[Effect of Bacillus pumilus ribonuclease on the paramagnetic centers of microbial cells].

Potential clinical application of Bacillus pumulus cytotoxic ribonuclease (binase) selectively inducing the death of tumor cells makes it imperative to investigate its effect on the normal human microflora. Flow cytometry was used to determine that binase concentration causing the apoptosis of cancer cells had no effect of the viability of Escherichia coli K12. The changes in the paramagnetic centers of E. coli K12 cells in the presence of nontoxic binase concentrations revealed by EPR spectroscopy included higher EPR signals from iron-containing proteins (including those from the Fe-S clusters) and of the Mn(II) hyperfine structure. The TMTH spin probe (N-(1-hydroxy-2,2,6,6-tetramethylpiperidine-4-il)-2-methylpropanamide hydrochloride) was used to reveal a twofold increase in the levels of reactive oxygen species (ROS) in the cells, which induced oxidative stress in the enzyme-treated bacteria. Inductively coupled plasma mass spectrometry revealed elevated contents of alkaline (Li, Na, K), alkali earth (Mg, Ca), transition (Cr, Mn, Fe, Cu, Zn), and post-transition metals (Bi, Pb) in the cells. Elevated levels of Cu and Zn (which impair the activity of the respiratory chain enzymes) and of Mn, which is known as a superoxide dismutase cofactor, confirmed development of the oxidative stress in bacteria.

[Probiotics for plants: NO-producing lactobacilli protect plants from drought].

After the inoculation of wheat roots with a suspension of the bacterium Lactobacillus plantarum, leveling of oxidative stress detected by the accumulation of H2O2 and MDA was found in leaves. Activation of catalase and increased integral antioxidant capacity in seedlings treated with NO-producing lactobacilli were detected during the determination of the contribution of bacterial NO to the plant stress reaction. Thus, for the first time, we have demonstrated that lactobacilli affect plant adaptive responses to stress by the involvement of nitric oxide.

[Ribonucleases as antiviral agents].

Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but molecular mechanisms of their antiviral activity remain unclear. The review observes the most known RNases which possess established antiviral effects, actually intracellular RNases (RNase L, MCPIPI protein, eosinophylic RNases) as well as exogenously applied ones (RNase A, BS-RNase, onconase, binase, synthetic RNases). Attention is given on two important but not always obligatory aspects in molecule of RNases, which have antiviral properties: catalytic activity and ability to the dimerization. The hypothetic scheme of virus elimination by exogenous RNases, that reflects possible types of interaction of viruses and RNases with a cell, is proposed. The evidence for RNases as classical components of immune defense which are perspective agents for development of new antiviral therapeutics is produced.

[Cellular targets of antitumor ribonucleases].

Some ribonucleases (RNases) produce selective toxic effect on the cancer cells. The mechanism of this antitumor activity remains largely unclear. The subject of this review is the RNases interaction with cellular components, resulting in the induction of apoptosis of tumor cells. Cell surface structures, which are potential acceptors of the exogenous RNase are discussed: acidic lipids and glycoproteins, heparansulfate-containing proteoglycans, actin, and RNA-associated proteins. Cell membranes of normal and malignant cells differ according to the composition of these components, which largely determines the selectivity of RNases for the latter. Different types of RNA are examined as intracellular targets of the RNases activity, evidence is presented demonstrating the possibility of exogenous RNases intervening in the process of RNA interference. The role of potassium channels, NF-kappaB-dependent.signaling pathway and various caspases in apoptosis induced by exogenous RNases is discussed. Evidence is also presented showing that the sensitivity of cells to exogenous RNases is linked to the expression of certain oncogenes, namely RAS, KIT, AML1-ETO. It is suggested that discovering the details of the mechanisms of RNases cytotoxic effect in malignant cells susceptible to their activity, will in the future serve as a foundation to developing new tools of targeted anticancer therapy.

[The C-terminus of transcription factor TnrA from Bacillus subtilis controls DNA-binding domain activity but is not required for dimerization].

The transcription factor ThrA, which belongs to the MerR transcription regulators, in Bacillus subtilis cells controls genes of nitrogen metabolism under conditions of nitrogen limitation. As all the DNA-binding proteins, it is present as a dimer in cells, but the dimerization site is still unknown. The multiple alignment of TnrA homologs from the other Bacilli allowed to identify the putative dimerization sites. Using the C-terminal truncated TnrA proteins it is established, that, in contrast to other MerR-proteins, the TnrA C-terminus does not participate in dimerization. The surface plasmon resonance has revealed that C-terminus truncations of TnrA do not inactivate its DNA-binding activity. By contrary, it increased an affinity to DNA, confirming that C-terminus controls the DNA-binding activity in a full-length TnrA.

