Proapoptotic action of alpha-tocopheryl succinate in rat thymocytes is conditioned by the inhibition of mitochondrial succinate dehydrogenase

It has been established that alpha-tocopheryl succinate in concenrations 10-100 microM inhibits in a dose-dependent manner the viability of primary culture rats thymocytes and causes the DNA internucleosomal degradation that testifies to apoptotic way of thymocytes destruction. These effects were accompanied by an enhanced production of intracellular superoxide. This is the first report demonstrating that apoptosis induced by alpha-tocopheryl succinate was accompanied by a dose-dependent inhibition of mitochondrial succinate dehydrogenase. Known apoptosis inducers--actinomicin D, staurosporin and hydrogen peroxide decreased a cell survival but neither induced any significant changes in succinate dehydrogenase activity which means that this effect is characteristic only of alpha-tocopheryl succinate and seems to be an important event triggering the apoptotic response by it. It was supposed that alpha-tocopheryl succinate might appear as a pseudosubstrate for mitochondrial succinate dehydrogenase leading to its inhibition, dysfunction of the mitochondrial electron transport chain, generation of reactive oxygen species and iduction of apoptosis.     

Formation of “Nonculturable” dormant forms of the phytopathogenic enterobacterium <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Reversible transition of the phytopathogenic gram-negative bacterium Erwinia carotovora, subsp. atroseptica, strain SCRI1043, to a dormant state was demonstrated; it was associated with a complete loss of cell ability to form colonies on the standard medium, i.e., with acquiring “non-culturability”. Entering of Erwinia cells to a nonculturable state occurred after long-term incubation (100–150 days) of the stationary-phase cell suspensions in either a fresh complete medium or in the carbon-free mineral medium or treatment with a chemical analogue of microbial anabiosis autoinducers (4 × 10⁻⁴ M of C₁₂-alkylhydroxybenzene, AHB). However, confocal laser microscopy of the cells stained with the Live/Dead BacLight kit revealed that the majority of E. carotovora cells (90%) from long-incubated suspensions retained membrane integrity. In these suspensions, round cells of smaller size prevailed, with the envelope, containing an electron-dense outer layer and an underlying layer of lower density; the cytoplasm was coarse-granulated. Detection of “nonculturable” E. carotovora cells by quantitative real-time PCR analysis (Q-PCR) with specific primers by using standard procedures of sample preparation was shown to be inefficient. A special procedure including cell washing from the incubation medium in the absence of growth stimulation was developed, which promoted recovery of the colony-forming ability of the cells (up to 10% of the initial CFU number) and improved cell detection by Q-PCR from the number of genomic copies. The results provided further insight into the ways of long-term survival of phytopathogenic bacteria under environmental changes and carbon starvation.     

The decline in antibodies to hepatitis C virus during antiviral therapy

The goal of the study was to analyze B-cell response to hepatitis C virus during antiviral therapy among responders and non-responders. The content of antibodies to individual structural and non-structural HCV proteins was investigated during two years in three groups of patients: initial responders, non-responders and a reference group (without therapy). Treated patients in all groups exhibited the decrease in antibodies to analyzed HCV proteins, but with different patterns. The first statistically significant differences in the decline of the virus-specific antibodies between initial responders and non-responders were observed within the first three months after the beginning of therapy. Some treated patients demonstrated the decrease in antibody levels to HCV proteins after the end of therapy.     

2'-O-methyl-modified anti-MDR1 fork-siRNA duplexes exhibiting high nuclease resistance and prolonged silencing activity

The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic-producing strains, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.