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Loss of function or/and death of cardiomyocytes is one of the major contributing factors in the development of heart failure. Cytosolic Hsp60 can directly interact and regulate activation of some kinases and sequestrate certain proapoptotic molecules to avoid the cardiomyocyte apoptosis. We assumed that Akt1 kinase, a downstream effector of PI3 kinase, can interact with Hsp60. Our aim was to clarify the interaction of Akt1 and Hsp60 and to investigate the Akt1 expression in normal and failing hearts in acute and chronic stress. The experimental mouse models of inducible myocarditis and DCM-like pathology were developed in our laboratory. Akt1 and phospho-Akt1 (pS473) expression were studied by Western blot analysis. Co-immunoprecipitation method was used to test complex formation of Akt1 and Hsp60. The interaction of Hsp60 and Akt1 was detected for the first time by co-immunoprecipitation method in normal myocardium and under pathology as well. There were no significant changes in the level of Akt1 expression in both myocardia. At the same time we observed significant decrease in Akt1 phosphorylation at the final stage of DCM-like pathology but not at experimental myocarditis. The final stage of heart failure in mouse model of DCM-like pathology was characterized by reduced level of phospho-Akt1/Akt1 (pS473; -26%; P0.05), whereas no differences were found in total Akt1 protein content. We suggest a possible involvement of cytoplasmic Hsp60 in regulation of Akt1 activity at heart failure progression.

Recently, it has been suggested that some heat shock proteins such as Hsp70 and Hsp60 are involved in autoimmune diseases including cardiospecific ones. In this work we focused on the involvement of another well known heat shock protein, Hsp90, and its novel co-chaperone, Sgt1, in dilated cardiomyopathy (DCM). We found that the level of autoantibodies against these two proteins was significantly higher in patients with DCM and ischemic heart disease than in sera of healthy donors. We have also analyzed the expression level and subcellular localization of Hsp90 and Sgt1 in diseased myocardia. Using Western blot we found changes in subcellular localization of Hsp90 in the left ventricle of DCM hearts while the total level of this protein remained unchanged. Regarding the Sgt1 protein, we found an increased level in DCM and no changes in subcellular localization. Taken together, our data suggest that Hsp90 and Sgt1 might be involved in the progression of heart failure and might serve as markers for cardiomyopathies of different origin.

Molecular chaperon Hsp60 and protein kinase p70S6K play an important functional role in the regulation of cardiomyocytes vital function or apoptosis. Aim. To study a possibility of in vivo complex formation between Hsp60 and p70S6K in cardiomyocytes. Methods. Co-immunoprecipitation, Western-blot analysis. Results. We have identified in vivo interaction between molecular chaperone Hsp60 and two isoforms of proteinkinase p70S6K in human myocardium, normal and affected by cardiomyopathy. Conclusions. The results obtained suggest a possible participation of molecular chaperon Hsp60 in regulation of p70S6K activity in stressinduced apoptotic signaling pathway in cardiomyocytes.

Aim. To investigate the levels of specific autoantibodies against tyrosyl-tRNA synthetase and its individual modules in the blood serum of people with heart failure caused by dilated cardiomyopathy, myocarditis and ischemic heart disease compared with healthy donors. Methods. Recombinant proteins were obtained using bacterial strains transformed with appropriate plasmid vectors and were purified by chromatography on Ni-NTA-agarose. The levels of specific autoantibodies were investigated by ELISA. Results. The increased levels of autoantibodies specific to tyrosyl-tRNA synthetase, its N-terminal catalytic module and non-catalytic C-module, were found in the blood serum of patients, compared with healthy donors. Conclusions. The results obtained demonstrate the possible role of tyrosyl-tRNA synthetase in adaptive changes of the myocardium in response to stress factors.

Phosphorylation is one of the most frequently occurring posttranslational modifications in proteins. It plays an essential role in transferring outside signals into a cell and regulates different cellular processes such as growth, metabolism, proliferation, motility and differentiation. Melusin is a stress response protein which strictly reacts to the threshold levels of mechanic stress and activates cardiomyocytes signaling pathways. The search for potential sites of melusin phosphorylation was performed using bioinformatic analysis of primary protein sequences. The comparative bioinformatic analysis of possible phosphorylation sites, evolutionary and structural motifs has identified Ser326,Ser329 and Ser334 as the most likely sites for phosphorylation of melusin by protein kinase CK2 in cardiamyocytes.

