SWEET transporters of Medicago lupulina in the arbuscular-mycorrhizal system in the presence of medium level of available phosphorus.

Arbuscular mycorrhiza (AM) fungi receive photosynthetic products and sugars from plants in exchange for contributing to the uptake of minerals, especially phosphorus, from the soil. The identification of genes controlling AM symbiotic efficiency may have practical application in the creation of highly productive plant-microbe systems. The aim of our work was to evaluate the expression levels of SWEET sugar transporter genes, the only family in which sugar transporters specific to AM symbiosis can be detected. We have selected a unique "host plant-AM fungus" model system with high response to mycorrhization under medium phosphorus level. This includes a plant line which is highly responsive to inoculation by AM fungi, an ecologically obligate mycotrophic line MlS-1 from black medick (Medicago lupulina) and the AM fungus Rhizophagus irregularis strain RCAM00320, which has a high efficiency in a number of plant species. Using the selected model system, differences in the expression levels of 11 genes encoding SWEET transporters in the roots of the host plant were evaluated during the development of or in the absence of symbiosis of M. lupulina with R. irregularis at various stages of the host plant development in the presence of medium level of phosphorus available for plant nutrition in the substrate. At most stages of host plant development, mycorrhizal plants had higher expression levels of MlSWEET1b, MlSWEET3c, MlSWEET12 and MlSWEET13 compared to AM-less controls. Also, increased expression relative to control during mycorrhization was observed for MlSWEET11 at 2nd and 3rd leaf development stages, for MlSWEET15c at stemming (stooling) stage, for MlSWEET1a at 2nd leaf development, stemming and lateral branching stages. The MlSWEET1b gene can be confidently considered a good marker with specific expression for effective development of AM symbiosis between M. lupulina and R. irregularis in the presence of medium level of phosphorus available to plants in the substrate.

[Investigation of the composition of biologically active substances in extracts of wild plants].

The article presents the research materials of composition and the properties of biologically active compounds of aqueous ethanolic extracts of wild plants. To obtain extracts, we used raw plants containing phenolic compounds and aromatic wild plants: the herb St. John's wort (Hypericum), thyme herba (Thymus vulgaris), yarrow (Achillea millefolium), oregano (Origanum vulgaris); leaves of sage (Salviae folium); rose hips (Rosae), hawthorn fructus (Crataegus) and fruits of mountain ash (Sorbus). The optimum composition of the mixtures used and time of extraction has been established: the ratio of alcohol and water in extracting mixtures 1:1 by volume; ratio raw material:extractant - 1:7 by weight. The total content of fenolic substances in extracts of herbaceous plants varied from to 15.5 to 24.4 mg/g, and in fruit extracts from 24.2 to 29.7 mg/g. Substances of phenolic nature, including gallic and ferulic acid, rutin, hesperidin, quercetin and apigenin were identified in the studied extracts using the HPLC. The analysis of flavonoid composition showed that rutin content in the investigated extracts varied from 0.56 mg/g up to 13,80 mg/g, of quercetin - from 0.52 to 1.36 mg/g; apigenin - from 0.44 to 1.44 mg/g; hesperidin from 2.44 to 32,72 mg/g. The content of phenolic acids varied from 0.16 to 1.44 mg/g (ferulic acid) and from 0.16 to 3.12 mg/g (chlorogenic acid). Total antioxidant activity of the studied phytoextracts (dilution 1:10) ranged from 142 to 230 µg/ml (in terms of ascorbic acid), which is consistent with the results of the quantitative analysis of flavonoids. The results of the studies of antimicrobial properties of phytoextracts showed that for E. coli the most active extracts were from thyme and yarrow, and against S. aureus - from St. John's wort. Extracts of St. John's wort and yarrow were effective against Rhizopus stolonifer.

Heat shock protein HSP40 family chaperone DNAJB6/MRJ expression analysis in blood cells obtained from patients with atopic dermatitis in different phases.

