Associations of cold receptor TRPM8 gene single nucleotide polymorphism with blood lipids and anthropometric parameters in Russian population.

We analyzed associations of single nucleotide polymorphisms rsl13004520 (R247T), rs11562975 (L250L), rs7593557 (S419N), rs11563208 (I1016I), and rs11563071 (V1058V) of the cold receptor TRPM8 (2q37.1) gene with blood plasma lipids and anthropometric parameters in Russian population (randomly chosen residents of Novosibirsk: 507 women and 459 men, mean age 57 years). The studied polymorphisms are localized in regions encoding NH2-terminal (R247T, L250L, S419N) and COOH-terminal (I1016I, V1058V) cytoplasmic domains of the channel. We showed association of single nucleotide polymorphism V1058V with the levels of total cholesterol and LDL and HDL cholesterol, and association of I1016I polymorphism with triglyceride content. Polymorphisms L250L and S419N correlated with anthropometric parameters (body mass index and waist and hip circumferences).

[HFE gene polymorphism in the population of Northern Asia].

Human HFE gene haplotype analysis with reference to IVS2(+4)t/c, IVS4(-44)t/c, IVS5(-47)a/g polymorphic sites was performed in different North Asian ethnic groups. Of the eight possible intronic haplotypes, TTG, TTA, CTA and CCA were identified. High frequency of the CCA haplotype appears to be a characteristic feature of all Asian native populations. Potential functional importance of IVS4(-44)t/c polymorphism is demonstrated. Patients presenting with iron overload syndrome are shown to have low frequency of IVS4(-44)c.

[All signs of metabolic syndrome in the hypertensive ISIAH rats are associated with increased activity of transcription factors PPAR, LXR, PXR, and CAR in the liver].

It is known that the metabolic syndrome (MS), which includes hypertension, dislipidemia, glucose intolerance, and obesity leads to cardiovascular diseases. The MS risk is growing catastrophically. Molecular mechanisms allowing to understand the reason of integrated dysfunctions, taking place at MS cases, have remained almost unstudied. The chronical stress plays a crucial role in MS development; therefore in the present work a hypertensive rat strain with Inherited Stress-Induced Arterial Hypertension (ISIAH) was used as a model. It was shown that ISIAH rat strain as compared with the control WAG rat strain is characterized by increased content of triglyceride, VLDL and LDL cholesterols, a decreased content of HDL cholesterol, a high level of apolipoprotein B-100, and decreased level of apolipoprotein A-I. The ISIAH rats body weight was higher as compared with WAG rats; ISIAH rats blood glucose content was higher too. Thus, strain hypertension for ISIAH rat is accompanied by dislipidemia, increased glucose content, and increased body weight, representing a whole set of MS signs. Since at MS cases the systemic abnormalities in lipid and carbohydrate metabolism take place, the functional activity of transcription factors (TFs) participating in integral regulation of lipid and carbohydrate metabolism genes in liver was measured. PPAR, LXR, PXR, CAR DNA-binding activity was increased in ISIAH rats, suggesting involvement of these TFs in MS development. Integrated investigation of PPAR, LXR, PXR, CAR regulatory mechanisms, signal transduction and transcriptional targets will provide insights into the pathogenesis of MS and offer valuable information for designing of drugs for MS treatment.

[Polymorphism of intron 2 of the SDF1 gene in Galloway, Hereford, and Russian Black Pied cattle].

The region of intron 2 of the SDF1 gene encoding a chemokine of the CXC subfamily has been resequenced in Galloway, Hereford, and Black Pied cattle. Five of the single-nucleotide polymorphisms (SNPs) that were earlier detected by other authors in various breeds of cattle in North America (99C/G, 128T/C, 206C/T, 267C/G and 313C/T) have been found. The 270insC polymorphic marker has proved to be monomorphic in Russian cattle breeds. Hereford cattle significantly differ from Galloway and Black Pied cattle in the frequencies of some SNP variants and their combinations. The number of SNP combinations in Hereford and Galloway cattle exceeds that in Black Pied cattle.

[Effect of hepatocarcinogenicity of estragole on the glucocorticoid-mediated induction of liver-specific enzymes and the activity of the transcription factors FOXA and HNF4 in the liver of mouse and rat].

The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.

[Polymorphism in the human 2'-5'-oligoadenylate synthetase genes (OAS), associated with predisposition to severe forms of tick-borne encephalitis, in populations from North Eurasia].

