A period of urgent postnatal adaptation as a critical stage of human ontogenesis

The infant's state immediately after delivery is characterized by critical shifts of homeostasis parameters: decompensated mixed acidosis, pathologic indices of gaseous blood condition (arterial hypoxemia and hypercapnia), spontaneous postnatal hypothermia. Qualitative change of functional systems after delivery is accompanied by expressed tense of adaptation and regulation mechanisms and by centralization of cardiac rhythm control. The use of mathematical analysis of cardiac rhythm made it possible to find out that the change of degree of adaptation tense in the process- of postnatal period obeys the exponential dependence; moreover, the duration of transitional process of healthy newborns is 1 hour, but it considerably increases in case of hypoxia. After comparison of cardiac rhythm indices of these two groups of newborns it has been pointed out that mechanisms of adaptation after delivery are equal and they are based on the growing activity of central regulation processes. The dynamics of transitional process such as the period of postnatal adaptation indicates the reserve possibilities of infant and helps to reveal pre-nosological forms of disadaptational syndrome.     

2,5-Diketopiperazines: A New Class of Poly(ADP-ribose)polymerase Inhibitors

We show for the first time that natural 2,5-diketopiperazines (cyclic dipeptides) can suppress the activity of the important anticancer target poly(ADP-ribose)polymerase (PARP). Cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) can interact with the key residues of the PARP-1 active site, as demonstrated using docking and molecular dynamics simulations. One of the amide groups of cyclo(L-Ala-L-Ala) and cyclo(L-Ala-D-Ala) forms hydrogen bonds with the Gly863 residue, while the second amide group can form a hydrogen bond with the catalytic residue Glu988, and the side chain can make a hydrophobic contact with Ala898. Newly identified diketopiperazine inhibitors are promising basic structures for the design of more effective inhibitors of PARP family enzymes. The piperazine core with two chiral centers provides many opportunities for structural optimization.     

Adaptation of molecular genetic methods to study microorganisms associated with fish

A modern molecular genetic method has been adapted to study microorganisms associated with fish. A procedure for total DNA isolation from various organs and tissues of fish has been developed. Besides fish freshly caught, frozen and salted specimens were used. A bacterial PCR product was shown to amplify with highly conservative primers at an annealing temperature of 70–72°C from fish gills, liver, intestine, and skin.     

Adapting molecular-genetic methods for studying the taxonomic diversity of microbial communities associated with macrophytes

The combination of molecular-genetic techniques used in the study is applied to investigate microorganisms associated with macrophytes. The method of enzymatic lysis with phenol-chloroform extraction is optimal for the total bacterial DNA isolation from both periphyton organisms and enriched cultures. Amplifying the total DNA on conservative primers at the two-step PCR regime is recommended. An analysis of the taxonomic diversity of the microbial community from biofilm on the reed grass and in the enriched cultures propagated on various growth media has been carried out. The results have revealed a high diversity of periphyton microorganisms associated with reed grass, including representatives of such phylogenetic lines as proteobacteria (α, β, γ, and δ subgroups), Bacteroidetes/Chlorobi, Chlamydiae/Verrucomicrobia, and cyanobacteria. The low diversity of sequences in enriched cultures is represented by dominating genotypes of Cellvibrio with a high percentage of homology and uncultivated bacilla.     

Interaction of yeast mitochondria with fatty acids and mitochondria-targeted lipophilic cations

The effect of fatty acids and mitochondria-targeted lipophilic cations (SkQ1, SkQ3, MitoQ, and C₁₂TPP) on tightly-coupled mitochondria from yeasts Dipodascus (Endomyces) magnusii and Yarrowia lipolytica was investigated. Micromolar concentrations of saturated and unsaturated fatty acids were found to decrease the membrane potential, which was recovered almost totally by ATP and BSA. At low, micromolar concentrations, mitochondria-targeted lipophilic cations are “relatively weak, mild uncouplers”, at higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their prooxidant and detergent action. At very low, not uncoupling concentrations, mitochondria-targeted lipophilic cations profoundly promote (potentiate) the uncoupling effect of fatty acids. It is conceivable that the observed uncoupling effect of lipophilic cations can be, at least partially, due to their interactions with the endogenous pool of fatty acids.     

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273–1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663–668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH₂ interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ^? being reduced back to SkQH₂ by heme b_H of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C₉ or C₁₃ of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (С₁₂TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since С₁₂TPP is specifically accumulated in mitochondria and increases the H⁺ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by С₁₂TPP.     

Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages.     

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

The cell cycle inhibitor p21^CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.