Suppression of base excision repair reactions by apoptotic 24kDa-fragment of poly(ADP-ribose) polymerase 1 in bovine testis nuclear extract

In this study, we examined the interaction of PARP1 and its apoptotic 24kDa-fragment with DNA duplexes mimicking different stages/pathways of base excision repair (BER) using a photocross-linking technique combined to in vitro functional assay. We found that endogenous PARP1 was photocross-linked to the gapped, nicked and flap containing DNA structures and its apoptotic 24kDa-fragment (p24), like PARP1, can interact with the same BER DNA intermediates. Effects of exogenous p24 on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. We showed that the interaction of p24 with DNA, as a whole, inhibited the BER reactions. However, gap filling and nick sealing catalyzed by the enzymes of the extract with DNA substrates characteristic for short patch (SP) BER pathway cannot be completely inhibited by p24. In contrast, binding of p24 to DNA duplex with a 5'-furan or a 5'-flap at the 5'-side of a nick inhibits strand-displacement DNA synthesis and activity of FEN1 in the repair of DNA via long patch (LP) BER pathway. Stimulation of the LP BER reactions induced by the addition of FEN1 or PCNA to the extract is suppressed by p24 thereby indicating that p24 can efficiently compete with these proteins of LP BER. Addition of pol beta to the extract can partially overcome the inhibitory effect of p24 and restore strand-displacement DNA synthesis. Thus, the apoptotic 24kDa-fragment of PARP1 may be considered as more efficient in inhibition of the LP than SP pathway and the effect may depend on the ratio of p24 to the repair enzymes catalyzing precise stages of BER.     

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.     

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 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.     

Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination]

Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.     

见血封喉乳汁脂溶性成分及其抗氧化活性研究

首次采用气相色谱.质谱联用技术(GC-MS)对见血封喉(Antiaris toxicaria(Pers.)Lesch.)乳汁的脂溶性成分进行了分析,共鉴定了27个化学成分,占其总量的91.7%.用清除DPPH自由基能力的方法测定了见血封喉乳汁脂溶性部位的抗氧化活性,结果显示出一定的抗氧化活性,SC50值为500μg ml-1.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.     

Sex ratio in early embryonal mortality in man

The problem of the functioning specificity of sex chromosomes during the early stages of embryogenesis in man and the associated problem of the sex ratio in spontaneous and induced abortions, as well as in newborns, remains open. We have conducted a cytogenetic examination of 342 spontaneous abortions divided into three clinical groups on the basis of the severity of the developmental disturbances of the embryo: spontaneous abortions sensu stricto with a developed embryo without any significant intrauterine delay of development (n = 100), nondeveloping pregnancies (n = 176), and anembryonic fetuses (n = 66). The frequency of chromosomal mutations in these groups was 22.0, 48.3, and 48.5%, respectively. Statistical analysis has demonstrated significant differences between the studied groups in the frequencies of the normal and abnormal karyotypes: the major contributions to these differences were associated with autosomal trisomy, triploidy, and 46,XY karyotype. The presence of 46,XY may reflect specific genetic mechanisms of prenatal mortality of embryos with normal karyotype, associated with sex and/or with the imprinting of X-chromosomes. The sex ratio in spontaneous abortions with normal karyotype was as follows: 0.77 for spontaneous abortions with well-developed embryos without any significant intrauterine delay of development; 0.60 for non-developing pregnancies; and 0.31 for anembryonic fetuses. An analysis of DNA from the embryos and their parents has demonstrated a low probability of contamination of cell cultures with mother cells as a possible source of prevalence of embryos with 46,XX karyotype among spontaneous abortions. Nondeveloping pregnancies and anembryonic fetuses showed statistically significant differences in the sex ratio (1.11) from the control group consisting of medical abortions. Differences in the sex ratio were due to an increasingly lower proportion of embryos with karyotype 46,XY (relative to the expected one) among the fetuses with an increased severity of developmental disturbances. The statistical "chances ratio" index also provided evidence that embryos with 46,XY karyotype had a higher propensity to produce a well-formed fetus as compared with the female embryos. We propose that the expression of genes of the maternal X-chromosome in XY embryos supports a more stable development during early embryogenesis as compared with XX embryos. In the latter case, normal development is coupled with the operation of an additional mechanism for compensation of the dose of X-linked genes. Operation of this mechanism increases the probability of disturbances in female embryos. A higher viability of XY embryos during the early stages of ontogenesis in man appears to explain their underrepresentation in samples of spontaneously aborted embryos and appears to be the major factor responsible for the deviation of the sex ratio from the theoretically expected value.