Heat shock protein HSP40 family molecular chaperone DNAJB6/MRJ expression has been analyzed in blood cells of patients with atopic dermatitis compared with healthy donors. Severity of disease was estimated according index SCORAD. Methods: Peripheral blood cells were separated using Percoll density gradient. Purified neutrophils and lymphocytes have been stained with antibodies to the heat shock protein DNAJB6/MRJ. Cells were analyzed using flow cytometry. Real time PCR method has been used to verify the bacterial contamination of the skin of patients with atopic dermatitis. Statistical analysis was performed by ANOVA. Results: Expression of DNAJB6/MRJ protein has been found to be elevated in all samples of cells obtained from patients with atopic dermatitis. The highest level of the DNAJB6/MRJ protein expression was shown in neutrophils at the acute phase of severe atopic dermatitis. DNAJB6/MRJ protein expression in lymphocytes of patients with atopic patients was less extensive compared with neutrophil level and was shown to be higher at subacute phase of disease. The DNAJB6/MRJ protein expression was found to be statistically significant higher in lymphocytes from atopic patients compared with healthy donors. The bacterial contamination of skin (verified by PCR) was shown to influence the DNAJB6/MRJ protein level in lymphocytes of atopic dermatitis patients. Conclusion: Expression of the heat shock protein DNAJB6/MRJ was elevated in neutrophils and lymphocytes of patients with atopic dermatitis compared with healthy donors. The highest level of the DNAJB6/MRJ protein was found to be in neutrophils at acute phase of severe atopic dermatitis and gradually decline as continue to the disease.

[Genetic mechanisms of E. coli cell stability to the antibiotics].

Mycotoxin Fumonisin B1 posseesses the weak DNA-damage activity for the E. coli strain defective in UvrA gene participating in DNA reparation. Fumonisin B1 in the concentration 10(-5) M increases a resistance of wild type E. coli cells to antibiotic doxycycline. Obtained results indicate the influence of fumonisin B1 on the genome of the cultured E. coli cells.

[Effect of thermal processing of Bacillus intermedius ribonuclease on its catalytic activity and biological properties]. / Vliianie termicheskoi obrabotki ribonukleazy Bacillus intermedius na ee kataliticheskuiu aktivnost' i biologicheskie svoistva.

Bacillus intermedius ribonuclease (binase), which is known to exert a growth-stimulating effect at low concentrations and a genotoxic effect at high concentrations, loses these abilities completely after exposure to 100 degrees C for 10 min, but retains approximately 96% of its catalytic activity and structural integrity. Other types of modification, such as photoinactivation and site-specific mutagenesis, gave rise to enzyme forms with unaltered structure but reduced (sometimes to trace amounts) catalytic activity. Genotoxicity was always proportional to the catalytic activity of the native enzyme, while a notable growth-stimulating effect may be exerted by enzymes with low activity. The loss of biological activity of thermoinactivated binase was related to the increase in the number of negatively charged groups on the enzyme surface, which led to a substantial decline in the adhesive properties of the enzyme.

[Genotoxic effects of metabolic derivatives of the new drug phosphabenzide]. / Genotoksicheskie éffekty metabolicheskikh proizvodnykh novogo lekarstvennogo preparata fosfabenzida.

Genotoxic action of four possible metabolites of the new tranquilizer phosphabenzide (acetylphosphabenzide, diphenylphosphinylacetic acid, phosphabenzide hydrazone with pyruvic acid, bis-1,2-(diphenylphosphinylacetyl)hydrazine) has been studied. These metabolites belong to slightly toxic phosphororganic compounds. The Ames Salmonella/microsomes tests performed on strains TA100 and TA98 showed that of these compounds only acetylphosphabenzide possessed mutagenic action. Metabolic activation of liver microsomes decreased the mutagenic effect. The mechanism of action of acetylphosphabenzide is likely to involve the formation of acetylhydrazine, capable of producing active electrophiles attacking DNA.

[Induction of the SOS-response with Bacillus intermedius RNAase]. / Induktsiia SOS-otveta RNKazoi Bacillus intermedius.

This paper deals with the induction of the SOS-functions of a bacterial cell by the exogenous ribonuclease of Bacillus intermedius. Two test systems were employed, which made it possible to quantitatively estimate SOS-response induction activity in gram-positive and gram-negative bacteria. It was established that the catalytically active enzyme elicits the SOS-response in the cells of Escherichia coli PQ 37 and causes prophage induction in the lysogenic strain Bacillus subtilis 168 phi 105 WT (a phenomenon related to the SOS-response). An enzyme with a His101Glu-substituted active center, exhibiting residual catalytic activity, failed to induce the SOS-response in an SOS-chromotest with E. coli PQ 37, indicating dependence of the SOS-inducing effect of the enzyme on its catalytic activity. It is suggested that the effect of active ribonuclease on membrane-bound and cytoplasmic RNA causes a change in the nucleotide pool and, as a consequence, elicits the SOS-response of the cell.

