[The development of kit for hybridization extraction of DNA and RNA of agents of hemotransmissive infections from serum and blood plasma].

To decrease dependence of effectiveness of isolation of nucleic acids of composition and amount of applied sample a kit was developed for hybridization extraction of DNA HBV RNA HCV and RNA HIV from blood serum in two formats--using up to 250 mkl and up to 1 ml of sample. This kit, in complex with kits for detection using polymerase chain reaction technique in real-time, forms a test characterized by high analytical sensitivity i.e.

MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain.

The most popular strategy for normalization of RT-qPCR data involves presenting them in comparison with expression of "housekeeping" genes. However, the required stable expression of the control genes is not always achievable. As an alternative, we used ribonucleoprotein phage particles as an exogenous internal control and demonstrated that this type of normalization provides a simple and reliable method for quantification in RT-qPCR experiments.

[Development of the high-throughput fluorescence assay detecting SNPs in hemostasis and folate metabolism genes for clinical use].

Genetic predisposition of an individual patient should be taken in account to choose the proper treatment. Implementation to clinical practice requires the development of rapid, high-throughput, and easy assays intended to detect single nucleotide polymorphisms. A detection kit intended to identify the hemostasis and folate cycle gene mutations G20210A FII, G1691A FV, G10976A FVII, G103T FXIII, C807T ITGA2, T1565C ITGB3, 5G(-675)4G PAI, G(-455)A FGB, C677T and A1298C MTHFR, A2756G MTR, A66G MTRR was suggested in this work.

Expression of renin-angiotensin system genes in brain structures of ISIAH rats with stress-induced arterial hypertension.

We studied the expression of genes encoding angiotensinogen (Agt), renin (Ren), angiotensin-converting enzyme (ACE), and type 1 angiotensin receptor (AT1A) in the hypothalamus and medulla oblongata of hypertensive ISIAH rats and normotensive WAG rats. The amount of Agt mRNA in the hypothalamus of young ISIAH rats was increased by 30% compared to WAG controls. In the medulla oblongata of young ISIAH rats, the levels of mRNA of Agt and AT1A receptor were enhanced by 60% and 24%, respectively, compared to WAG rats.

Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days).

[Renin-angiotensin system gene expression in the kidney and in the heart in hypertensive ISIAH rats].

The content of mRNA of renin-angiotensin system (RAS) genes in the kidney and heart of hypertensive ISIAH and normotensive WAG rats was measured by the real-time PCR. Statistically significant decrease of RAS gene mRNA was registered in the kidney of ISIAH rats, including Ren (by 45%), Ace (43%), AT1A (34%), COX-2 (50%). In the myocardium AT1A mRNA expression decreased by 28% while Ace mRNA expression increased by 80%.

Brain proteoglycans in postnatal development and during behavior decline in senescence-accelerated OXYS rats.

Proteoglycans (PG) are involved in the brain development as well as in the pathogenesis of age-related neurodegenerative disorders but underlying mechanism remains poorly understood. We showed that senescence-accelerated OXYS rats are suitable model of age-related cerebral dysfunctions. Here the content and composition of PG in the postnatal development and during behavioral decline were examined in the brain of OXYS and Wistar(control) rats at the ages of 1, 7, 14 days and 1, 2 and 14 months.

[Development of an ultrasensitive mismatch tolerant PCR assay for the qualitative and quantitative determination of HIV-1 RNA].

A real-time fluorescent polymerase chain reaction was used to develop a RealBest HIV RNA kit that was clinically suitable for the detection of HIV-1 RNA and for the estimation of virus load in plasma and serum samples. Due to the selection of a highly conserved target region and to the experimental study of the impact of different primer-template and probe-template mismatches on RT-PCR with subsequent selection of the optimum oligonucleotide set, the developed assay can detect and measure the concentration of all subtypes of HIV-1, group M. The assay provides a high reproducibility and sensitivity and a wide dynamic range of virus loads (20 to 10 million IU/ml of plasma or serum).

Gene expression for the renin system in the myocardium of hypertensive ISIAH rats.

The concentration of mRNA for angiotensin-converting enzyme (ACE) and angiotensin type 1A receptor (AT1A) in the myocardium of hypertensive ISIAH rats and normotensive WAG rats was measured by real-time PCR. Gene expression of the angiotensin type 1A receptor in the myocardium of 4-month-old ISIAH rats was lower than in WAG rats. The content of mRNA for angiotensin-converting enzyme in the myocardium of adult ISIAH rats was elevated by 80%.