The levels of Hsp60, Hsp70 and Hsp90 in rats’ gastric mucosa cells lysates upon the development of experimental chronic atrophic gastritis (CAG) were investigated by the immunoblot-analysis. The quantities of Hsp60 and Hsp70 at the early (the first two weeks) and late (the 5–6 weeks) stages of disease progression were reliably higher in comparison with normal mucosa. It was also observed an increase in Hsp90 expression at the end of the first week of CAG. The increase in Hsp expression in gastric mucosa was shown upon activation of apoptosis, generation of reactive oxygen species, and gastric atrophy development.

More than 12.1 million people with hypertension (32.2% of the adult population) were registered in Ukraine according to the official statistics on 1 January 2011. The etiopathogenesis of AH is not fully established. Hsp60 is the molecular chaperon/chaperonin, and it's expression significantly increases in response to different kinds of stress (emotional stress, infections, smoking etc). Elevated blood pressure is a mechanical stress to the endothelium and it can induce expression of heat-shock protein 60 (Hsp60) on the endothelial cell surface. Endothelial cells in the vessel wall can be damaged by (auto) immune reactions to Hsp60 present on the cell surface. Elevation of anti-Hsp60 in the circulation is associated with the presence and severity of coronary heart disease, atherosclerosis development, pathological changes in the small vessels of the brain etc etc. Specificity of the anti-Hsp60 antibodies and their role in the pathogenesis of AH has not been established. The aim of this work was to identify the level of anti-Hsp60 antibodies in the sera of patients with AH. 128 patients with AH were examined. To define level of anti-Hsp60 antibodies the sera 39 patients with AH, including 12 clinically healthy individuals (the family history are included the AH cases)--1 group, 19 patients with stage 2--2 group and 8 patients with stage 3--3 group were examined. The control group included 112 blood donors. Anti-Hsp60 antibodies in sera were determined by ELISA and immunobloting (Western-blotting). Recombinant piotein GroEL Escherihia coli (prokaryotic homologue of human Hsp60) and human Hsp60 were used as antigens. Average of levels of antibodies against GroEL and human Hsp60 in the serum of all groups twice exeeded the value of the control (P0.001). Antibodies to prokaryotic Hsp60 were prevailed in patients with AH. The seropositive serum to Hsp60 were detectived in patients, that had the risk of the AH complications by ELISA and immunoblotting. In addition, highly reactive IgG anti-Hsp60 antibodies purified by affinity chromatography from human sera of patients with AH recognized GroEL and human Hsp60 in immunoblotting. Elevated levels of anti-Hsp60 antibody in sera of patients with AH stage 3 correlated with pronounced changes in the target organs such as a massive recurrent hemorrhage into the retina, acute ischemic stroke, cardiosclerosis and angionephrosclerosis. It may indicate the involvement of anti-Hsp60 antibodies in the development of the target organ damage.

Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.

The data concerning DNA-Topoisomerase I (Topo-I) from human placenta as an autoantigen at the thyroid cancer are presented. A new effective scheme of the Topo-I purification using the High-Trap columns is described, as well as the enzyme physico-chemical properties and its immunoreactivity in the ELJSA and immunoblotting. It was shown that 46 % of the sera from patients suffering the thyroid cancer were immunoreactive against Topo-I, 36 % of autoantibodies from thyroid cancer patients sera recognized sequential determinants on the Topo-I molecule. The highest levels of immunoreacivity were observed in the sera of thyroid cancer patients accompanied by autoimmune thyroiditis. We suggest a possible linkage between the autoimmune processes and malignization of the thyroid gland cells. Представлены данные по изучению ДНК-топоизомеразы I (Топо 1), выделенной из плаценты человека, как аутоантигена при раке щитовидной железы. Описана новая эффективная схема очистки Топо I с применением High-Trap-колонок, фи­ко-химические свойства фермента и его иммунореактивность, определенная в реакции ELISA и иммуноблоттинге. Показано, что Топо I-положительными оказались 46 % сыво­роток пациентов с раком щитовидной железы; из них 36 % содержали аутоантитела против Топо 1, распознающие анти­генные детерминанты секвенционного типа. Отмечено, что наиболее иммунореактивными были сыворотки пациентов с раком щитовидной железы и сопутствующим аутоиммунным тиреоидитом. Высказано предположение о возможной связи аутоиммунных процессов со злокачественной трансформацией клеток щитовидной железы. Представлено дані з вивчення ДНК-топоізомерази І (Топо І), виділеної з плаценти людини, як аутоантигена при раку щитовидної залози. Описано нову еффективну схему очищення Топо І з використанням High-Trap-колонок, фізико-хімічні властивості ферменту і його імунореактивність, визначену в реакції EUSA та імуноблотингом. Показано, що Топо I-позитивними виявилися 46 % сироваток пацієнтів з раком щито­видної залози; з них 36 % містили аутоантитіла проти Топо І, які розпізнавали антигенні детермінанти секвенційного типу. Відмічено, що найбільш імунореактивними були сиро­ватки пацієнтів з раком щитовидної залози і супутнім аутоімунним тиреоїдитом. Зроблено припущення щодо мож­ливого зв'язку аутоімунних процесів із злоякісною трансфор­мацією клітин щитовидної залози.

