Animal Models of Mitochondrial Diseases Associated with Nuclear Gene Mutations.

Mitochondrial diseases (MDs) associated with nuclear gene mutations are part of a large group of inherited diseases caused by the suppression of energy metabolism. These diseases are of particular interest, because nuclear genes encode not only most of the structural proteins of the oxidative phosphorylation system (OXPHOS), but also all the proteins involved in the OXPHOS protein import from the cytoplasm and their assembly in mitochondria. Defects in any of these proteins can lead to functional impairment of the respiratory chain, including dysfunction of complex I that plays a central role in cellular respiration and oxidative phosphorylation, which is the most common cause of mitopathologies. Mitochondrial diseases are characterized by an early age of onset and a progressive course and affect primarily energy-consuming tissues and organs. The treatment of MDs should be initiated as soon as possible, but the diagnosis of mitopathologies is extremely difficult because of their heterogeneity and overlapping clinical features. The molecular pathogenesis of mitochondrial diseases is investigated using animal models: i.e. animals carrying mutations causing MD symptoms in humans. The use of mutant animal models opens new opportunities in the study of genes encoding mitochondrial proteins, as well as the molecular mechanisms of mitopathology development, which is necessary for improving diagnosis and developing approaches to drug therapy. In this review, we present the most recent information on mitochondrial diseases associated with nuclear gene mutations and animal models developed to investigate them.

Hemocompatibility of Galactomannan and Galactoglucomannan Sulfates in In Vitro Experiments.

We identified compounds that do not independently provoke aggregation of human platelets and do not affect hemolysis of human erythrocytes in vitro: lacking anticoagulant activity sulfated galactoglucomannan (polydispersity 1.43; degree of sulfation 0.66) in concentrations ≤0.2 mg/ml; exhibiting anticoagulant activity (in concentrations up to 0.002 mg/ml) sulfated galactoglucomannan (polydispersity 1.5; degree of sulfation 1.81) and galactomannan obtained by sulfation with the sulfamic acid-urea complex (polydispersity 2.75; degree of sulfation 1.25) and galactomannans obtained by sulfation with chlorosulfonic acid in 1,4-dioxane (polydispersity 1.61/22.27; degree of sulfation 1.00/0.74).

Study of the Blood Compatibility of Sulfated Organosolv Lignins Derived from Abies sibirica and Larix sibirica Wood Pulp.

The effects of sulfated organosolv lignins derived from fir (Abies sibirica) and larch (Larix sibirica) (SLf and SLl; 4-3-7.5% sulfur, median-weight molecular mass 2960-4888 Da), on human blood/plasma clotting, platelet aggregation, and erythrocyte hemolysis were studied in vitro. Antithrombin activities of the samples were below 2 U/mg. Specimens of SLf (sulfur content 6.5, 6.6, and 7.5%, molecular weights 3503, 3487, and 3580 Da, respectively) and SLl (4.3 and 6.3%, 2960 and 3497 Da) in a concentration of 0.01 mg/ml did not prolong the blood clotting time, did not provoke human platelet aggregation, did not destroy erythrocyte membranes, and could be used for construction of drug delivery systems. The SLf sample (6.5%, sulfur, 3503 Da) in concentrations from 0.09 to 1.82 mg/ml did not stimulate platelet aggregation, reduced ADP-induced platelet aggregation, and 2-fold prolonged the blood/plasma clotting time 2-fold in comparison with control and could be used for creation of biomaterial with clot-resistant surface.

RNA (C5-cytosine) Methyltransferases.

The review summarizes the data on pro- and eukaryotic RNA (C5-cytosine) methyltransferases. The structure, intracellular location, RNA targets, and catalytic mechanisms of these enzymes, as well as the functional role of methylated cytosine residues in RNA are presented. The functions of RNA (C5-cytosine) methyltransferases unassociated with their methylation activity are discussed. Special attention is given to the similarities and differences in the structures and mechanisms of action of RNA and DNA methyltransferases. The data on the association of mutations in the RNA (C5-cytosine) methyltransferases genes and human diseases are presented.

Results of program acute myeloid leukemia therapy use in National Medical Research Center for Hematology of the Ministry of Health of Russian Federation.

