M. L. Filipenko

Increase of tyrosine hydroxylase and dopamine transporter mRNA levels in ventral tegmental area of male mice under influence of repeated aggression experience

Tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA levels in the ventral tegmental area (VTA) of midbrain were measured by multiplex RT-PCR in male mice with repeated experience of social victories (winners) and social defeats (losers) in 10 daily agonistic confrontations. Two independent experiments revealed enhanced TH and DAT mRNA levels in VTA of the winners in comparison with the losers and controls (animals after 5 days of individual housing). A positive correlation between DAT and TH mRNA levels was shown.

Repeated experience of social defeats increases serotonin transporter and monoamine oxidase A mRNA levels in raphe nuclei of male mice

Serotonin transporter (SERT) and monoamine oxidase A (MAOA) mRNA levels in the raphe nuclei area of the midbrain were measured by the multiplex reverse transcription-polymerase chain reaction method in male mice with repeated experience of social victories (winners) and defeats (losers) in ten daily agonistic confrontations. Experiments revealed enhanced SERT and MAOA mRNA levels in the losers compared with the winners and controls. It has been supposed that SERT and MAOA genes are involved in enhancement of serotonin inactivation in response to the increase of serotonergic activity shown earlier in the losers. A positive correlation between MAOA and SERT mRNA levels in the raphe nuclei area of the midbrain was shown.

Molecular Implications of Repeated Aggression: Th, Dat1, Snca and Bdnf Gene Expression in the VTA of Victorious Male Mice

Background It is generally recognized that recurrent aggression can be the result of various psychiatric disorders. The aim of our study was to analyze the mRNA levels, in the ventral tegmental area (VTA) of the midbrain, of the genes that may possibly be associated with aggression consistently shown by male mice in special experimental settings. Methodology/Principal Findings The genes were Th, Dat1, Snca and Bdnf; the male mice were a group of animals that had each won 20 daily encounters in succession and a group of animals that had the same winning track record followed by a no-fight period for 14 days. Increased Th, Dat1 and Snca mRNA levels were in the fresh-from-the-fight group as compared to the controls. Increased Th and Dat1 mRNA levels were in the no-fight winners as compared to the controls. Significant positive correlations were found between the level of aggression and Th and Snca mRNA levels. Conclusions Repeated positive fighting experience enhances the expression of the Th, Dat1 and Snca genes, which are associated with brain dopaminergic systems. The expression of the Th and Dat1 genes stays enhanced for a long time.

Polymorphic variants of folate metabolizing genes (C677T and A1298C MTHFR and C1420T SHMT1 and G1958A MTHFD) are not associated with the risk of breast cancer in the West Siberian Region of Russia

Breast cancer is one of the most widely distributed cancers in women. We investigated the role of allele variants in the folate metabolizing genes MTHFR (C677T and A1298C alleles), SHMT1 (C1420T allele), and MTHFD (G1258A allele) as a possible factor in predisposition to breast cancer. We determined allele and genotype frequencies of single nucleotide polymorphisms (SNPs) in the case (850 women with sporadic form of breast cancer) and control (810 healthy women) groups. None of the polymorphisms were significantly associated with breast cancer risk. To increase the statistical power of our study, we conducted a meta-analysis which included published genotype data and the results of our work. The meta-analysis revealed no significant association between the studied SNPs and breast cancer risks either.

Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system

Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.

Genetic variation of Mycobacterium tuberculosis circulating in Kharkiv Oblast, Ukraine

BackgroundA persistent increase of tuberculosis cases has recently been noted in the Ukraine. The reported incidence of drug-resistant isolates of M. tuberculosis is growing steadily; however, data on the genetic variation of isolates of M. tuberculosis circulating in northern Ukraine and on the spectrum and frequency of occurrence of mutations determining resistance to the principal anti-tuberculosis drugs isoniazid and rifampicin have not yet been reported.MethodsIsolates of M. tuberculosis from 98 tuberculosis patients living in Kharkiv Oblast (Ukraine) were analyzed using VNTR- and RFLP-IS6110-typing methods. Mutations associated with resistance to rifampicin and isoniazid were detected by RFLP-PCR methods, and also confirmed by sequencing.ResultsWe identified 75 different genetic profiles. Thirty four (34%) isolates belonged to the Beijing genotype and 23 (23%) isolates belonged to the LAM family. A cluster of isolates belonging to the LAM family had significant genetic heterogeneity, indicating that this family had an ancient distribution and circulation in this geographical region. Moreover, we found a significant percentage of the isolates (36%) belonged to as yet unidentified families of M. tuberculosis or had individual non-clustering genotypes. Mutations conferring rifampicin and isoniazid resistance were detected in 49% and 54% isolates, respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene were predominant among drug-resistant isolates. An association was found for belonging to the LAM strain family and having multiple drug resistance (R = 0.27, p = 0.0059) and also for the presence of a mutation in codon 531 of the rpoB gene and belonging to the Beijing strain family (R = 0.2, p = 0.04).ConclusionsTransmission of drug-resistant isolates seems to contribute to the spread of resistant TB in this oblast. The Beijing genotype and LAM genotype should be seen as a major cause of drug resistant TB in this region.