Animal and fungal cells (in contrast to prokaryotes) contain two distinct sets of related aminoacyl-tRNA synthetases (aaRSs) encoded by nuclear genes and functioning in cytosol and mitochondria. The structural differences between mitochondrial and cytoplasmic enzymes may reflect the functional adaptation to fulfil mitochondrial processes in addition to protein synthesis. Mitochondrial import of nuclearencoded tRNAs has been described in yeast, plants and protozoans but it has not been observed in mammalian cells. Ifs established that mitochondrial lysyl-tRNA synthetase (MSK) plays a prominent role in the transport of tRNA into yeast mitochondria for complementation o f mitochondrial tRNAs genes mutations. We tried to identify MSK homologues in mammalian cells with the help of monospecific antibodies against pre-MSK by ELISA and Western-blot analysis. We have identified cross-reactive proteins in mitochondrial and cytoplasmic fractions of mammalian cell lysates. These data, together with the results of cross-aminoacylation on mitochondrial and cytoplasmic tRNAs, suggest the presence of common antigenic determinants in the mitochondrial and cytoplasmic lysyl-tRNA synthetases from higher animals. Клітини еукаріот на відміну від прокаріот містять дві різні групи аміноацил-тРНК синтетаз, які кодуються ядерним геномом та функціонують в цитозолі і мітохондріях. Струк­турні відмінності між ферментами мітохондрій і цитоплаз­ми можуть бути відображенням функціональної адаптації до процесів, які відбуваються в мітохондріях, крім участі в біосинтезі білка. Імпорт цитозольних тРНК у мітохондрії описано для дріжджів, рослин і найпростіиіих, однак він не спостерігався в клітинах ссавців. Виявлено, що мітохондріальна лізил-тРНК синтетаза (MSK) відіграє провідну роль у транспорті тРНК у мітохондрії дріжджів для комплемен­тації мутацій мітохондріальних генів тРНК За допомогою моноспецифічних антитіл проти npe-MSK ми зробили спробу ідентифікувати гомологи MSK у клітинах ссавців методами ELISA і Вестерн-блотинга. В цитоплазматичних і міто­хондріальних фракціях лізатів клітин ссавців нам вдалося виявити білки, які мають імунологічний перехрест з MSK Разом з результатами перехресного аміноацилювання ці дані дають підставу для припущення щодо наявності спільних антигенних детермінант у мітохондріальних і цитоплазма­тичних лізил-тРНК синтетаз ссавців. Клетки эукариот (в отличие от клеток прокариот) содержат две различные группы аминоацил-тРНК синтетаз, кодируе­мых ядерным геномом и функционирующих в ц и то золе и митохондриях. Структурные отличия между ферментами митохондрий и цитоплазмы могут быть отражением функци­ональной адаптации к процессам, происходящим в митохонд­риях помимо участия в биосинтезе белка Импорт цитозольных тРНК в митохондрии описан у дрожжей, растений и простейших, однако не наблюдался в клетках млекопитаю­щих. Установлено, что митохондриальная лизил-тРНК син­тетаза (MSK) играет ведущую роль в транспорте тРНК в митохондрии дрожжей для комплементации мутаций митохондриальных генов тРНК. С помощью моноспецифических антител против npe-MSK мы попытались идентифицировать гомологи MSK в клетках млекопитающих методами ELISA и Вестерн-блотинга. В цитоплазматических и митохондриальных фракциях лизатов клеток млекопитающих нам удалось обнаружить белки, имеющие иммунологический перекрест с MSK В совокупности с результатами перекрестного аминоацилирования эти данные дают основание предположить нали­чие общих антигенных детерминант у митохондриальных и цитоплазматических лизил-тРНК синтетаз высших живо­тных.

The main functional compartments of molecular chaperone Hsp60 are mitochondria and cytoplasm. Up to 30 % of Hsp60 are located in cytoplasm of cardiomyocytes. The interaction between molecular chaperone Hsp60 and proapoptotic Bax protein in the cytoplasmic fraction from normal human heart tissue has been revealed by co-immunoprecipitation in contrast to myocardium affected by dilated cardiomyopathy, where this interaction has not been observed

