0.35 ng/ml for Tn I, 0.1 ng/ml for NT proBNP.Median admission FABP was insignificantly higher than level measured 2 weeks later (4.17 vs 4.03 ng/ml, p=0.069). FABP exceeded ULN in 38 (55.1%) patients and in 35 (50.7%) patients at admission and in 2 weeks, respectively (p=0.65). Median admission NT proBNP was significantly higher than 2 weeks level (13.23 vs 6.02 ng/ml, p0.0001). Median admission and 2-weeks levels of Tn I were similar and greatly lower than ULN. There were 27 all cause deaths (39.1%) during follow up. Median admission levels of TnI, FABP and NT proBNP were similar in patients who died and survived. Two weeks NT proBNP was significantly higher in patients who died (8.65 vs 3.62 ng/ml, p=0.012). ROC curve derived cut-off levels of FABP and NT proBNP (3.31 ng/ml and 3.5 ng/ml, respectively) were used in univaritate regression analysis. According to this analysis FABPor= 3.31 ng/ml was related to occurrence of death (OR 3.54; 95% CI 1.03-12.17, p=0.044). FABP and variables with p0.1 (age, history of MI and diabetes, regular treatment with nitrates, signs of aortic stenosis, pulmonary rales at admission, and 2 weeks level of NT proBNPor = cut-off) were included into multivariate logistic regression model. Independent predictors of death were aortic stenosis (OR 31.67; 95% CI 6.11-164.00) and NT proBNPor= 3.5 ng/ml (OR 5.75; 95%CI 1.69- 19.52).In this group of patients hospitalized due to worsening of HF admission values of neither FABP nor other biomarkers studied were predictors of death during about 1 year of follow up. FABP level after 2 weeks of hospital stay was related to occurrence of death but as predictor was inferior to NT-proBNP measured at the same time point.

To predict motor activity in the newborn, electromyographic (on rats) and ultrastructural (on chick embryos) studies were performed after administration of donor babies' sera taken in the first days of life. Morphological and electromyographic parameters were compared with late clinical symptoms. A rating schedule was developed with regards to biological test results, which allows motor disorders to be detected in the newborn.

Two forms of phosphodiesterase (F1 and F2) with different regulatory properties have been isolated from the soluble fraction of human brain cortex. F1 is the Ca(2+)-calmodulin-dependent phosphodiesterase and its activity is inhibited by calmodulin antagonists (W-7, TFP, tamoxifen) via a mechanism typical for the majority of Ca(2+)-calmodulin-dependent enzymes. F2 is activated by micromolar concentrations of cGMP (7-14-fold) and by Ca2+ ions (1.5-3-fold) in the absence of exogenous calmodulin. F2 contains a tightly bound Ca(2+)-binding component (apparently calmodulin) which does not dissociate from the enzyme in the presence of EGTA. The mechanism of calmodulin antagonists action on F2 is different from that for F1.

The kinetic characteristics of two forms of soluble Ca(2+)-calmodulin-activated phosphodiesterase (PDE I and PDE II) from human brain are presented. Both PDE forms display a higher affinity for cGMP than for cAMP: the differences in the Km values of PDE II for the both cyclic nucleotides are less pronounced. For the both enzyme forms cGMP competitively inhibits cAMP hydrolysis. Activation of PDE I by Ca(2+)-calmodulin is characterized by increasing Vmax without any changes in Km for both substrates. In case of PDE II the Ca(2+)-calmodulin-dependent increase in Vmax is accompanied by decreasing Km for cAMP and cGMP. Pyrazidol and nitropyrazidol inhibit Ca(2+)-calmodulin induced activity with no effect on basal PDE activity. The data obtained exclude competition of the both compounds with the substrate and the activator for the catalytic site and the allosteric Ca(2+)-calmodulin-specific site. It is suggested that the allosteric effect of these compounds on the activity of PDE involves a site in the enzyme which is distinct from the calmodulin-binding site.

The effects of the antidepressant pyrazidol (pirlindole) and its nitro analog on cGMP-dependent phosphodiesterase from the soluble fraction of human brain cortex have been studied. Pirlindole inhibits the cGMP-induced activity of phosphodiesterase (IC50 = 1585 microM) without influencing the basal activity; its action is noncompetitive in respect of the substrate and the activator. Nitropirlindole is a more potent inhibitor of cGMP-dependent phosphodiesterase (IC50 = 115 microM) competing with the substrate for the catalytic site (K(i) = 70 microM). There is no direct interaction of pirlindole (pyrazidol) and its nitro analog with the cGMP-specific site in phosphodiesterase.

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.

The paper provides the reported data on the incidence, localization, clinical manifestations, course, diagnosis and treatment of dermoid cysts. Describes three cases of dermoid cysts verified by computed tomography and surgery. Presents a case of an atypical dermoid cyst with an outbreak of the cyst contents to the brain ventricles and origination of purulent meningitis. Shows the high information content of computed tomography in respect to intravital diagnosis of that type of pathology.

As many as 61 patients with neurogenic syncopes were examined. Of these, 46 demonstrated well-defined signs of the hyperventilation syndrome within the structure of the clinical manifestations. The remaining 15 patients had no disorders indicated. The control group comprised 18 healthy persons. Thorough analysis of factors provoking syncopal, pre- and postsyncopal conditions, studies into the psychic and vegetative spheres including the respiratory system--all this made it possible to define a number of features characteristic of patients entering the groups under study. It has been shown that hyperventilation bears the function of the leading factor in the pathogenesis of vegetative disorders. In addition to chronic hyperventilation sequels, those disorders entail the occurrence of syncopal conditions. During treatment, of paramount importance is the correction of the impaired respiratory pattern, the reduction of neuromuscular excitability along with the use of psychotropic and vegetotropic remedies.

The role of hyperventilation-induced shifts in the pathogenesis of different kinds of autonomic disorders (autonomic paroxysms, syncopal states, allergic signs) was investigated in 156 patients. This role should be considered in therapeutic interventions, especially in respiratory training aimed at correcting the respiratory pattern in order to achieve psychoautonomic stabilization. Distinct pathogenic mechanisms of combined hyperventilation and autonomic disorders are discussed.