AIM: To analyze treatment results of 172 patients with acute myeloid leukemia (AML) aged 18-60 years in National Medical Research Center for Hematology of MHRF. MATERIALS AND METHODS: Inductive and consolidation program for 139 (80%) patients was based on a standardized protocol: 4 courses "7+3" with different anthracycline use (2 courses of daunorubicin, idarubicin, mitoxantrone) and continuous use of cytarabine on the second inductive course. In 20% of patients cytarabine courses at the dose of 1 g/m2 2 times a day for 1-3 days combined with idarubicin and mitoxantrone were used as two consolidation courses. Allogenic bone marrow transplantation was performed in the first complete remission (CR) period in 40% of patients. RESULTS: The frequency of CR achievement in all patients was 78.6%, refractory forms were observed in 13.9% of patients, early mortality - in 7.5% of patients. Seven-year overall survival (OS) rate was 40.7%, relapse free survival (RFS) - 43.2%. When estimating effectiveness depending on cytogenetic risk group it was demonstrated that 5-year OS and RFS in patients with translocation (8; 21) cannot be considered as satisfying, it accounted for 50 and 34%, respectively. At the same time in patients with 16th chromosome inversion (inv16) these characteristics accounted for 68.6 and 63.5%. Acquired results forced reconsidering of the consolidation program in AML patients of this subgroup. The median time to allogenic blood stem cells transplantation (allo-BSCT) in patients with first CR was 6.5 months that was taken as a reference point in landmark analysis of patients in whom allo-BSCT was not performed. Landmark analysis showed that in AML patients of favorable prognosis group allo-BSCT does not significantly reduce the probability of relapse (0 and 36%) and does not influence RFS (33 and 64%). In patients of border-line and poor prognosis allo-BSCT significantly reduces relapse probability (26 and 66%; 20 and 100%) and significantly increases a 7-year RFS (68.7 and 30%; 45.6 and 0%). Allo-BSCT also results in significant RFS increase and reduces the probability of relapse (25 и 78%) in patients in whom CR was achieved only after the second induction course. At the same time allo-BSCT does not influence patients who achieved CR after the first treatment course: 55 and 50%. CONCLUSION: Multivariate analysis showed that cytogenetic risk group (HR=2.3), time of CR achievement (HR=2.9), and allo-BSCT transplantation (HR=0.16) are independent factors for disease relapse prognosis after achieving CR.

Effect of Xylan Sulfates on Coagulation of Human Blood Plasma.

Sulfated derivatives of xylan (isolated from Bétula pubéscens wood) with average molecular weight ~34 kDa, sulfur content of 11.3-17.5%, a degree of substitution of 0.74-1.64 are anticoagulants of direct type of action. Antithrombin and antifactor Xa activities in three tested xylan samples did not differ and reached 30.8-31.8 and 13.5-14.3 U/mg, respectively.

Dose Dependence of the Anticoagulant Effect of Intravenously Administered Cellulose Sulfate.

Experiments on rabbits showed that increasing the dose of intravenously administered cellulose sulfate from wheat straw (dynamic viscosity 3.4 cP, sulfur content 14.1%) increased plasma clotting time in some coagulation tests and plasma anticoagulant activity. When cellulose sulfate was administered in the dose of 1 mg/kg, plasma clotting time in the presence of the anticoagulant (5 min after administration) was ~3-fold higher than after saline administration.

Antitumor effect of arabinogalactan and platinum complex.

The article presents the results of investigation of antitumor properties of platinum-arabinogalactan complex. We showed the ability of the complex to inhibit the growth of Ehrlich ascites tumor cells. It is found that the distribution of the platinum-arabinogalactan complex is not specific only for tumor cells in mice. The complex was found in all tissues and organs examined (ascites cells, embryonic cells, kidney, and liver). The mechanism of action of the arabinogalactan-platinum complex may be similar to cisplatin as the complex is able to accumulate in tumor cells.

New composites of betulin esters with arabinogalactan as highly potent anti-cancer agents.

Betulin and its esters are the natural compounds with high in vitro cytotoxicity toward many cancer cells. However, the poor water solubility of these compounds has limited their applications. We prepared new composites of betulin esters using two methods, namely ball-milling of the mixtures of betulin esters with arabinogalactan and preparation of thin films of these mixtures by evaporating the aqueous solutions. These composites revealed higher water solubility as compared with the initial substances without losing the structural integrity and functionality. As a result, the new composites have shown much higher inhibitory effects against different cancer cell lines such as Ehrlich ascites carcinoma cells and lung carcinoma cells (A549) in comparison with the initial substances. The cell viability studies based on Annexin V and Propidium iodide probes have confirmed the high proapoptotic effect of betulin ester derivatives against cancer cells.

