Peculiarities of interaction of monoclonal antibody B2 with polycyclic aromatic hydrocarbons and peptide-mimotope of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene

In order to determine the possible mechanisms of interactions of monoclonal antibody B2 with haptens and early synthesized peptide-mimotope of benzo[a]pyrene using the phage display method, amino acid sequences of variable fragments of heavy and light chains are determined and a model of Fab-fragment is constructed. The structure of the antibody active center is determined using molecular docking with polycyclic aromatic hydrocarbons. It is identified that the active center of monoclonal antibody B2 brings the two pockets of binding. The correlation between preliminarily obtained experimental data on the cross-reactivity of monoclonal antibody B2 with some ligands and calculated bond energy is found. It is shown that synthetic peptide-mimotope of benzo[a]pyrene is weak competing with the conjugate of benzo[a]pyrene for binding with monoclonal antibody B2. The immunization of mice with the conjugate of peptide and bovine serum albumin results in creation of antibodies to benz[a]anthracene and anthracene but not to benzo[a]pyrene. The model of peptide-mimotope of benzo[a]pyrene from pIII protein of bacteriophage is built. It is determined that tryptophan included into peptide composition can be exposed on the surface and be available for antibody. The data of modeling obtained in this study can be applicable for further optimization as both the structure of peptide-mimotope of benzo[a]pyrene and for active center of monoclonal antibody B2.     

Effect of point substitutions in the MnSOD, GPX1, and GSTP1 genes on the risk of familial and sporadic breast cancers in residents of the Altaĭ region of the Russian Federation

The frequencies of the polymorphic gene variants MnSOD Ala9Val, GPX1 Pro198Leu, and GSTP1 Ile105 Val were estimated in female residents of Altai krai with breast cancer. The frequency distributions of the genotypes for all genes studied in both patients and control subjects fit the Hardy-Weinberg equilibrium. The estimated frequencies of the genotypes for the studied genes in the control group did not differ from those earlier reported for Caucasoid women living in Europe. The T(rs1050450) allele of the GPX1 gene was demonstrated to protect against sporadic breast cancer (OR = 0.74 (95% CI = 0.58-0.94), p = 0.012). Carriers of the genotype combination MnSOD CC + GPX1 CC were found to have a 1.6 times higher risk of sporadic breast cancer compared to the control group (OR = 1.59 (1.05-2.41), p = 0.0258). The polymorphic loci GSTP1 (rs1695) and MnSOD (rs4880) were not found to be significantly associated with the risk of familial or sporadic breast cancer.     

Novel DNA glycosylases from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.     

New approach to synthesis of peptide immunomimetic of benzo[a]pyrene

A novel approach to discovery of the peptide with an internal immunological image of carcinogen is suggested in this work. The hybridomas producing monoclonal antibodies (mAb) B2 against benzo[a]pyrene and benz[a]antracene were prepared. Polyclonal Ab against benzo[a]pyrene (Bp) and benz[a]antracene (Ba), antracene, chrysene, and pyrene were also prepared. We identified the related peptide-presenting phage specificity binding to mAT B2 and polyclonal Ab against Bp from 12 large random peptide libraries using phage display method. ICR mice were immunized with specific binding of positive phage clone for studying its immunogenicity. The Bp antibodies were found in the blood serum. All positive phage clones carried the sequence. Thus, the unique peptide with an internal immunological image of Bp may be the new candidate for anticarcinogenic vaccine.     

Existence of cardiac PNMT mRNA in adult rats: elevation by stress in a glucocorticoid-dependent manner

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that synthesizes epinephrine from norepinephrine. The aim of this study was to determine potential PNMT gene expression in the cardiac atria and ventricles of adult rats and to examine whether the gene expression of this enzyme is affected by immobilization stress. PNMT mRNA levels were detected in all four parts of the heart, with the highest level in the left atrium. Both Southern blot and sequencing verified the specificity of PNMT detected by RT-PCR. Single immobilization for 2 h increased gene expression of PNMT in both atria and ventricles. In atria, this effect was clearly modulated by glucocorticoids, because either adrenalectomy or hypophysectomy prevented the increase in PNMT mRNA levels in response to immobilization stimulus. This study establishes, for the first time, that PNMT gene expression occurs in cardiac atria and also, to a small extent, in ventricles of adult rats. Immobilization stress increases gene expression in atria and ventricles. This increase requires an intact hypothalamus-pituitary-adrenocortical axis, indicating the involvement of glucocorticoids.     

The C terminus of annexin II mediates binding to F-actin

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II C-terminal amino acid residues, LLYLCGGDD, participate in F-actin binding.     

Regulation of annexin A2 by reversible glutathionylation

The annexin A2-S100A10 heterotetramer (AIIt) is a multifunctional Ca(2+)-dependent, phospholipid-binding, and F-actin-binding phosphoprotein composed of two annexin A2 subunits and two S100A10 subunits. It was reported previously that oxidative stress from exogenous hydrogen peroxide or generated in response to tumor necrosis factor-alpha results in the glutathionylation of Cys(8) of annexin A2. In this study, we demonstrate that AIIt is an oxidatively labile protein whose level of activity is regulated by the redox status of its sulfhydryl groups. Oxidation of AIIt by diamide resulted in a time- and concentration-dependent loss of the ability of AIIt to interact with phospholipid liposomes and F-actin. The inhibitory effect of diamide on the activity of AIIt was partially reversed by dithiothreitol. In addition, incubation of AIIt with diamide and GSH resulted in the glutathionylation of AIIt in vitro. Mass spectrometry established the incorporation of 2 mol of GSH/mol of annexin A2 subunit at Cys(8) and Cys(132). Glutathionylation potentiated the inhibitory effects of diamide on the activity of AIIt. Furthermore, AIIt could be deglutathionylated by glutaredoxin (thiol transferase). Thus, we show for the first time that AIIt can undergo functional reactivation by glutaredoxin, therefore establishing that AIIt is regulated by reversible glutathionylation.