2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes which play an important role in the antiviral defense in mammals. In human genome three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) located within OAS2 and OAS3 genes are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russian population. In current study we investigated the distribution of three of that SNPs (OAS3rs2285932 (C/T Ile438Ile), OAS3rs2072136 (G/A, Ser567Ser) and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform)) in seven populations from North Eurasia: Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakasses, Tuvinians and Shorians) and Arctic Mongoloids (Chukchi). Differences between populations in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. We found that these frequencies correlate with the ethnicity of the populations and with their supposed differential exposure to TBE virus. Particularly, the lowest frequencies of G/G genotype for OAS3 gene rs2072136 SNP (that according to our previously obtained data is associated with predisposition to severe forms of TBE) were found in Altaians, Khakasses, Tuvinians and Shorians who may highly contact with TBE virus in places of their habitation. Thus, data obtained allow to suppose that TBE virus might act as a selection factor for particular OAS genes variants in Central Asian Mongoloids.

Polymorphism of 2'-5'-oligoadenylate synthetase (OAS) genes, associated with predisposition to severe forms of tick-borne encephalitis, in human populations of North Eurasia.

2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes playing an important role in antiviral defense in mammals. In the human genome, three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously, we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) of OAS2 and OAS3 are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russians. In the current study, we investigated the distribution of three of the above SNPs, OAS3 rs2285932 (C/T, Ile438Ile), OAS3 rs2072136 (G/A, Ser567Ser), and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform), in seven populations of North Eurasia: Caucasians (Russians, Germans from Altai region), Central Asian Mongoloids (Altaians, Khakass, Tuvinians, and Shorians), and Arctic Mongoloids (Chukchi). Interpopulational differences in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. These frequencies correlated with the ethnicity of the populations and with their supposed differential exposure to the TBE virus. In particular, the lowest frequencies of G/G genotype for OAS3 SNP rs2072136 (which, according to our earlier results, is associated with predisposition to severe forms of TBE) were found in Altaians, Khakass, Tuvinians, and Shorians, who commonly contact with the TBE virus in their habitation regions. Thus, the data obtained suggest that the TBE virus might act as a selection factor for particular OAS variants in Central Asian Mongoloids.

Correlation between hepatocarcinogenic effect of estragole and its influence on glucocorticoid induction of liver-specific enzymes and activities of FOXA and HNF4 transcription factors in mouse and rat liver.

It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.

[The spectrum of mutations in the low-density lipoprotein receptor gene in the Russian population].

The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49-years-old and had the highest level of total serum cholesterol in this age group. Seven previously non-described mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.

Increased interaction of proteins in nuclear extract from mouse liver and lung tumors with TATA-containing oligonucleotide.

Increased TATA-binding activity of proteins in nuclear extracts from murine hepatocarcinoma HA-1 and murine Lewis lung adenocarcinoma was demonstrated. The dependence of the amount of formed complexes on protein concentration, displacement of labeled 32P-TATA-containing oligonucleotide by its unlabeled analog, and weak interaction with an oligonucleotide containing damaged TATA box confirm specificity of the formed complexes.

[Frequency of chromosomes carrying endogenous retroviruses in the populations of domestic pig and wild boar].

Statistical analysis of the frequency of chromosomes carrying three types (A, B, and C) of porcine endogenous retroviruses (PERV) was based on molecular-genetic testing of populations of domestic pigs and wild boars. Domestic pigs were shown to have higher frequency of PERV and haplotypes containing two and three types of PERV than wild boars.

[Comparative analysis of mitochondrial and nuclear DNA topoisomerase I from maize].

We conducted a comparative study of the properties of topoisomerase I isolated from maize nuclei and mitochondria. We found that nuclear and mitochondrial enzymes possess different ability to bind single stranded DNA. Study of the enzyme activity dependence on Mg2+ demonstrated an absolute dependence of the mitochondrial topoisomerase activity. Contrary, nuclear enzyme activity was not absolutely dependent but stimulated by the magnesium cation. Mitochondrial topoisomerase formed covalent bond with the 5'-end of the cleaved DNA what is unique property of prokaryotic topoisomerase I. Nuclear enzyme bound covalently to the 3'-end like all eukaryotic topoisomerases I. The search through databases revealed genes which could encode mitochondrial topoisomerase I in the genomes of higher plants. Using both cDNA sequencing and in silico methods we demonstrated an existence of the ortholog gene in the maize genome. This gene shares significant homology with prokaryotic topoisomerase I genes that may explain differences in the properties of the mitochondrial and nuclear enzyme. Data obtained is of a significant interest both from the point of view of plant organelle evolution and mitochondrial genome expression mechanisms study.

