Study of the quaternary structure of rat 1-Cys peroxiredoxin

Peroxiredoxins (Prx) are a family of antioxidant proteins with peroxidase activity. The ability of 1-Cys Prx to self-associate was studied with the use of native PAGE and Western blotting. Two protein bands corresponding to monomeric and dimeric forms were detected in the preparation of the recombinant 1-Cys Prx subjected to native PAGE, with dimers being more abundant. The third band corresponding to the oligomeric form was detected after incubation of the recombinant 1-Cys Prx with DTT, although monomers and dimers were also observed. These results indicate that monomeric, dimeric, and oligomeric states of the protein are likely to be interchangeable. Native PAGE in combination with Western blot analysis revealed that self-association of 1-Cys Prx also occurred at physiologically relevant concentrations in vivo. The native 1-Cys Prx existed in the monomeric and dimeric forms in rat olfactory epithelium, with monomers being more common. The structural sensitivity of the recombinant 1-Cys Prx to imidazole was shown.     

A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation.

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.     

Seasonal changes in proteolytic activity of calpains in striated muscles of long-tailed ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

Seasonal changes in proteolytic activity and content of calpains in striated muscles of the longtailed ground squirrel Spermophilus undulatus were studied by casein zymography and Western blotting analysis. The results testify to hyperactivation of calpain proteases in the skeletal muscles of awakened animals during the “winter” activity. The observed changes are discussed in the context of adaptation of skeletal muscles of long-tailed ground squirrels to hibernation.     

Three-dimensional ultrastructural and immunohistochemical study of immature neurons in the subgranular zone of the rat dentate gyrus

The present study is devoted to three-dimensional ultrastructural organization of mitotically dividing immature neurons in dentate gyrus using biophysical approaches. In adult vertebrate brain, cell proliferation persists throughout life mainly in dentate gyrus of the hippocampus (DG) and olfactory bulb. Neurogenesis has been demonstrated using tagged thymidine analogues incorporated into the S phase of the cell cycle, but these may also detect repaired DNA in postmitotic neurons. Recent retroviral labelling has shown that neuronal progenitors/neuroblasts divide and produce functional neurons. Providing ultrastructural evidence of mitotically active cells has proven problematical, not only because of technical issues of identifying dividing cells at electron microscope level, but also because it is difficult to demonstrate unequivocally that neurons identified in the electron microscope are really post mitotic. However by characterising post mitotic cells labelled with BrdU and doublecortin and comparing these with post mitotic cells reconstructed in 3-dimensions from ultrathin serial sections, we have been able to illustrate individual mitotic elements and phases of cells within the GC layer of adult rat dentate gyrus. Here we show dividing cells in metaphase within clusters of immature GCs in subgranular zone (SGZ). These reconstructions provide ultrastructural confirmation that cells expressing doublecortin (DCX), a microtubule-associated protein expressed in migrating neurons, localize as clusters in the subgranular zone (SGZ) of dentate gyrus (DG) in the hippocampus during all animal life. Such DG cells with clear synaptic specializations, somatic spines and basal dendrites are exclusive to immature GC that appear to re-enter the cell cycle, suggesting the possibility that newly generated neurons within the DG might arise not only from precursors, but also from clusters of immature GC.     

The role of peroxiredoxin 6 in neutralization of X-ray mediated oxidative stress: effects on gene expression,preservation of radiosensitive tissues and postradiation survival of animals

Peroxiredoxins are redox-sensing multifunctional enzymes, among them peroxiredoxin 6 (Prx6) can neutralize the most broadest range of hydroperoxides and play an important role in maintaining the redox homeostasis of the cell. In the present study, radioprotective and signaling regulatory effects of Prx6 were demonstrated and characterized. Intravenously administered exogenous Prx6 protects the organism of mice from the destructive action of ionizing radiation in the lethal dose range of 5–10?Gy. Dose reduction factor of 1.4 Prx6 injection reduces the severity of radiation-induced leuko- and thrombopenia in irradiated animals, also preventing the destruction of epithelial cells in the small intestine. Injecting exogenous Prx6 also as its mutated form of Prx6–C47S lacking peroxidase activity affects the expression of genes involved in antioxidant response, DNA reparation, apoptosis and inflammatory processes, in bone marrow cells both in intact animals and in those subjected to ionizing radiation. The radioprotective properties of Prx6 are based, on the one hand, on the capability for ROS neutralization, and on the other hand – on the potentiality for activation of reparation processes of the cell under oxidative stress conditions. Prx6 can be considered as a potentially perspective radioprotective agent for the reduction of risks from the damaging action of ionizing radiation on the mammalian organism.     

The Effect of the Structure of Fluorescently Labeled Nucleotide Derivatives on the Efficiency of Their Incorporation in DNA in the Polymerase Chain Reaction

The efficiency of fluorescence DNA labeling was estimated for four fluorescent 2′-deoxyuridine 5′-triphosphate derivatives differing in the orientation of the main dye axis, which passes through the polymethine chain, relative to the linker connecting the dye to the nucleotide. To estimate the polymerase chain reaction (PCR) rate, real-time PCR was run with two commercial hot-start DNA polymerases possessing 5′→3′ exonuclease activity in the presence of an intercalating dye. The efficiency of the test compound incorporation in the PCR product was estimated via a quantitative analysis of the amplification product by agarose gel electrophoresis. The fluorescently labeled product was then hybridized on a biological microchip and the ratio of signals from perfect match and mismatch duplexes was determined. The incorporation efficiency and discrimination between perfect match and mismatch duplexes were found to depend on the relative orientation of the dye and the linker between the dye and pyrimidine base, as well as on the presence of hydrophilic groups in the dye. Compounds that are efficiently incorporated in a growing DNA strand and show a high specificity in hybridization analysis were identified using biochips.     

Time-dependent cGMP-activated conductance of detached patches of ROS plasma membrane

cGMP markedly increases the cationic conductance of the 'inside-out' patches of rod outer segment plasma membrane when applied to the inner side. The cGMP-activated conductance of some patches was shown to be time-dependent. The data obtained suggest that the change of cGMP concentration in the near membrane layer underlies this phenomenon rather than the change in the channel's activity. The hydrolysis and, probably, the desorption of the nucleotide are responsible for this.