[Study of Dj-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients].

Two isoforms of Dj-1 protein were identified using a proteomic study in tissue specimens from two groups of patients with confirmed benign prostate hyperplasia (BPH) and prostate cancer (PCa). Dj-1 was also found in the cell lines PC-3, DU-145, LNCaP, BPH-1, and the lowest level of Dj-1 was found in BPH-1. Immunochemical study (ELISA) of serum levels of Dj-1, Bcl-2, IGF-1 and IGFBP-3 proteins revealed statistically significant distinctions between two groups of patients (p=0,004, Mann-Whitney test) only for Dj-1. Taken together, these data suggest that Dj-1 protein is a perspective biomarker candidate for PCa.

[Mass-spectrometric identification of interaction sites between cytochrome P450 2B4 and NADPH cytochrome P450 reductase].

We determined the interaction sites of the cytochrome P450's protein-partners: 2B4 (d-2B4) and NADPH-cytochrome P450 of reductase (d-Fp). While in operation, these proteins are forming the complexes. We used 4-4'-dithio(bisphenyl)azide linker for non-specific covalent coupling of d-2B4 complexes with d-Fp in Emulgen-913-monomerized system. Covalently-linked peptides in this complex were identified with ESI-MS/MS. Several sites of these proteins' binding with each other were revealed. Based on them, a model of intermolecular protein interactions was created. The model includes 5 cross-linker-stabilized contact sites of d-2B4 with d-Fp involving the following peptides of d-2B4 and d-Fp: (1) d-2B4423-433 and d-Fp 102-109; (2) d-2B4324-336 and d-Fp570-585; (3) d-2B4327-336 and d-Fp452-464; (4) d-2B4 192-197 and d-Fp456-464; (5) d-2B4 134-139 and d-Fp406-425. Herein, in the latter two cases, the peptides of d-Fp are located in their inter-domain slit and stabilize protein-protein complex via nanoprobe cross-linker; therefore, the formation of d-2B4/d-Fp complexes in these sites may involve aminoacid residues d-Fp456-464 and d-Fp406-425 surrounding inter-domain slit.

[The mechanism of action of reactivating factor from Luteococcus japonicus subsp. casei].

Reactivating factor (RF) from Luteococcus japonicus subsp. casei was shown to be constitutively synthesized and to act a by one-step mechanism, being activated independently from stress. Cell reactivation (reversion of a cell's ability to form macrocolonies) might be ensured by the membrane mechanism of RF action, which is proved with the dependence of antistress activity from the condition of the cytoplasmic membrane and with the form of concentration dependence. The incubation of UV-treated L. casei suspension with RF increased the number of cells with intact barrier membrane (1.6-1.8-fold increase compared to RF-untreated cells) and the number of colony-forming cells. Cross defensive and reactivating RF effects on both L. casei and yeast Saccharomyces cerevisiae cells were described. Bacterial and yeast's RF compete for membrane receptors. Matrix Assisted Laser Desorption/Ionization time-of-flight (MALDI-TOF) spectrometry revealed that RF ofL. casei contained two major peptides of 5.8 and 7.6 kDa, while RF of S. cerevisiae was represented by a single peptide of 5.8 kDa. The presence of 5.8 kDa peptide in RF from bacteria and yeasts might ensure cross responses in these organisms.

[A study of protein profile changes in differentiating human myoblasts].

The changes in the protein profile in cultured human myoblasts after induction of differentiation was studied by proteomic techniques (a combination of O'Farrell two-dimensional electrophoresis and subsequent protein identification by MALDI-TOF MS and MS/MS analyses). Forty-one proteins have been identified, 25 of which were present in both proliferating and differentiating myoblasts, which allows them to be considered as myoblast housekeeping proteins. The changes in the distribution of some isoforms of tropomyosins, S100 proteins, cofilin, etc. have been revealed. The possible role of these changes in the cell protein profile in the realization of the program of skeletal muscle cell differentiation is discussed.

[Clinicobiochemical study of diagnostic value of the panel including 10 potential protein markers of prostatic cancer].

We tested diagnostic value of the panel of 10 specially selected proteins--potential markers of prostatic cancer. A double blind method and proteomic technologies were used in complex clinicobiochemical examination of 20 patients with benign and malignant tumors. The same diagnosis were obtained by clinicomorphological criteria and protein markers in 13 (65%) cases. The highest diagnostic efficacy was achieved in prostatic cancer--11 cases (79%) vs 14 by clinicomorphological criteria.