[Genotoxic effects of tonarol]. / Issledovanie genotoksicheskikh éffektov tonarola.

Genotoxic effects of 2,6-di-tret-butyl-4-methylphenol (tonarol) were studied using four test systems: the Ames test, the SOS chromotest, the cytogenetic test with rootlets of onion (Allium cepa), and the in vivo micronucleus test. Tonarol did not affect gene mutation induction in Salmonella typhimurium tester strains, the SOS response in the Escherichia coli strain PQ37, chromosomal aberrations in cells of onion (Allium cepa) rootlets, and micronuclei in erythrocytes of peripheral blood of CBA x C5713 L/G mice. Tonarol induced cell division in A.

SOS-inducing ability of native and mutant microbial ribonucleases.

The results of genotoxicity testing of microbial ribonucleases from Bacillus species with different catalytic activity obtained by site-directed mutagenesis in SOS chromotest are reported. At the concentrations 0.1-1 mg/ml, the induction factor for wild-type bacillar binase, barnase and mutant Arg58Lys binase with 100% activity was found to be significantly higher than 1.5 (1.8-2.8). Mutant RNases having decreased catalytic activity (binases with replacements Lys26Ala, Arg61Gln, His101Glu) or through natural inhibitor barstar inactivated wild-type RNase exhibited no SOS-inducing potency. The ability of native bacillar RNases and mutant enzymes possessing high catalytic activity comparable with the activity of wild-type RNase to cause the SOS response indicates that genotoxicity is mediated through the probable cleavage of cellular RNA.

[Dependence of the intensity of 2,4,6-trinitrotoluene-induced mutagenesis on the physiological state of tester strains of Salmonella typhimurium]. / Zavisimost' velichiny indutsirovannogo 2,4,6-trinitrotoluolom mutageneza ot fiziologicheskogo sostoianiia testernykh shtammov Salmonella typhimurium.

The dependence of the mutagenic potential of 2,4,6-trinitrotoluene (TNT) on the growth phase of the Salmonella typhimurium tester strains TA100 and TA98 is shown. The mutagenic potential proved to be maximal for log-phase cultures, being higher with strain TA100 than TA98. The amount of TNT absorbed by bacteria in the first minutes of contact varied, depending on culture age, and was the largest at the beginning of the exponential growth phase. A direct relation between the number of microbial cells and the amount of TNT absorbed from the medium was shown. The analysis of infrared (IR) spectra of cells after a 3-min contact with TNT, demonstrated that, at physiological temperatures, 2.4% of the original amount of TNT was present in cells of the tester strain in an unaltered form, while, at a lowered temperature, this value was 10.3%. Thus, the retardation of physiological processes in the cell changes the intensity of mutagen absorption and transformation. It is emphasized that an accurate assessment of mutagenicity should take into account the physiological state of tester bacteria.

[2,4,6-trinitrotoluene and 2,4-diamino-6-nitrotoluene: the absence of recA-dependent mutagenesis?]. / 1,4,6-Trinitrotoluol i 2,4-diamino-6-nitrotoluol: otsutstvie recA-zavisimogo mutageneza?

The genotoxicity of 2,4,6-trinitrotoluene (2,4,6-TNT) and its amino derivative, 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT), was studied using the Escherichia coli tester strain PQ37 in the SOS chromotest. The compound 2,4,6-TNT, without metabolic activation, virtually failed to induce an SOS effect in cells of the tester bacteria. Consequently, mutagenic activity of 2,4,6-TNT, which was shown earlier in the Ames test, does not depend on SOS mutagenesis. It was demonstrated that metabolic activation with the microsomal S9 human placenta fraction results in a threefold increase in the induction factor of the SOS effect caused by 2,4,6-TNT. The absence of the SOS-inducing activity of 2,4-DA-6-NT, regardless of the presence of a microsomal activating mixture, is shown. Thus, 2,4-DA-6-NT does not belong to metabolites of 2,4,6-TNT, responsible for the genotoxicity of this compound.

Bacterial ribonuclease: mutagenic effect in microbial test-systems.

Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, AraR-assay; the prophage induction test; and the DNA-repair test. The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward AraR-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution). Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity. Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coli strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain. The importance of the roles of both of enzyme catalytic activity and native structure is emphasized. A proposed mechanism for exogenous ribonuclease action is discussed. Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis.