[Hepatitis C virus genotyping using 5 nuclease real-time PCR and probes with oligodeoxyinosine linkers].

Hepatitis C virus (HCV) infection is a major cause of severe liver disease including liver cirrhosis and hepatocellular carcinoma. Genotyping became fundamental in the clinical assessment of patients with hepatitis C because the genotype of the hepatitis C virus determines the chance of therapeutic response and duration of treatment. We developed a new real-time PCR assay for genotyping of HCV with increased specificity due to a novel approach to dual-labeled probe design using oligodeoxyinosine linkers.

Short oligonucleotide tandem ligation assay for genotyping of single-nucleotide polymorphisms in Y chromosome.

We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads.

[Development of uncompetitive exogenous internal amplification control for real-time PCR based on UFA method].

An uncompetitive exogenous internal amplification control method (EIAC) was developed on the basis of short synthetic DNA segment, whose amplification can be detected in real time by UFA spectroscopy principle. The EIAC was shown to be useful as internal control in diagnostic test systems based on DNA or RNA detection by multiplex real-time PCR. It can be applied to assess the quality of extracted DNA or RNA, and also to detect and study the factors causing PCR inhibition and earlier plateau effect.

Decontamination of polymerase chain reaction reagents using DEAE-cellulose.

Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity.

Renin system of the kidney in ISIAH rats with inherited stress-induced arterial hypertension.

The renal renin system was studied in ISIAH rats with inherited stress-induced arterial hypertension. The expression of genes for renin (Ren1) and cyclooxygenase (Cox-2) was evaluated in renal tissue of ISIAH and WAG rats (normotensive control). Basal gene expression for Ren1 and Cox-2 in ISIAH rats was much lower than in WAG rats.

Age-related changes in proteoglycan composition in rat brain.

Brain proteoglycans play an important role in learning and memory processes and in the pathogenesis of neurodegenerative disease. Brain proteoglycans in Wistar rats and early aging OXYS rats at the age of 1, 2, and 14 months were compared. Proteoglycan content in OXYS rats aged 1 and 2 months is 2-fold lower than in Wistar.

Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions.

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared.

Proteoglycans and human breast cancer.

Comparative analysis of proteoglycans in the control and tumor tissue of human mammary gland revealed disorders in the biosynthesis of dermatan sulfate proteoglycans in tumor cells. Using the methods of reverse transcription-PCR and Western blotting for the analysis of decorin expression we showed that these disorders were paralleled by reduced expression of the core protein and changes in the structure of proteoglycan carbohydrate chains and could be a cause of malignant degeneration of mammary gland cells.

Neuroendocrine profiling in inherited stress-induced arterial hypertension rat strain with stress-sensitive arterial hypertension.

The functions of the hypothalamic adrenal cortical and sympathetic adrenal medullary systems were studied in rats with inherited stress-induced arterial hypertension (ISIAH strain). A characteristic feature of the ISIAH strain is an increase in arterial blood pressure measured both under basal conditions and after restraint stress in particular. In the control ISIAH rats, the basal plasma ACTH concentration was slightly lower than that in the normotensive Wistar albino Glaxo (WAG) rats, and no differences were found in plasma corticosterone.

[Cytoplasmic male sterility and restoration of pollen fertility in higher plants].

The review deals with cytoplasmic male sterility (CMS) in higher plants: impairment of the pollen formation resulting from interaction of the nuclear and mitochondrial genomes. The information on the known nuclear restorer-of-fertility genes and their effects on the expression of CMS-associated mitochondrial loci are considered. Heteroplasmy of mtDNA in plants and its potential association with CMS inheritance, as well as possible mechanisms of the observed direct and reverse association between altered expression of the CMS-inducing mitochondrial genome, metabolic defects, and pollen sterility are discussed.

Functional activity of the sympathoadrenal system in hypertensive NISAG rats.

NISAG rats with stress-induced arterial hypertension are characterized by hyperactivity of the sympathoadrenal system under rest conditions and during stress exposure.

[Structural and transcriptional variation of mitochondrial DNA in pollen-sterile agamospermous sugar beet (Beta vulgaris L.) progeny].

The cytoplasm status according to mitochondrial sequence tags was determined in agamospermous sugar beet progenies characterized by unstable manifestation of cytoplasmic male sterility. The detected variations in the ratios of homologous sequences related to the N and S mtDNA types did not correlate with pollen phenotypes of the plants. Polymerase chain reaction allowed semiquantitative evaluation of these variations and their detection.

A new approach to revealing point mutations in DNA analyzed by colorimetric detection.

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect.