Biochemistry (Moscow) (v.74, #4)

Carcinogenesis: Evolution of concepts by A. V. Lichtenstein (353-361).
Cancer is considered as an unintended consequence of internal imperfection of multicellular organisms: Darwinian evolution “does not foresee the future and does not plan for it”, it is forced to handle only anything that it has at a given moment “at hand”, which makes inevitable compromises and restrictions. In this case, there are a number of founding dogmas including mutagenesis as the main driving force of carcinogenesis; the environment as the main source of mutagenic effects; tumor monoclonality; cancer cell multistage transformation as Darwinian process of successive mutation—selection cycles. Recent discoveries complicate, supplement, and sometimes transform into an opposite fixed concepts. As a result, a new “image” of carcinogenesis is formed as a biological phenomenon whose conservation is indicative of its evolutionary utility.
Keywords: carcinogenesis; mutagenesis; epigenetics; transformation; differentiation

T-cadherin activates Rac1 and Cdc42 and changes endothelial permeability by E. V. Semina; K. A. Rubina; P. N. Rutkevich; T. A. Voyno-Yasenetskaya; Y. V. Parfyonova; V. A. Tkachuk (362-370).
In the present study, expression of T-cadherin was shown to induce intracellular signaling in NIH3T3 fibroblasts: it activated Rac1 and Cdc42 (p < 0.01) but not RhoA. T-Cadherin overexpression in human umbilical vein endothelial cells (HUVEC) using adenoviral constructs induced disassembly of microtubules and polymerization of actin stress fibers, whereas down-regulation of endogenous T-cadherin expression in HUVEC using lentiviral constructs resulted in micro-tubule polymerization and a decrease in the number of actin stress fibers. Moreover, suppression of the T-cadherin expression significantly decreased the endothelial monolayer permeability as compared to the control (p < 0.001).
Keywords: T-cadherin; Rho GTPases; endothelial cell permeability; microtubules; actin

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as ΔΨ (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP4− and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low ΔΨ value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the ΔΨ-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.
Keywords: ATP/ADP-antiporter; membrane potential; mitochondria; Tetrahymena pyriformis ; uncouplers; DNP; intracellular adaptation

Correlation between hepatocarcinogenic effect of estragole and its influence on glucocorticoid induction of liver-specific enzymes and activities of FOXA and HNF4 transcription factors in mouse and rat liver by V. I. Kaledin; M. Yu. Pakharukova; E. N. Pivovarova; K. Yu. Kropachev; N. V. Baginskaya; E. D. Vasilieva; S. I. Ilnitskaya; E. V. Nikitenko; V. F. Kobzev; T. I. Merkulova (377-384).
It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.
Keywords: hepatocarcinogens; estragole; tryptophan oxygenase; tyrosine aminotransferase; glucocorticoid induction; HNF4; FOXA

Effect of solvent phase transitions on enzymatic activity and structure of laccase from Coriolus hirsutus by E. V. Stepanova; T. V. Fedorova; O. N. Sorokina; V. V. Volkov; O. V. Koroleva; A. T. Dembo (385-392).
The effect of solvent phase transitions on catalytic activity and structure of the active site of laccase produced by the Basidiomycetes Coriolus hirsutus 072 was studied. As shown by small-angle X-ray scattering, laccase exists in solution as a mixture of monomeric and aggregated particles in the percent ratio 85: 15. This ratio did not change on phase transitions. A complex nature of laccase activity dynamics during thawing and further heating to 20°C was shown. Spontaneous oxidation of T1 copper center in the temperature range 12–20°C was not observed. According to spectral data, the structure of laccase active sites including all copper centers of types T1, T2, and T3 changes during the phase transition.
Keywords: laccase; phase transitions; small angle X-ray scattering (SAXS); EPR spectra; active site

In the present study we have investigated the characteristics of folding and unfolding pathways of two model proteins, ovalbumin and α-lactalbumin, monitored through the changes in surface hydrophobicity using fluorescence and circular dichroism spectroscopy. In the unfolding process, it was observed that ovalbumin and α-lactalbumin followed a three state transition pathway involving an intermediate state having high surface hydrophobicity. The intermediate state has also been characterized by circular dichroism spectroscopy, and it was found that the intermediate retained almost the same secondary structure as the native proteins, and therefore it can be referred to as molten globule state. The refolding process was monitored using fluorescence and circular dichroism spectroscopy, and it was observed that the refolding of α-lactalbumin was reversible and proceeded through the accumulation of similar type of intermediates as observed during its unfolding pathway. However, on refolding from the guanidine hydrochloride-denatured state, ovalbumin reached a different folded state.
Keywords: surface hydrophobicity; protein unfolding; folding intermediate; fluorescence and circular dichroism spectroscopy; extrinsic fluorescence spectroscopy

Design of a novel interleukin-13 antagonist from analysis of informational structure by A. N. Nekrasov; L. E. Petrovskaya; V. A. Toporova; E. A. Kryukova; A. V. Rodina; E. Yu. Moskaleva; M. P. Kirpichnikov (399-405).
Interleukin-13 (IL-13) is one of the cytokines involved in the development of Th2-type immune response. It plays an important role in the pathogenesis of asthma and other allergic diseases. Two deletion forms of IL-13 were constructed on a basis of informational structure analysis and expressed in E. coli cells. They were found to differ in ability to stimulate proliferation of TF-1 cell line. Deletion variant 146 (DV146) completely lacks such activity, whereas DV148 provides about 50% of the proliferation stimulation. The simultaneous addition of DV146 with full-length IL-13 suppresses proliferation depending on the concentration of the deletion form. Thus, the designed protein acts as an antagonist of IL-13.
Keywords: ANIS method; informational structure; deletion variant; IL-13 antagonist; TF-1 proliferation