[Composition of the oil-slime microbial community determined by analysis of the 16S rRNA gene].

Analysis of the 16S rRNA genes of the cultured microorganisms of industrial oil-slime revealed predominance (-85-90%) of the Gammaproteobacteria in the community of aerobic heterotrophs and specific oil-slime degraders. Relation of the isolated strains with members of the genera Pseudomonas, Stenotrophomonas, and Enterobacter was established. Analysis of the same gene in the total DNA from the oil-slime revealed greater microbial diversity (-20 operative taxonomic units determined by T-RFLP) than in the cultured part of the community, which included -12 different colony types. Three major restriction fragments were found, with their total area -50%. These results demonstrated the low morphological and phylogenetic diversity of the oil-slime bacterial community.

[Binase penetration into alveolar epithelial cells does not induce cell death].

Microbial ribonucleases possess a broad spectra of biological activities, demonstrating stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. The mechanisms of their penetration into the cells are not clear so far. This research is aimed to the study of Bacillus intermedius RNase (binase) penetration in alveolar lung epithelial cells--pneumocytes of type II. Using immunofluorescence we have shown for the first time have internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in pneumocytes MLE-12, which also derived from type II cells. However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. The obtained results testified the higher sensitivity of tumor cells towards binase compared with normal cells, and also showed that penetration of the enzyme into alveolar cells did not directly correlated with the cell death.

[Manganese in atherogenesis: detection, origin, and role].

The role of transition metal ions in atherogenesis is controversial; they can participate in the hydroxyl radical generation and catalyze the reactive oxygen species neutralization reaction as cofactors of antioxidant enzymes. Using EPR spectroscopy, we revealed that 70% of the samples of aorta with atherosclerotic lesions possessed superoxide dismutase activity, 100% of the samples initiated Fenton reaction and demonstrated the presence of manganese paramagnetic centers. The sodA gene encoding manganese-dependent bacterial superoxide dismutase was not found in the samples of atherosclerotic plaques by PCR using degenerate primers. The data obtained indicates the perspectives of manganese analysis as a marker element in the express diagnostics of atherosclerosis.

[Induction of the NO synthesis in lactobacilli under stress conditions].

An increase in the nitric oxide (NO) biosynthesis in Lactobacillus plantarum 8P-A3 cells takes place under strong stress influence, which leads to a considerable decrease in the microbial cell viability: heating at 70 degrees C and 80 degrees C, prolonged cultivation, toxic effect of hexylresorcinol. The factors, which do not lead to cell death, such as heating at 60 degrees C, 50 microg/ml homoserine lactone, Bacillus intermedius 7P ribonuclease (binase) in concentrations up to 300 microg/ml, do not induce NO synthesis. The activation of the NO biosynthesis in response to stress treatment evidences to universality of key-mechanisms of stress response in cells differing in the level of their organization as well as to important role of nitric oxide in them.

[The role of AmtB, GlnK and glutamine synthetase in regulation of transcription factor TnrA in Bacillus subtilis].

The nitrogen is a macroelement for all alive cells, from bacteria to animals. Although NH3/NH4+ are highly toxic to animal, they are the preferred source of nitrogen for the most microorganisms and are assimilated by glutamine synthetase in the GOGAT cycle. The nitrogen limitation triggers a number of regulatory processes and activates many genes, providing the utilizing of alternative nitrogen sources. In Bacillus subtilis the genes of nitrogen metabolism are regulated by the transcription factor TnrA. In a cells it is bound to AmtB-GlnK proteins, the interaction with Glutamine synthetase (GS) represses its DNA-binding activity. Here we show the lack of AmtB leads to the nitrogen deficiency in a cell and, consequently, the increased expression of TnrA-dependent genes. In the lack of GlnK the transcription factor TnrA is constitutive bound to GS, the TnrA activity is repressed even under nitrogen limit conditions. Apparently, the TnrA activity is subjected to permanent repression by GS. In the absence of GS, the TnrA activity is strongly higher in compare to control, even under nitrogen limitation, when GS is active. These data allow to suggest that TnrA activity is regulated by the competitive binding to GlnK and GS.

[Stress response genes expression analysis of barley Hordeum vulgare under space flight environment].