The autoantibodies directed against actin and myosine have been detected in the blood sera of patients with dilated cardiomyopathy (DCMP) at the level that was sufficiently higher than in healthy donors. While comparing of the immunogeneicity of these pro­ teins we have stated that myosine from affected by DCMP myocardium have been more immunogenic then from normal one. In the case of actin the immunogeneicity have been higher for the protein from normal myocard. The investigations of actin by limited proteolysis may suggest that the differencies in immune properties are not connected with primary structure but with structures of higher levels. Introduction. Nowadays the presence of autoimmune processes during the development of various pathologies including heart diseases is obvi­ ous. At the same time the functional activity of autoAbs wasn't exami­ ned enough. The investigation of structural and functional properties of autoantibodies (aAbs) directed against antigens from heart and vessels, the establishment of their role while the origin and development of heart diseases is necessary not only for theoretical but for practical aims also, in the context of developing of novel therapeutic and diagnostic prepa­ rations (1). The aim of our investigation was to study the molecular mechanisms of autoantibodyg enesis directed against the most important tissue-speci­ fic antigens — actin and myosin, during development of dilated cardio­ myopathy (DCMP). In recent studies the aAbs directed against adenine nucleotide translocator (ANT) (2), the mitochondrial enzyme from myo­ cytes were detected; the aAbs directed against another mitochondrial en­ zyme, branched chain keto acid dehydrogenase (BCKD) (3) were also described at DCMP and myocarditis. The laboratory of Limas (USA) during recent years studied the structure and properties of aAbs directed against p-adrenoreceptor (4—8); these aAbs were detected in 30—40 % of patients with DCMP. The presence of aAbs directed against myosin, tropomyosin, vimentin, laminin at some heart diseases such as postmyo- cardial infarction syndrome, dilated cardiomyopathy, infectious myocar­ ditis, and doxorubicin (Adriamycin) cardiotoxicity (9)' is occasionally accompanied by the deposition of antibodies within the myocardium, par­ ticularly on the sarcolemma. The present paper is a first stage of study of aAbs, their properties and role at DCMP progressing, which are directed against the most abundant heart antigens — actin and myosin. The method described (10) for purifing of preparative quantities of antigens from human myocardium (pathomorphological material) enabled us to obtain the mentioned above antigens as from normal myocardium as from myocardium, affected by DCMP. The presence of aAbs in the sera of patients with DCMP was not unexpected because many facts suggested the presence of cellular and humoral immunity in the development of DCMP {4). These data concern © L. L. Sidorik, N. V. Rodnin, V. I. Bobyk, D. V. Ryabenko, A. V, Veberov, T. Yu. Tkachenko G. Kh. Matsuka, 1996

Investigation of antibodies to the DNA-topoisomerase-1, the marker for autoimmune process in the blood serum, was conducted in 22 patients with chronic fibrous-degenerative pancreatitis, operated on in the clinic. The trustworthy raising of the antibodies titer in comparison with such in healthy donors was noted. In comparative analysis of the index in patients before and after the operation it was established, that in 12 of them after the standard draining operation it did not change within up to 12 months, in 10 patients, after Frey operation, performed in our modification, it had lowered almost twice.

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins – molecular chaperons (HSPs) – are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HSPs are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.

ATP activity of actomyosin in dilated cardiomyopathy (DCMP) was studied together with impact on the above activity of tropomyosin-troponin regulatory complexes recovered from the normal myocardium and cardiac muscle of a DCMP patient. Recordable in DCMP was a striking decline (1.5-fold) in ATP activity of actomyosin. But no significant difference was to be seen in sensitivity to Ca2+ ions of actomyosins obtained from the normal myocardium and cardiac muscle of the DCMP patient. The cardial tropomyosin-troponin regulatory complex from the DCMP patient's myocardium was shown to be endowed with somewhat more manifest activity compared to the analogous complex recovered from the normal myocardium.

Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl-tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested. The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods. pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes. It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.

The PI3K/PDK/Akt/mTOR/p70S6K signaling pathway is primary associated with the activation of insulin receptors and is important for cardiomyocytes survival. p70S6K is a key regulator of the speed and efficiency of protein biosynthesis within the cell. Recently the pro-apoptotic protein BAD has been identified as a new target for р70S6K1. BAD is inactivated in normal cardiomyocytes by р70S6K1 phosphorylation which prevents the cardiomyocytes apoptosis. Aim. To study possible changes in р70S6K1 expression and/or cellular localization at heart failure progression – in DCM- affected human myocardia and murine hearts with experimental DCM-like pathology. Methods. Western-blot analysis and immunohystochemistry. Results. The substantial decrease in р70S6K1 level was observed at the final stage of pathology progression and in the dynamics of DCM pathogenesis as well. For the first time relocalization of the protein to the connective tissue was shown according to the Western-blot results. Conclusions. The data obtained allow us to understand a possible role of р70S6K1 in the regulation of stress-induced apoptotic signaling in cardiomyocytes.