[Effect of Point Mutations in the Polymerase Genes of the Influenza A/PR/8/34 (H1N1) Virus on the Immune Response in a Mouse Model].

The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

[Local antibody immune responses in influenza patients and persons vaccinated with seasonal, pre-pandemic, and pandemic live attenuated influenza vaccines].

Mucosal immunity is one of the most important factors of human anti-influenza defense. The data about local immune responses in influenza A (H3N2) patients and in persons vaccinated within 2000-2009 with different seasonal LAIVs, A (H1N1)pdm2009 LAIV, and A (H5N2) LAIV are discussed. The influenza infection resulted in the larger quantities of local IgA and IgG conversions than seasonal LAIV vaccination. 56% of young (18-21 y.o.) persons had high titers (> or = 1:64) of IgA to A (H1N1)pdm2009 virus before its circulation. 19% of persons had anti A (H5N2) IgA before vaccination. Two-fold vaccination with A (H1N1) pdm2009 and A (H5N2) LAIVs resulted in local antibody conversions in 54% and 27% of volunteers, respectively. Both these vaccines increased local IgA avidity. The number of antibody conversions after vaccination with seasonal LAIVs was in inverse dependence on their titers before vaccination. These results make it possible to conclude that the intensity of local antibody immune response to any LAIV depends on the state of local immunological memory, particularly on the presence of the crossreactive antibody-secreting B cells.

In vitro studies of changes in human endothelial cell functions under the effect of imiquimod.

Imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) is an active immunomodulator with antiviral effects. In addition to its stimulatory effect on cell-mediated immunity, in vivo studies have detected its antiviral and antiangiogenic effects. Possible direct effect of imiquimod on endothelial cells remains not studied. We have shown that imiquimod inhibited proliferation and migration of human endothelial cells (EA.hy 926 strain) in vitro and induced apoptosis (but not necrosis) of endothelial cells and production of IL-6 cytokine. These results suggest that imiquimod inhibits angiogenesis via direct modulation of endothelial cell function.

[Anticoagulant activity of extracts from cedar bark, anthocyanidins of spruce and birch bark, and cellulose of aspen, fir, and wheat straw].

We have investigated in vitro the anticoagulant (AC) activity of proanthocyanidins from the bark of birch, cedar, spruce, pine, and larch; sulfated arabinogalantan and dihydroquercetin from larch wood; extracts from birch, cedar, and spruce; microcrystalline cellulose (MCC) from aspen and fir wood and wheat straw; and methylcellulose (MC) from aspen wood. The AC properties of the investigated substances are related mostly to their antithrombin activity. The AC activity increases with the content of sulfur in MCC of wheat straw, MC of aspen wood, and arabinogalañtan of larch wood. The maximum AC activity was observed in samples of sulfated MCC from fir wood and wheat straw. Their antithrombin activity (134 +/- 8 and 96 +/- 6, respectively) is worth of carrying out model tests in vivo.

Symmetry of electrostatic interaction between pyrophosphate DNA molecules.

We study chiral electrostatic interaction between artificial ideal homopolymer DNA-like molecules in which a number of phosphate groups of the sugar-phosphate backbone are exchanged for the pyrophosphate ones. We employ a model in which the DNA is considered as a one-dimensional lattice of dipoles and charges corresponding to base pairs and (pyro)phosphate groups, respectively. The interaction between molecules of the DNA is described by a pair potential U of electrostatic forces between the two sets of dipoles and charges belonging to respective lattices describing the molecules. Minima of the potential U indicate orientational ordering of the molecules and thus liquid crystalline phases of the DNA. We use numerical methods for finding the set of minima in conjunction with symmetries verified by the potential U . The symmetries form a non-commutative group of 8th order, S . Using the group S we suggest a classification of liquid crystalline phases of the DNA, which allows several cholesteric phases, that is polymorphism. Pyrophosphate forms of the DNA could clarify the role played by charges in their liquid crystalline phases, and open experimental research, important for nano-technological and bio-medical applications.

[Effect of sutent and celecoxib on the properties of endothelial cells in vitro].