[Antimutagenic activity of the enzyme preparation "binase" in microbial test systems]. / Antimutagennaia aktivnost' fermentnogo preparata "binaza" v mikrobnykh test-sistemakh.

The occurrence of microorganisms and the rates of terminal biogenic reduction of sulfates and synthesis of methane in stratal waters in deposit 302 of Bashkir Carboniferous deposition at the Romashkinskoe oil field were studied. It was shown that deposit 302 is a dynamic, highly reduced ecosystem containing sulfates and hydrogen sulfide in considerable concentrations, in which active biogenic processes occur. Sulfate reduction is a dominating anaerobic process by which the organic constituents of oil are transformed. The sulfate-reducing microflora is quite varied and characterized by high metabolic potentials. Enriched cultures, which can oxidize many organic substances, such as benzoate, acetate, ethanol, or lactate, at the expense of reduction of sulfates and ferric ion, were isolated from the samples extracted from deposit 302. It was suggested that the sulfate-reducing microflora might be responsible not only for sulfate reduction in the stratum but also for mobilization of part of insoluble iron oxides in the oil trap rock. The findings indicate that the dissimilation sulfate- and iron-reducing bacteria can contribute to the geochemistry of organic and mineral compounds in underground ecosystems.

[Mutagenic activity of 2,4,6-trinitrotoluene: the role of metabolizing enzymes]. / Mutagennaia aktivnost' 2,4,6-trinitrotoluola: rol' metaboliziruiushchikh fermentov.

Mutagenic activity of 2,4,6-trinitrotoluene (2,4,6-TNT) and one of its derivatives, 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) was studied in the Ames test using the Salmonella typhimurium tester strains TA98, TA100, TA100NR (deficient in classical nitroreductases), and TA100/1, 8DNP (deficient in O-acetyltransferase). The number of revertants was significantly increased by 2,4,6-TNT, in comparison with that of spontaneous revertants. Base pair substitutions were preferentially induced by 2,4,6-TNT. The mutagenic effect of this compound was virtually altered by in vitro metabolic activation with the S9 fraction of human placenta. Neither 2,4,6-TNT nor 2,4-DA-6-NT exhibited mutagenic activity in the strains TA 100NR and TA 100/1,8DNP. Based on the results obtained, it is concluded that the mutagenic potential of 2,4,6-TNT is determined by its metabolites that form during bacterial metabolism. Bacterial nitroreductases and O-acetyltransferases are the key activators of this compound to form the ultimate mutagens.

[Genotoxicity of the preparation "binase" in tests on Salmonella: Ames test and ARA-test]. / Genotoksichnost' preparata "binaza" v testakh na Salmonella: test Eimsa i Ara-test.

The present work deals with mutagenicity determination of enzyme sample "binase" (Bacillus intermedius ribonuclease) in microbial test-systems: Ames test and Ara-test. The weak mutagenic effect of "binase" high concentration was established in both tests by induction of forward Ara-mutations and Histidine-reverse mutations. A metabolic activation is seen to remove this effect.

[An evaluation of the genotoxicity of nonionogenic surface-active substances]. / Otsenka genotoksichnosti neionogennykh poverkhnostno-aktivnykh veshchestv.

The mutagenic activity of 16 industrial nonionic surfactant samples has been investigated in Ames-test without metabolic activation and with the use of mouse liver microsomal fraction in vitro. The genotoxical estimation of their possible biodegradation products is given. The mutagenicity of a number of high molecular polyethylene glycoles related to nonionic surfactants is shown.

[The mechanisms of the manifestation of the genotoxic effects of binase]. / Mekhanizmy proiavleniia genotoksicheskikh éffektov binazy.

The high concentrations of commercial sample of Bacillus intermedius 7P ribonuclease (binase) showed a weak mutagenic effect in Ames test and Ara-test. It has been established that binase induces the SOS-functions of microbial cell and prophage induction. The way of exogenic enzyme action through activation of RecA protein is proposed.

[Assessment of the genotoxicity of the "Binase" enzyme preparation]. / Otsenka genotoksichenosi fermentnogo preparata "Binaza".

"Binase" enzyme sample (a microbial ribonuclease) has been tested for mutagenicity in a set of tests. The set included Ames test Salmonella/microsome, Escherichia coli Rec-test, bacteriophage induction assay, DNA-repair synthesis in lymphoid cells. "Binase" is shown to possess a small genotoxic effect at high concentrations. Both animal and plant S-9 fractions eliminated the effect.