Transcriptome of barley Hordeum vulgare grown aboard International Space Station (ISS) was analyzed by means of microarray. It was revealed 500 genes with mRNA level, changed more than two folds in space environment. Among them are genes encoding stress response proteins, videlicet Heat Shock Proteins (HSP), Pathogenesis-Related Proteins (PR) and Antioxidant Proteins. Further analysis of these genes by real time PCR showed enhanced transcription level of Reactive oxygen Species (ROS) scavenging genes. The mRNA level of superoxide dismutase (sod) was 6 folds higher in space environment when compare to Earth conditions. Glutamyl transferase gene expression was enhanced 24 times in space. Transcription of catalase gene (cat) was increased 18 times and of ascorbate peroxidase was increased 3 times in space in comparison with ground control. For the first time it was shown that space flight environment may induce oxidative stress in plants.

[Isolation of Campylobacter fetus from persons with obstetric-gynecological infections].

Campylobacter fetus subspecies fetus is a cause of different obstetric-gynecological diseases. It is a first time when rate of infection with Campylobacter was studied and connection between the infection and development of chronic gynecologic diseases and pathology of labor was established. Bacteria were isolated and identified in 36.0% +/- 0.7 of studied women admitted to inpatient clinics. It was established that Campylobacter fetus subspecies fetus can cause abnormalities in placenta functions as well as different inflammatory processes during pregnancy.

[ResD-ResE two-component system positively regulates gene expression of bacilli guanyl-specific ribonucleases].

The role of the ResD-ResE two-component signal transduction system in regulation of the bacilli guanyl-specific ribonucleases genes expression was studied. The proteins with the homology to the Bacillus subtilis ResD and ResE regulatory proteins were found in all sequenced genomes of the Bacillus. Using the B. subtilis strains deficient in the genes for these proteins it was shown that the ResD-ResE signal transduction system positively regulates the expression of the genes for B. intermedius, B. pumilus, and B. thuringiensis ribonucleases in the B. subtilis host cell. The data obtained in this work indicate that regulatory system similar to the B. subtilis ResD-ResE two-component signal transduction system also functions in other representatives of the Bacillus genus.

[Inhibition of functional activity of macrophages in vitro by dimer RNase Bacillus intermedius].

The influence of cross-linked by dimethylsuberimidate dimeric RNAse from Bacillus intermedius on peritoneal rat macrophages has been investigated in vitro. It has been shown that dimeric RNase with concentrations of 0.5-40.0 mg/ml decreases the functional activities of macrophages. This is manifested in the inhibition of the phagocyte function of macrophages and suppression of the fusion of phagosomes with lysosomes. The change in the cytoplasmatic membrane surface structure induced by the dimers, which is stronger than that induced by monomers, has been demonstrated using atomic force microscopy. The role of membrane properties modification in the inhibition effect of RNase dimers on the functional activities of macrophages is discussed.

[Start codon in the serine proteinase gene from Bacillus intermedius].

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.

[Genotoxicity of Helicobacter pylori deltaPAI in DNA comet assay].

Chronic infection with Helicobacter pylori is a factor inducing development of cancer diseases. The mechanism of its action on the "host" DNA is still not clear. In present study we investigate genotoxical potential of two strains of H. pylori: wild type H. pylori P12 and PAI-deficient mutant H. pylori deltaPAI. DNA damage was detected by DNA comet assay in gastric adenocarcinoma (AGS) and epithelial adenocarcinoma (HeLa) cells under neutral conditions using Tail Moment as a quantitative parameter. It was shown, that infection of AGS and HeLa cells with both strains H. pylori at different multiplicity of infection (20-500) for 6 h and infection of AGS for 12 h did not induce DNA damage. Our results revealed a significant dose-dependent increasing of Tail Moment in the AGS cells after infection with mutant H. pylori deltaPAI for 24 h, while genotoxity of wild type H. pylori P12 under the same conditions was not observed.

[Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions: changes in morphological and physiological characteristics].

Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions results in altered morphological and physiological characteristics of the cells. M. gallisepticum populations in a complete nutrient medium contain pear-shaped vegetative cells (d approximately 0.3 microm; l approximately 0.8 microm) with pronounced polar and cytoskeleton-like structures. Such mycoplasma cells are able to induce damage in a bacterial genome, causing an SOS response of the test strain (Escherichia coli PQ37). In a starvation medium, M. gallisepticum produces nanoforms, small coccoid cells (d approximately 0.15-0.2 microm) without either polar or cytoskeleton-like structures. Unlike vegetative cells, nanoforms do not induce genome damage. Alleviation of unfavorable growth conditions results in a reversion of nanoforms to typical vegetative cells.