We studied the anti-angiogenic properties of sutent (SU11248) and celecoxib in human endothelial cell line EA. hy 926 in vitro. Sutent 0.05-0.5 microg/ml suppressed their proliferation and migration depending on dose while celecoxib did the same at 5.0 microg/ml. We were the first to demonstrate that endothelial cell incubation was followed by increase in 5'-nucleotidase activity in the presence of sutent while celecoxib did not produce such effect. It may be suggested that elevated 5'-nucleotidase concentration at the membranes of endothelial cells might in turn contribute to the pool of extracellular adenosine to stimulate antiinflammatory effect. Our data also contribute to the knowledge about the anti-angiogenic properties of sutent and celecoxib.

[Anticoagulant activity of arabinogalactane sulfate and cedar bark extract studied in vitro].

We have studied in vitro the ability of the Siberian cedar crust (SCC) extract (Pinus sibirica Du Tour) and arabinogalactan sulphate (AGS) extracted from wood of Siberiam pine-tree (Larix sibirica Ledeb.) to increase the human blood plasma coagulation time and also to inhibit the amydolytic activity of thrombin (aIIa) and the coagulation factor Xa (aXa). A method has been developed by means of which SCC increases the aXa activity by a factor of 3.7 and the aIIà activity by a factor of 2.5. The AGS preparation increased the blood plasma coagulation time in the test for activated partial thromboplastin time. An effective concentration, at which the time of plasma coagulation was increased by a factor of 2 (in comparison to the control) was 2.94 +/- 0.33 mg/ml. AGS did not exhibit the ability to inhibit the Xa activity.

Cross-linking of Escherichia coli formamidopyrymidine-DNA glycosylase to DNA duplexes containing photoactivatable phenyl(trifluoromethyl)diazirine groups.

New reactive analogs of substrates for DNA repair enzyme E. coli Fpg protein containing the residues of 8-oxoguanine and photoactivatable phenyl(trifluoromethyl)diazirine groups were synthesized. Their substrate properties were investigated. Using photocross-linking technique, we established the presence of contacts of two nucleosides located near the oxoG with amino acids from the Fpg protein. The cross-linking efficiency achieved 10%.

On the involvement of the water-polaron mechanism in energy trapping by reaction centers of purple bacteria.

A locus for binding a mobile water molecule was searched for in the immediate vicinity of the special pair in the reaction center. Using the PROTEUS PC-program (a part of the GRASP package) atomic structures of the reaction centers were analyzed in purple bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides. In both structures the loci for binding mobile water molecules were found at the distance of about 4.5 A from the middle of the special pair in the reaction center. The reorientation of a hydrogen atom of this water molecule in the electric field of the excited special pair required energy of no less than 40 MeV that corresponded to predictions of the water-polarization model of trapping of electron excitation which was developed by M. V. Fok and one of the authors of this article.

[Experimental and theoretical study of temperature dependence of steady-state parameters of delayed luminescence induction in leaves of higher plants]. / Eksperimental'noe i teoreticheskoe issledovanie temperaturnoi zavisimosti statsionarnogo znacheniia induktsii zamedlennoi liuminestsentsii list'ev vysshikh rastenii.

The temperature dependence of delayed luminescence of Hibiscus rosa sinensis leaves was studied in the temperature range from -25 degrees C to degrees C. Temperature dependence of the steady-state delayed luminescence intensity has two maxima, at -10 and at +35 degrees C. For the theoretical modeling, the mathematical model of plant photosynthesis developed earlier was used. The temperature dependence of the delayed fluorescence induction was obtained by introducing into the model the temperature dependences for 12 rate constants derived from the data on the effect of temperature on different stages of photosynthesis. The theoretical temperature dependence of the steady-state delayed luminescence was shown to have the same shape as the experimental one.

[Influence of the sucrose outflow from leaves of higher plants on delayed luminescence induction in photosynthesis]. / Vliianie ottoka sakharozy iz list'ev vysshikh rastenii na induktsiiu zamedlennoi liuminestsentsii pri fotosinteze.

The effect of the transport of sucrose from leaves of higher plants on the width of the spectra of induction of delayed luminescence was studied. It was shown that the duration of the induction period decreases when the sucrose outflow from the leaves is limited by cooling the leaf petiole for two hours under light. It was concluded that the accumulation of sucrose in the conducting tissues of the leaf stimulates the increase in the CO2 fixation rate on rellumination after dark adaptation.