[The study of aberrant methylation in blood leukocytes of liquidators of the Chernobyl accident].

The study of aberrant methylation of CpG islands in the promoter regions of genes (P16/CDKN2A, P14/ARF, RASSF1A, GSTP1) in blood leukocytes of liquidators of the Chernobyl accident (n = 83, 38-76 years of age) and control subjects of two groups (n = 48, age ≤ 35 and n = 65, age > 35) was carried out using methylation-sensitive restriction endonuclease analysis followed by PCR. The total number of AciI sites in the analyzed fragments ranged from 2 to 7 for different genes. Only 1 subject (2.1%) from the control group (healthy young individuals, age ≤ 35) has methylation of the studied CpG--dinucleotides of RASSF1A gene. Promoter methylation of at least one of the genes analyzed was observed in 28.92% liquidators and significantly exceeded (p = 0.016) such rate in a one-age (> 35 years of age) control group (12.31%). A significantly elevated frequency (p = 0.023) of individuals with abnormal methylation of GSTP1 gene in the group of liquidators as compared to the control group was revealed. The occurrence of promoter methylation of RASSF1A gene significantly correlated with aging both in the control group (r = 0.214; p = 0.023) and in the liquidators of the Chernobyl accident (r = 0.230; p = 0.036). No similar trend was found for other genes. Multiple regression analysis showed that the growth in the number of methylated loci of a set of genes p16, p14 and GSTP1 is exclusively due to the fact of exposure (OR = 7.32, 95% CI = 2.49-25.83, p-value = 2.7 x 10(-5)). The results obtained demonstrate for the first time the reality of the radiation-induced aberrant methylation of CpG islands in promoters of genes involved in the basic protective, functions of cells in the human body in remote periods after radiation exposure.

[Role of the genetic factors, detoxication systems and oxidative stress in the pathogenesis of endometriosis and infertility (review)].

The aim of this paper is to provide a systematic review of the role of the genetic factors, detoxication systems and oxidative stress in the pathogenesis of endometriosis and infertility. Endometriosis and infertility are still both the most uncommon diseases in gynecology. Many aspects of female reproductive function are strongly influenced by genetic factors, and numerous studies have attempted to identify susceptibility genes for disorders affecting female fertility such as polycystic ovary syndrome, endometriosis, fibroids, cancer (ovarian, vulvar, cervical), premature ovarian failure, recurrent pregnancy loss and pre-eclampsia. The most solid evidence linking specific polymorphisms to endometriosis is showed by the studies investigating a phase II detoxification enzyme. No data were found concerning influences of the genetic factors on the female infertility. Contrary, a lot of studies devoted to the genetic factors of male infertility are presented. It's known that endometriosis associated with increased systemic oxidative stress. The implication of increased systemic oxidative stress in disease progression or the association with other oxidative stress-related pathologic conditions needs to be addressed in further studies. The majority of studies suggest a reduced antioxidant capacity in infertile wome with endometriosis. In the present review we discussed the role of the genetic factors in the pathogenesis of endometriosis and infertility. NAT2 polimorphism, xenobiotic methabolism and exogenous factors are somehow related with these diseases. An altered balance between pro-oxidant antioxidant activities may have an impact on folliculogenesis and adequate embryo development.

[Allelic variants of polymorphic genes associated with a higher frequency of chromosome aberrations].

Genotypic associations were studied for the frequency of chromosome aberrations in human peripheral blood lymphocytes. Cytogenetic analysis (1000 metaphase plate per individual) and genotyping at 19 sites of genes involved in detoxification and DNA repair were performed in a sample of 83 Chernobyl liquidators and a matched control sample of 96 volunteers. In either sample, the frequency of chromosome aberrations was higher in carriers of the minor alleles of the XPD gene (sites 2251T > G and 862G > A) and the positive genotypes of the GSTM1-GSTT1 genes. The highest frequency of chromosome aberrations was observed in carriers of a combined genotype including at least one minor allele of the XPD sites + at least one insertion in the GSTM1-GSTT1 genes. The high-risk genotype, which had a prevalence of 64%, was strongly associated with a higher frequency of chromosome aberrations in both volunteers (OR = 6.9, P = 0.008) and Chernobyl liquidators (OR = 5.6, P = 0.002).

[The relationship between genotypes and frequencies of chromosome aberrations in human lymphocytes under irradiation in vivo and in vitro].