[Determination of "amino acids conflicts" and amino acids substitutions in primary structures of 41 human proteins by use the proteomic technologies].

Proteomic studies of some human tissues and organs (skeletal muscles, myometrium, motor zone of the brain, prostate), and also cultivated myoblasts revealed 41 of 300 identified proteins, in which the present of certain variants of amino acids ("conflicts") was recognized at several positions. Among the 93 registered amino acids "conflicts", seven cases represented the results of the protein polymorphisms caused by corresponding substitution of individual amino acid. Moreover, among prostate proteins the proteomic analysis revealed two isoforms of prostate-specific antigen, formed due to alternative splicing. Thus, our results have shown, that proteomic technologies allow to specify effectively the features of primary structures and to characterize various kinds of polymorphism in many human proteins.

[Comparative transcriptomic and proteomic analysis of the fetal and adult human liver].

The transcriptome and proteome comparative analysis of the fetal liver (9.5-10.5 weeks of gestation) and normal adult liver was developed in the present investigation. We used 44k microarrays of Agilent company and 2D-GE MALDI-TOF in transcriptome and proteome approaches, respectively. The top lists of expression genes and proteins for fetal liver obtained by these methods were compared. Transcriptome analysis confirmed proteome data only partially, but these two semi-quantitative approaches established the interdependences between expression levels of genes and proteins. The discrepancies between data obtained by two approaches were discussed. The unique feature of the fetal liver cell content is the combination of hemopoietic and nonmaturated liver cells. Fetal liver changes its hemopoietic role during embryogenesis towards the detoxicative tissue being able to metabolize different substances and produce wide serum of proteins. The status of fetal liver as the hemopoietic tissue was entirely characterized by transcriptome analysis having registered embryonic and adult hemoglobins, insufficient cytochrome-dependent detoxification system and highly developed anti-apoptotic potential.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[Identification of AGR2 protein, a novel potential cancer marker, using proteomics technologies].

A comparative analysis of the proteins in prostate tissues of the patients operated for hyperplasia (n = 7) or cancer (n = 5) was performed aiming to search for protein diagnostic markers. Differences in several minor proteins were detected using two-dimensional electrophoresis according to O'Farrel; among them, an additional protein with a molecular weight of 19 kDa and an isoelectric point of 9.0 was observed in four of the cancer cases. Mass spectrometry allowed this protein to be identified as the androgen-induced secreted protein AGR2. The possibility of using AGR2 as a diagnostic marker of prostate cancer is discussed.

[Proteomic analysis of two isogenic Vibrio cholerae of the classical biovar with the alternative expression of virulence genes].

At carrying out the proteomic analysis of two isogenic Vibrio cholerae Dakka35 of the classical biovar itwas revealed, that toxigenic (1 type) and nontoxigenic (2 type) clones differ from each other not only the expression ofgenes of exopolysaccharide, motility, and soluble haemagglutinin/protease, but also change of activity about other 60 genes. Among 11 identified proteins 5 are the enzymes participating in a metabolism cells. Besides it is revealed, that clones 2 types of Dakka35 strain synthesize in a more level of OmpU and TolC proteins, which provide their more significant stability to action of bile in comparison with clones of 1 type. It was shown, that bile serves as a signal from an environment for switching of gene expression of the genes, coding production of factors as virulence, and carrying out protective function of bacterial cell.

[New approaches to molecular diagnosis of prostatic cancer].

We compared prostatic proteins in patients operated for adenoma or cancer. 630 protein fractions were obtained from each tissue sample after fractionation by two-dimentional electrophoresis according to O'Farrell. Comparison of the samples from adenoma and cancer showed their difference by 7 proteins among which were isoforms of glyceraldehydes-3-phosphate-dehydrogenase, alpha-collagen and several little known proteins. Most of the cancer patients had the protein with molecular mass 19 kDa and isoelectric point 9.0. By the results of mass-spectrometry this protein was identified as androgen-induced secreted protein AGR2. This protein is considered a potential oncomarker. Prospects of some postgenome technologies for detection of new diagnostic markers of prostatic cancer are discussed.