Chaperone Skp from Yersinia pseudotuberculosis exhibits immunoglobulin G binding ability by E. V. Sidorin; R. H. Ziganshin; G. A. Naberezhnykh; G. N. Likhatskaya; E. V. Trifonov; S. D. Anastiuk; O. V. Chernikov; T. F. Solov’eva (406-415).
A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM−1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.
Keywords: chaperone Skp; immunoglobulin-binding protein; immunoglobulin G; Fc fragment of IgG; computer modeling

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Salmonella enterica O47 and studied by sugar analysis along with one- and two-dimensional 1H- and 13C-NMR spectroscopy. The following structure of the linear ribitol phosphate-containing repeating unit of the O-polysaccharide was established: $$ o 2) - D - Ribitol - 5 - P - (O o 6) - alpha - D - Galp - (1 o 3) - alpha - L - FucpNAm - (1 o 3) - eta - D - GlcpNAc - (1 o , $$ where FucNAm stands for 2-acetimidoylamino-2,6-dideoxy-L-galactose. About 10% of Gal is O-acetylated at position 4 and another minor O-acetyl group is present at an undetermined position. Functions of the S. enterica O47 antigen biosynthetic genes were tentatively assigned by comparison with gene databases and found to be in agreement with the O-polysaccharide structure. A comparison of the O-antigen gene clusters of S. enterica O47 and E. coli O145 suggested their close evolutionary relationship.
Keywords: Salmonella enterica ; lipopolysaccharide; bacterial polysaccharide structure; acetimidoyl group; ribitol phosphate; O-antigen gene cluster

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca2+-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria by O. V. Krestinina; D. E. Grachev; I. V. Odinokova; G. Reiser; Yu. V. Evtodienko; T. S. Azarashvili (421-429).
The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-32P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca2+ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of FoF1-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of FoF1-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca2+- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.
Keywords: brain mitochondria; peripheral benzodiazepine receptor; permeability transition pore; FoF1-ATPase subunit c ; PBR/TSPO

Analysis of mitochondrial DNA somatic mutations in OXYS and Wistar strain rats by U. N. Rotskaya; I. B. Rogozin; E. A. Vasyunina; N. G. Kolosova; B. A. Malyarchuk; G. A. Nevinsky; O. I. Sinitsyna (430-437).
Rats of the OXYS strain are sensitive to oxidative stress and serve as a biological model of premature aging. We have compared spectra of somatic mutations in a control region of mtDNA from the liver of the OXYS rat strain and of Wistar rats as a control. The majority of nucleotide substitutions in the mutation spectra were represented by transitions: 94 and 97% in the OXYS and Wistar rats, respectively. It was shown that 40% of somatic mutations in the control region of mtDNA from Wistar rats were significantly consistent with the model of dislocation mutagenesis. No statistical support for this model was found for mutations in the control region of mtDNA from OXYS rats. The mutation frequency in the ETAS section was higher in the OXYS strain rats than in Wistar rats. These results suggest different mechanisms of mutagenesis in the two rat strains under study.
Keywords: OXYS rats; Wistar rats; somatic mutations; mtDNA; liver

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14α-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.
Keywords: cytochrome P450; colloidal gold nanoparticles; bioelectrochemistry; drug metabolism

Inactivation of genes encoding superoxide dismutase modifies yeast response to S-nitrosoglutathione-induced stress by O. V. Lushchak; N. Z. Nykorak; T. Ohdate; Y. Inoue; V. I. Lushchak (445-451).
Antioxidant enzymes can modify cell response to nitrosative stress induced, for example, by nitric oxide or compounds decomposing with its formation. Therefore, we investigated the effects of S-nitrosoglutathione (GSNO) on cell survival, activity of antioxidant enzymes, and concentrations of reduced and oxidized glutathione in parental and isogenic strains defective in Cu,Zn- or Mn-superoxide dismutases (Cu,Zn-SOD and Mn-SOD, respectively), or in both of them. Stress was induced by incubation of the yeast with 1–20 mM GSNO. The strains used demonstrated different sensitivity to GSNO. A Cu,Zn-SOD-defective strain survived the stress better than the parental strain, while the double mutant was the most sensitive to GSNO. The ·NO-donor at low concentrations (1–5 mM) increased SOD activity, but its high concentrations (10 and 20 mM) decreased it. The activity of catalase in all strains was enhanced by GSNO. Inhibition of protein synthesis by cycloheximide did not prevent the activation of SOD, but it prevented the activation of catalase. These facts suggest that SOD was activated at a posttranslational level and catalase activity was enhanced via de novo synthesis. A GSNO-induced increase in oxidized glutathione level in the studied yeast strains might account for cell killing by GSNO due to the development of oxidative/nitrosative stress.
Keywords: yeast; superoxide dismutase; S-nitrosoglutathione; nitric oxide; markers of oxidative stress

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll BA molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll BA to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll BA. Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the BA molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.
Keywords: bacterial photosynthesis; reaction center; photochemical charge separation; site-directed mutagenesis; bacteriochlorophyll; Rhodobacter sphaeroides

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism by K. B. Shumaev; S. A. Gubkina; E. M. Kumskova; G. S. Shepelkova; E. K. Ruuge; V. Z. Lankin (461-466).
The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.
Keywords: free radicals; malondialdehyde; methylglyoxal; modification of amino acids