The data on the variability of an elevated level of the frequencies of chromosome aberrations for a group of liquidators of the Chernobyl Nuclear Station accident depending on genotypes by candidate loci are presented. The genotyping was carried out by sites, which previously showed the associations with the cytogenetic variability in control experiments. It was shown that, for a group of liquidators heterozygote by site SOD2 C47T, the control level of the frequency of chromosome aberrations is not exceeded significantly. At the tendency level, the frequency of aberrations for liquidators was reduced for double homozygotes by deletions of genes GSTM1-GSTT1 and for homozygotes by the minor allele of site CYP1A1 T606G that is in an accordance with the results of experiments with the control sampling. The elevated level of chromosome aberrations for liquidators, as a whole, is observed for genotypes, which are characteristic of an elevated level of spontaneous aberrations, and it does not completely correspond to genotypes with the elevated radiosensitivity of chromosomes.

[Polymorphism of repair genes and cytogenetic radiation effects].

For 99 healthy volunteers, the frequencies of spontaneous and y-induced (1 Gy in vitro) chromosome aberrations in blood lymphocytes were compared with the results of PCR-genotyping by 8 repair genes: XRCC1, XPD, ERCC1, APEXI, RAD23B, OGG1, ATM, Tp53 (in all, 10 polymorphic sites). The frequency of spontaneous aberrations of chromosome type increased additively with the number of copies of minor allele of excision repair gene XPD variant *2251G and *862A D (p = 0.025). The frequency of gamma-induced chromosome aberrations proved to be elevated for the carriers of a minor allele OGG1*977G (p = 0.011). The significantly elevated number of gamma-induced chromosome aberrations was also observed for the carriers of major alleles XRCC1*G1996 and XRCC1*C589 (p = 0.002).

[Somatic mutagenesis in human lymphocytes depending on genotypes for detoxification and oxidative response loci].

Associations of polymorphism of seven detoxification genes and three genes of oxidative response with the frequency of chromosome aberrations in human peripheral blood lymphocytes were studied. The genotyping data were correlated with the frequencies of spontaneous and gamma-induced (1 Gy in vitro) chromosome aberrations estimated for a group of healthy donors (97 males under 25 years of age) by analyzing 500-1000 metaphase cells per individual. The spontaneous level of aberrations of the chromosomal type was reduced in homozygotes for the GSTM1 locus deletion, and especially in double homozygotes for deletions of the GSTM1 and GSTT1 genes. The frequency of gamma-induced chromosome aberrations was reduced in G/G homozygotes for the minor allele of the poorly studied CYP1A1 T606G site: 0.094 +/- 0.006 against 0.112 +/- 0.002 for T allele carriers (P = 0.004). Linkage of the T606G site with well known and functionally important sites of the CYP1A1 gene (A4889G, T3801C) was analyzed.

[The effect of polymorphism of genes of xenobiotics detoxication on the frequencies of spontaneous and induced chromosome aberrations in human lymphocytes].

Here presented the data on the frequencies of chromosome aberrations in lymphocytes of peripheral blood of 97 volunteers depending on genotypes by genes of xenobiotics detoxication before and after gamma-irradiation with dose of 1 Gy in vitro. The frequencies of aberrations were estimated by analyzing not less than 500-1000 metaphases per person. The data of cytogenetic analysis were compared with the results of PCR-genotyping of loci GSTM1, GSTT1, GSTP1, CYP1A1, CYP2D6, NAT2, and MTHFR. The significant differences by the frequencies of aberrations between "single-locus" genotypes were not found except for GSTM1 locus, for which the enhanced frequency of spontaneous aberrations of chromosome type in "positive" genotypes compared to "zero" ones, i.e., homozygotes by deletion (p = 0.04) was observed. The minimum frequency of spontaneous aberrations of chromosome type was recorded for carriers of double homozygotes by deletion of GSTM1-GSTT1: 0.0006 +/- 0.0003 against 0.0027 +/- 0.0003 for the rest of genotypes (p = 0.016 by the Mann-Witney test). The frequency of gamma-induced chromosome aberrations was correlated with the total amount of minor alleles in loci GSTP1, NAT2, and MTHFR (r = 0.25 at p = 0.0065).

[The association of TCR-mutant cells with DNA polymorphism in women residing in radiation polluted regions of the Russian Federation].

