[Analysis of gene expression pattern in peripheral blood leukocytes during experimental heat wave]. / Analiz izmeneniia ékspressii genov v leikotsitakh krovi cheloveka vo vremia ispytaniia, modeliruiushchego usloviia teplovoi volny.

The conditions of Moscow 2010 summer heat wave were simulated in an accommodation module. Six healthy men aged from 22 to 46 years stayed in the module for 30 days. Measurements of gene expression in peripheral blood leukocytes before, during and 3 day after simulated heat wave were performed using qRT-PCR. We observed a shift in the expression level of certain genes after heat exposure for a long time, and rapid return to the initial level, when volunteers leaved the accommodation module. Eight genes were chosen to form the "heat expression signature". EGR2, EGR3 were upregulated in all six volunteers, EGR1, SIRT1, CYP51A1, MAPK9, BAG5, MNDA were upregulated in 5 volunteers.

Structural investigations of recombinant urokinase growth factor-like domain.

Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two ß-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel ß-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the ß-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹5N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.

Altered gene expression pattern in peripheral blood leukocytes from patients with arterial hypertension.

The role of various inflammatory mechanisms and oxidative stress in the development of atherosclerosis and arterial hypertension (AH) has been increasingly acknowledged during recent years. Hypertension per se or factors that cause hypertension along with other complications lead to infiltration of activated leukocytes in the vascular wall, where these cells contribute to the development of vascular injury by releasing cytokines, oxygen radicals, and other toxic mediators. However, molecular mechanisms underlying leukocyte activation at transcriptional level in AH are still far from being clear. To solve this problem we employed cDNA microarray technology to reveal the differences in gene expression in peripheral blood leukocytes from patients with AH compared with healthy individuals. The microarray data were verified by a semi-quantitative RT-PCR method. We found 25 genes with differential expression in leukocytes from AH patients among which 21 genes were upregulated and 4 genes were downregulated. These genes are implicated in apoptosis (CASP2, CASP4, and CASP8, p53, UBID4, NAT1, and Fte-1), inflammatory response (CAGC, CXCR4, and CX3CR1), control of MAP kinase function (PYST1, PAC1, RAF1, and RAFB1), vesicular trafficking of molecules among cellular organelles (GDI-1 and GDI-2), cell redox homeostasis (GLRX), cellular stress (HSPA8 and HSP40), and other processes. Gene expression pattern of the majority of genes was similar in AH patients independent of the disease stage and used hypotensive therapy, but was clearly different from that of normotensive subjects.

Gene expression analysis to identify mRNA markers of cardiac myxoma.

cDNA expression arrays were used to identify mRNA expression markers for cardiac myxoma. The RNA profile analysis suggests that cardiac myxoma should be considered as a stand-alone tissue rather than a pathological modification of particular normal tissue. The analysis reveals a set of genes which are highly and steadily expressed in cardiac myxomas and can serve as an mRNA expression markers of the tumour. Marker status of selected genes was confirmed by reverse transcriptase polymerase chain reaction analysis. Genes MIA (melanoma inhibitory activity) and PLA2G2A (phospholipase A2, group IIA) show the highest specificity as cardiac myxoma markers, since they have more than 10-fold higher RNA level in cardiac myxomas than in any one of 15 normal tissues tested. Among markers of myxoma at least three are participants of phospholipid metabolism: ANXA3, PLA2G2A, and phospholipid transfer protein. Tissue inhibitor of metalloproteinase 1 and secretory leucocyte protease inhibitor are inhibitors of proteases degrading extracellular matrix proteins and participating in cell proliferation regulation. MIA, SPP1, fibromodulin are modulators or participants of the interaction between extracellular matrix proteins and their cell surface receptors. SOX9 is a transcription factor required for chondrocyte differentiation. Calretenin (CALB2) is an intracellular calcium-binding protein with poorly understood function.

Properties of antigenomic hepatitis delta virus ribozyme that consists of three RNA oligomer strands.

