CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the Nterminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

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.

The tumor-associated antigens, up-to-date methods for their identification and immunotherapeutics strategies for cancer treatment have been reviewed. The main attention has been focused on the antigens identified by SEREX (Serological identification of antigens by recombinant expression cloning) metho dology and the categories of tumor-associated antigens (i. e. identified by SEREX approach) have been listed. The main immunotherapeutic approaches have been classified, the successes of active specific immunotherapy based on immunodominant peptides of tumor-associated antigens have been described.

In this study two cDNA expressing libraries generated from thyroid papillar carcinomas were screened using SEREX approach. Thirty positive cDNA clones representing seventeen different genes were identified from both libraries. It is important to note, that three of them were isolated previously by other laboratories in SEREX screens of various types of human cancer. These include transcription factor NZF, a-catenin and ВАС RPU-a protein with unknown function. Moreover, we identified a whole panel of novel potential tumor-associated antigens, which would be further investigated. We are particularly interested in more detailed analysis of cathepsin H and transducer of ErbB2 (TOB2), which are differentially expressed in various types of human cancer. We will analyse the frequency of autoantibodies against identified antigens in sera of patients with various malignancies and healthy donors by heterologous screening. It is expected that among the clones isolated in this study, there might be novel cancer-associated markers.

The In sti tute of Mo lec u lar Bi ol ogy and Ge net ics, NAS of Ukraine 150, Ac a de mi cian Zabolotny Str., Kyiv, 03143, Ukraine The method for ob tain ing and pu ri fi ca tion of re com bi nant chap eron GroEL (prokaryotic homolog of eukaryotic chap eron Hsp60), in cluding the pro tein ex pres sion in E.coli cells, pre cip i ta tion with sat u rated am mo nium sul phate from a producent lysate, gel-fil tra tion on Sephacryl-300 col umn and ion-ex change chro ma tog ra phy on MonoQ HR 5/5 col umn, is de scribed. The method al lows ef fec tive ob tain ing a pre para tive amount of the GroEL pro tein of 95% pu rity. The re com bi nant pro tein was used for the anti-GroEL an ti bod ies pro duc tion and syn the sis of an af fine col umn.The im mu no logic cross-re ac tiv ity of polyclonal af fine-pu ri fied anti-GroEL an ti bod ies and mem bers of Hsp60 fam ily from dif fer ent mam ma lian or gans and cells-from mouse to hu man, has been re vealed. This al lows us ing these an ti bod ies for re search of the Hsp60 ex pres sion al ter ations and cell lo cal iza tion at hu man au to im mune and cancer pathologies.