Using flow-cytometric method the frequency of lymphocytes beaming mutations at T-cell receptor (TCR) locus was assessed in women residing in radiation polluted regions of Bryansk and Tula Districts. Simultaneously genotyping of the 8 polymorph loci for genes involved in detoxication of xenobiotics and oestrogen metabolism was carried out. The increased TCR-mutant cell frequency was found to be characteristic of homozygotes of the low activity appropriated enzymes for 3 loci (HFE187, GSTM1 and MTHFR) at least. This tendency was statistically significant in case of deletion polymorphism of the GSTM1 gene: TCR-mutant cell frequency of the homozygous carriers of a deletion at the GSTM1 locus was (4.63 +/- 0.18) x 10(-4) while it was (4.05 +/- 0.15) x 10(-4) in other groups of persons. The greatest mutant cell frequency was observed in carriers of the minor allele 4889G of the locus CYP1A. More often the increased values of the TCR-mutant cells (outside range "3sigma") were determined in women with genotypes A/G or G/G of the locus CYP1A1 (25%) than in carries of the normal genotype A/A (1.6%) (OR = 20.6; p = 0.0002). The comparison of the groups of women with reproductive system diseases reveals significant elevation in the mean TCR-mutant cell frequency in inhabitants of the most radiation polluted region among others.

[Genotype dependence of cytogenetic and epidemiological characteristics in the liquidators of the accident at the ChNPP].

The dependence of the level of unstable chromosome aberrations and nononcological diseases on the genotype in 57 liquidators of the ChNPP accident was studied. Candidate genes presumably affecting radiosensitivity were highly polymorphic loci of xenobiotic detoxication genes (glutathione-S-transferases GSTM1, GSTT1, GSTP1) and the 5,10-methylenetetrahydrofolate reductase gene (MTHFR) involved in DNA methylation and synthesis. An increased frequency (0.014 +/- 0.001 per cell) of unstable chromosome aberrations, including radiation-specific dicentrics and centric rings (0.0015 +/- 0.0002 per cell), has been found to be preserved in the group of liquidators examined in 2006-2007. No associations of polymorphism for each of the studied genes with cytogenetic parameters were revealed. Increased frequencies of chromosome aberrations were recorded in homozygous carriers of a deletion at the GSTM1 locus in combination with homozygosity for minor alleles at the MTHFR and GSTP1 loci (p = 0.00002 and p = 0.0233, respectively). The number of homozygous carriers of the minor allele GSTP1 was increased among patients with chronic obstructive pulmonary disease and in liquidators with acute circulation disturbances (p = 0.014 and p = 0.04, respectively). Double homozygotes for GSTM1 and GSTT1 deletions were significantly more frequent among subjects with benign tumors (cysts, polyps, p = 0.015) and with benign thyroid tumors (p = 0.017). This genotype has proved to be protective for patients with severe cardiovascular diseases (acute circulation disturbances, p = 0.027).

[Spatial distribution and species composition of prosthecate bacteria in Lake Baikal].

From the water column of Lake Baikal, several strains of prosthecate bacteria belonging to the genera Caulobacter and Brevundimonas were isolated. In this article, the methods applied for their isolation and cell number determination are described; the occurrence frequency and spatial distribution of these microorganisms in the lake are demonstrated. Characterization of the species composition of cultivable and uncultivable prosthecate bacteria was carried out using the methods of traditional and molecular microbiology, respectively. A comparative phylogenetic analysis of the DNA sequences of uncultivable bacteria, which showed homology to the members of the alpha subclass of proteobacteria, was carried out. It was demonstrated that the lake water column is inhabited by uncultivable alpha-proteobacteria of uncertain phylogenetic affinity, in addition to representatives of the species Caulobacter vibrioides and C. leidyi, which were detected by traditional microbiological methods.

Comparative effects of limited tryptic hydrolysis on physicochemical and structural features of seed 11S globulins.

The effect of the limited proteolysis by trypsin on selected seed storage 11S globulins (broad bean and pea legumins, glycinin and helianthinin) was studied by high-sensitive differential scanning calorimetry, fluorescence spectroscopy and analysis of proteolysis kinetics. Different behaviour of glycinin and helianthinin, on one hand, and broad bean and pea legumins, on the other, were observed: in the first group changes in the physicochemical characteristics of the proteins due to their limited proteolysis are more pronounced in comparison with the second one, in relation with the extent of primary structure modifications. The differences observed have been evaluated in relation with the amino acid sequence features of the four 11S globulin studied and agree with the literature data concerning the protein structural changes in the course of the limited proteolysis.

Personal sampler for monitoring of viable viruses; modelling of outdoor sampling conditions.

A new personal bioaerosol sampler has recently been developed and verified to be very efficient for monitoring of viable airborne bacteria, fungi and viruses. The device is capable of providing high recovery rates even for microorganisms which are rather sensitive to physical and biological stresses. However, some mathematical procedure is required for realistic calculation of an actual concentration of viable bioaerosols in the air taking into account a rate of inactivation of targeted microorganisms, sampling parameters, and results of microbial analysis of collecting liquid from the sampler. In this paper, we develop such procedure along with the model of aerosol propagation for outdoor conditions. Combining these procedures allows one to determine the optimal sampling locations for the best possible coverage of the area to be monitored. A hypothetical episode concerned with terrorists' attack during music concert in the central square of Novosibirsk, Russia was considered to evaluate possible coverage of the area by sampling equipment to detect bioaerosols at various locations within the square. It was found that, for chosen bioaerosol generation parameters and weather conditions, the new personal sampler would be capable to reliably detect pathogens at all locations occupied by crowd, even at distances of up to 600 m from the source.