A three-strand ribozyme, a derivative of antigenomic hepatitis delta virus (HDV) ribozyme, which consists of subfragments of 16 (L), 17 (S), and 33 nucleotides (B), has been constructed. The ternary B-L-S complex formed by the subfragments in stoichiometric ratio was able to catalyze a self-cleavage reaction. Kinetics of this reaction exhibited biphasic behavior and the same parameters as in the case of natural cis-ribozyme. Study of kinetics of reaction initiated by adding various reaction components and the study of binary complex formation between subfragments B and L, B and S, and also ternary B-L-S complex formation revealed that: 1) in the presence of Mg2+, B and S form a stoichiometric complex, L and S do not form complex at all, while B and L form 2 types of complexes, probably B-L and 2B-L; and addition of S subfragment prevented the formation of the latter complex; 2) the reaction initiated by S subfragment proceeds much slower than that initiated by other components pointing to the possibility that in the absence of S L may form a nonproductive complex with B, which is slowly displaced by S followed by productive ternary complex formation. Dissociation constants for binary B-L, B-S and ternary B-L-S complexes have been estimated.

Size distribution of the urokinase mRNA decay intermediates in different tissues and cell lines.

Many genes, particularly those encoding the products participating in the regulation of transcription, replication and tissue remodeling, produce short-lived mRNA. It has been commonly accepted that once mRNA is disintegrated, the degradation process is so rapid that the decay intermediates cannot be detected. In the present study we verified this postulate and focused our attention on the quantification of the decay products of the urokinase-type plasminogen activator (uPA) mRNA that belongs to short-lived mRNAs. Using a previously described modified quantitative RT-PCR method, we have shown that intact uPA mRNA coexists in normal human tissues, Jurkat and 5637 cells with a great abundance of its degradation products. The uPA mRNA decay products were not detected in T24P cells. The content of intact uPA mRNA in normal tissues was as low as 5% of the total amount of its poly(A)(+) fraction. The size distribution of the mRNA decay products suggests that the mRNA is digested by exonucleases or/and non-specific endonuclease with cut sites evenly distributed along the mRNA chain. Different decay degrees were demonstrated for subpopulation of the uPA mRNA molecules with intact 3' and 5' ends.

The increase in gene expression induced by introduction of rare codons into the C terminus of the template.

Short oligodeoxynucleotides (oligos) possessing two tandem Arg codons followed by TGA stop codon were inserted near the 3' end of a modified cat gene. It was found that while being decoded in vivo, the AGGAGGTGA oligo increased the yield of gene product and, in addition, caused -1 frameshifting. The 3-10-fold increase of the yield of the polypeptide was accompanied by increased accumulation of corresponding mRNA, indicating a protection from messenger decay. Transformation of the cells by a plasmid overproducing tRNA(4Arg) gene compensates for all the anomalies.

Application of poly(A)+RNA patterns method for searching of differentially expressed genes.

Poly(A)+RNA composition differences for normal, fetal and cirrhotic human liver before and after retinoic acid-induced differentiation of the F9 embryonal carcinoma cell line were analyzed by a novel poly(A)+RNA patterns method. The method is based on the polyacrylamide gel electrophoretic analysis of short cDNA termination products, synthesized by reverse transcriptase using poly(A)+RNA as a template, a set of short 5'-end labeled primers, three natural and one terminator deoxyribonucleotide. A number of known differentially expressed genes and some unknown ones were then identified by direct sequencing of the differentially represented bands excised from a gel and searching a complementary mRNA target sites in Genbank database.

3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase.

Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E. coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses. It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase. Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.

Some aspects of DNA polymerase functioning.

The group of DNA polymerases was studied using some new nucleoside 5'-triphosphate analogs with termination substrate properties. Among DNA polymerases tested the least specific appeared reverse transcriptases of retroviruses and the most specific were DNA polymerases alpha type from high eucaryotes including mammalians.

Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases.

A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates. ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E. coli DNA polymerase I. dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above-mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested.

Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters.

Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.