Polymorphisms in folate-metabolizing genes and risk of non-Hodgkin's lymphoma.

We investigated the role of single nucleotide polymorphisms (SNPs) in the folate-metabolizing genes MTHFR, MTR, MTRR, MTHFD, CBS and SHMT in regulating genetic susceptibility to Non-Hodgkins lymphoma (NHL). We determined the allele and genotype frequencies in the case group (146 patients with NHL) and the control group (540 blood donors). A significant association with NHL was observed only for MTHFD1 G1958A (allele G OR=1.382, P=0.05; genotype GA OR=2.316, P=0.01; genotype GG OR=2.153, P=0.03). After additional stratification of case and control groups according to sex and tumor type association of MTHFD1 G1958A with NHL was observed only in high-grade NHL subgroup (allele G OR=1.664, P=0.01) and in women subgroup (allele G OR=2.043, P=0.009). Meta-analysis for SNPs in the MTHFR, MTR, MTRR and SHMT revealed a reducing effect of the MTR 2756G allele on the risk of NHL (OR=0.902; 95% CI 0.821-0.991, P=0.03).

Increase in Expression of Brain Serotonin Transporter and Monoamine Oxidase A Genes Induced by Repeated Experience of Social Defeats in Male Mice

Serotonin transporter and monoamine oxidase (MAO) A are involved in the inactivation of serotonin. The former is responsible for serotonin re-uptake from the synapse, whereas the latter catalyzes serotonin deamination in presynaptic terminals. Expression of serotonin transporter and MAO A genes was investigated in raphe nuclei of midbrain of CBA/Lac male mice with repeated experience of social victories or defeats in 10 daily aggressive confrontations. The amount of cDNA of these genes was evaluated using multiplex RT-PCR. Two independent experiments revealed that the defeated mice were characterized by significantly higher levels of serotonin transporter and MAO A mRNAs than the control and aggressive animals. Increased expression of MAO A and serotonin transporter genes is suggested to reflect the accelerated serotonin degradation in response to activation of the serotonergic system functioning induced by social stress. Significant positive correlation between MAO A and serotonin transporter mRNA levels suggests common pathways of regulation of transcriptional activity of these genes.

Association of SNPs of CD40 Gene with Multiple Sclerosis in Russians

Multiple sclerosis (MS) is a serious, incurable neurological disease. In 2009, the ANZgene studies detected the suggestive association of located upstream of CD40 gene in chromosome 20q13 (p = 1.3×10−7). Identification of the causal variant(s) in the CD40 locus leads to a better understanding of the mechanism underlying the development of autoimmune pathologies. We determined the genotypes of rs6074022, rs1883832, rs1535045, and rs11086996 in patients with MS (n = 1684) and in the control group (n = 879). Two SNPs were significantly associated with MS: rs6074022 (additive model C allele OR = 1.27, 95% CI = [1.12–1.45], p = 3×10−4) and rs1883832 (additive model T allele OR = 1.20, 95% CI = [1.05–1.38], p = 7×10−3). In the meta-analysis of our results and the results of four previous studies, we obtain the association p-value of 2.34×10−12, which confirmed the association between MS and rs6074022 at a genome-wide significant level. Next, we demonstrated that the model including rs6074022 only sufficiently described the association. From our analysis, we can speculate that the association between rs1883832 and MS was induced by LD, whereas rs6074022 was a marker in stronger LD with the functional variant or was the functional variant itself. Our results indicated that the functional variants were located in the upstream region of the gene CD40 and were in higher LD with rs6074022 than LD with rs1883832.

Polymorphisms in the folate-metabolizing genes MTR, MTRR, and CBS and breast cancer risk

Alterations in the nucleotide sequences of folate-metabolizing genes can increase the risk of malignant transformation. The aim of our study was to investigate the association of three single-nucleotide polymorphisms (SNPs) in the folate-metabolizing genes - A2756G MTR, A66G MTRR, and 844ins68 CBS - which have putative functional significance in breast cancer risk. The allele and genotype frequencies of the SNPs were determined in a case group (840 women with sporadic breast cancer) and a control group (770 women). No statistically significant association of studied SNPs with breast cancer was revealed. A meta-analysis, which included data obtained from the literature and the present research, did not reveal any statistically significant associations of these SNPs with breast cancer. The results obtained provide evidence that these SNPs are not involved in the development of breast cancer.