[Study of the binding of nuclear proteins from Plasmodium berghei strains with different chloroquine sensitivity to oligonucleotides corresponding to regulatory elements of multidrug resistance (mdr1) gene].

Using electrophoretic mobility shift assay we first revealed in the nuclear extracts of the rodent malaria parasite Plasmodium berghei (P. berghei) proteins, which bind specifically to the double-stranded oligonucleotides reproducing the binding sites of the transcription factors of AP1 family, NF-IL6 and SP1 involved in the up-regulation of human multidrug resistance (mdr1) gene and to the oligonucleotide corresponding to the element responsive for the stimulation by serum (SRE). The nuclear proteins isolated from the P. berghei strains with various chloroquine sensitivity bound differently to the most of the oligonucleotide probes used. Mutations in the consensus sequences of AP1, NF-IL6 and SRE led to the loss of some DNA-protein complexes, suggesting the existence of malaria parasite nuclear proteins, whose DNA-binding domains are similar to DNA-binding domains of NF-IL6, SRF1, and AP1 family members. These proteins exhibit greater activities in chloroquine resistant strains. The results obtained denote profound alterations in the plasmodium regulatory apparatus occurred as the result of selection on chloroquine resistance.

Activation of constitutive androstane receptor under the effect of hepatocarcinogenic aminoazo dyes in mouse and rat liver.

Selective increase of DNA-binding activity of constitutive androstane receptor was detected in rat and mouse liver in response to aminoazo dyes exhibiting hepatocarcinogenic activity for these species (ortho-aminoazotoluene for mice and 3'-methyl-4-dimethylaminobenzene for rats). Competition of azo dyes with 3H-5alpha-androst-16-ene-3alpha-ol (a well-known ligand of constitutive androstane receptor) for binding to liver cell cytosol proteins was studied. Ortho-aminoazotoluene and 3'-methyl-4-dimethylaminobenzene were better competitors for cytosol proteins from mouse and rat liver, respectively.

Differences in binding of nuclear proteins from P. berghei strains differing by chloroquine resistance to oligonucleotides corresponding to mdr1 gene regulatory elements.

Proteins specifically reacting with AP1, MEF1, NF-IL6, and SP1 transcription factor binding sites were detected for the first time in nuclear extract of P. berghei (rodent malaria parasite) using gel retardation assay. P. berghei strains with different chloroquine resistance exhibited appreciable differences in the pattern of nuclear protein binding to the majority of the studied sites, which attests to changes in the plasmodial regulatory system during chloroquine resistance selection.

[Recognition of the potential SF-1 binding sites by SiteGA method, their experimental verification and search for new SF-1 target genes].

The SF-1 (Steroidogenic Factor-1) is a transcription factor known as a key regulator of the steroidogenic gene expression. SF-1 is required for the development and functioning at all levels of the hypothalamic-pituitary-gonadal and adrenal axis. Also it plays an essential role in sex determination. SF-1 is a member of the nuclear receptor superfamily and it activates gene expression by binding to DNA in a monomeric form. Here, we report the results of potential SF-1 binding sites identification by using the SiteGA recognition method. The SiteGA method was implemented using a genetic algorithm (GA) involving a iterative discriminant analyses of local dinucleotide context characteristics. These characteristics were compiled not only over the core binding sites region but over its flanks as well. Developed SiteGA method is characterized by considerably better recognition accuracy when compared to that for the weight matrix method. The experimental tests demonstrated that 83% of the sites recognized by the SiteGA method in the regulatory regions of steroidogenic genes, indeed, interact with the SF-1 factor. We also estimated the density of predicted sites in regulatory region of genes, the members of different functional groups and developed the criterion to search for new SF-1 target genes in genome sequences.

Lung cancer-associated SNP at the beginning of mouse k-ras gene intron 2 is essential for transcription factor binding.

The C to T substitution in position 311 n. p. of K-ras gene intron 2 in mice resistant to lung cancer (M. spretus) attenuates NF-Y transcription factor binding site in comparison with sensitive ICR mice (M. musculus). Appreciable differences between ICR and M. spretus in general pattern of binding of nuclear proteins to K-ras gene DNA within 248-332 n. p. fragment are demonstrated.