[Multiyear monitoring of the ecological status of industrial zones of Moscow using a test line of Drosophila melanogaster]. / Mnogoletnii monitoring za ékologicheskim sostoianiem promzony g. Moskvy s primeneniem test-linii Drosophila melanogaster.

For 12 years, recombination rate was assessed in Drosophila melanogaster males exposed on a thermoelectric power station (TPS) of Moscow. In 1994, experiments were also carried out on another Moscow TPS for comparison. The recombination frequency in exposed males was two- or threefold higher than in the control sample. Recombination frequencies observed in different years did not significantly differ from each other. Likewise, no significant difference was observed for the results obtained on two TPSs. A dramatic increase in recombination frequency in flies exposed on TPS was considered as an adequate response to high concentrations of effective mutagens discharged by TPS.

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.

Profiling of genes expressed in human monocytes and monocyte-derived dendritic cells using cDNA expression array.

Using a human cDNA expression array, we obtained expression profiles of 588 genes in CD14+ monocytes and monocyte-derived dendritic cells (DCs). Overall, 22 genes were upregulated, and nine genes were downregulated in DCs of both samples from two different individuals. Many of the genes that were upregulated in DCs encode proteins that are related to differentiation, cell structure, migration, termination of cell cycle as well as proliferation, e.g. tumour necrosis factor-alpha (TNF-alpha), tumour necrosis factor receptor II (TNFRII), thymosin beta-10, epithelial discoidin domain receptor 1, replication factor C, putative transcription factor DB1, alpha catenin, transforming growth factor-beta 1, prohibitin, p53-regulating protein and neu differentiation factor. Among the downregulated genes in DCs were genes that encode proteins of cell cycle regulation: mitotic growth and transcription activator, platelet-derived growth factor receptor-beta subunit, interleukin 2 receptor (IL-2R)-gamma subunit, IL-7R-alpha subunit, leucocyte interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR). Semi-quantitative reverse transcription-polymerase chain reaction method confirmed the upregulated expression levels in DCs for TNFRII, TNF-alpha, alpha catenin and downregulation of IFN-gamma, GM-CSFR on four different donor samples of DCs and monocytes. Moreover, our data show the presence of a 'switch-on' step for the TNF-alpha and TNFRII gene expression in immature DCs for further differentiation into mature DCs.

Expression of renin-angiotensin system genes in immature and mature dendritic cells identified using human cDNA microarray.

Using a cDNA glass microarray, the expression of 1081 genes in immature and mature dendritic cells (DCs) of two different individuals has been studied. The upregulation of mRNA transcripts of genes encoding the transcription factor ZFM1, Mos proto-oncogene serine/threonine-protein kinase, B-cell-specific transcription factor, preB-cell growth stimulating factor, ets translocation variant 6, and epidermal growth-factor-like CRIPTO was for the first time detected in DCs. Using semiquantitative RT-PCR analysis the upregulation of the transforming growth factor-alpha, integrin alpha 6 and ZFM 1 transcription factor in mature DCs was confirmed in samples from four different individuals. On the other hand, the downregulation of renin-binding protein transcript was detected in mature DCs using a cDNA microarray. For the first time, the expression of renin-angiotensin system genes was evaluated during maturation of DCs in samples from four donors by semiquantitative RT-PCR. A possible role of the renin-angiotensin system in DCs is discussed.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4+ Valpha24 natural killer T cells expressing CD40L.

Human Valpha24 natural killer T (Valpha24NKT) cells are activated by alpha-glycosylceramide-pulsed dendritic cells (DCs) in a CD1d-dependent and T-cell receptor-mediated manner. There are two major subpopulations of Valpha24NKT cells, CD4- CD8- Valpha24NKT and CD4+ Valpha24NKT cells. We have recently shown that activated CD4- CD8- Valpha24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of Valpha24NKT cells is currently limited. We aimed to investigate whether CD4+ Valpha24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4+ Valpha24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4+ Valpha24NKT cells, but not with resting CD4+ Valpha24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb. Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Valpha24NKT cells. The apoptosis of DCs from normal donors, triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4+ Valpha24NKT cells by virtue of apoptosis of DCs.