Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

Detection of concealed "illegitimate" nuclei in tetrad analysis of the diploid progeny of heterokaryons in Saccharomyces cerevisiae

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiae were continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1 mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an "illegitimate" hybrid after fusion. An extraneous spore (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.     

Deuterium Oxide Enhances <Emphasis Type="Italic">Escherichia coli</Emphasis> SOS Response Induced by Genotoxicants

It has been demonstrated that deuterium oxide enhances the SOS response of Escherichia coli cells induced by chemical genotoxicants and mutagens. This demonstrates that the heavy nonradioactive hydrogen isotope deuterium can be considered to be a comutagen.     

Shell Microstructure of <Emphasis Type="Italic">Rhynchonella loxiae</Emphasis> Fischer (Brachiopoda,Rhynchonellida) from the Upper Volgian of Moscow Region

The shell microstructure of Late Jurassic rhynchonellids of the family Rhynchonellidae from the boreal basin of the Russian Platform is studied for the first time. The studied rhynchonellids differ in the shell microstructure from the Late Jurassic and Early Cretaceous rhynchonellids of the families Cyclothyrididae, Praecyclothyrididae, Basiliolidae, and Norellidae from the warm-water Mediterranean basin. The revealed peculiarities of shell structure of Rhynchonella loxiae (type species of Rhynchonella) may be added to the diagnosis of Rhynchonella and should be considered in characterizing the family Rhynchonellidae.     

Inhibition of Mycobacterium tuberculosis strains H37Rv and MDR MS-115 by a new set of C5 modified pyrimidine nucleosides

Two sets of pyrimidine nucleoside derivatives bearing extended alkyloxymethyl or alkyltriazolidomethyl substituents at position 5 of the nucleobase were synthesized and evaluated as potential antituberculosis agents. The impact of modifications at 3′- and 5′-positions of the carbohydrate moiety on the antimycobacterial activity and cytotoxicity was studied. The highest effect was shown for 5-dodecyloxymethyl-2′-deoxyuridine, 5-decyltriazolidomethyl-2′-deoxyuridine, and 5-dodecyltriazolidomethyl-2′-deoxycytidine. They effectively inhibited the growth of two Mycobacterium tuberculosis strains in vitro, laboratory H37Rv (MIC₉₉ = 20, 10, and 20 μg/mL, respectively) and clinical MDR MS-115 resistant to five top antituberculosis drugs (МIC₉₉ = 50, 10, and 10 μg/mL, respectively).     

The use of a novel reagent, Cu-FL, for determination of NO synthesis stimulated by the medicinal leech salivary cell secretion in human endothelial cell (HUVEC) and rat cardiomyocyte cultures

Using a novel NO-specific reagent, the complex of Cu²⁺ with a fluorescein derivative (Cu-FL), stimulation of NO production by the medicinal leech salivary cell secretion (SCS) has been demonstrated for the first time in cultures of human endothelial cells (HUVEC) and rat cardiomyocytes (RCM). NO was detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in the culture fluid by the fluorescence method. SCSstimulated NO synthesis in HUVEC but not in RCM cells was accompanied by NO release into the intercellular space thus determining its subsequent distribution. Localization of intracellular NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In the endothelial cells SCS-activated nitrosylation processes, estimated by the increase of nitrite-ion content in the culture medium. It is therefore important to use Cu-FL, rather than Griss-reagent, during the first hour of analysis of NO synthesis. It is possible that the NO-depended mechanism of the SCS action on endothelial cells may be a factor responsible for the positive effect of SCS during hirudotheraphy.     

Effect of environment on the adaptive abilities of fry roach Rutilus rutilus (L.) (Cyprinidae) during early ontogenesis

In recent years a considerable decrease in the abundance of predatory fishes has been observed in spawning tributaries of the Rybinsk Reservoir caused by their intensive catching. The lack of encounters with predators before the downstream migration of young fish hampers the development of necessary skills of defensive behavior in the absence of predation experience. As a result, after downstream migration, the juveniles are incapable of adapting to the predation pressure in the reservoir and are subjected to intensive elimination. The adaptive potential of the roach Rutilus rutilus L. was experimentally studied in siblings raised from the larvae to the late fry stage both in the presence and absence of a predator. It has been found that the fry that was raised under different conditions differed in their adaptive potential in new environment conditions.     

Comparative Genomic Analysis of Mycobacterium tuberculosis Drug Resistant Strains from Russia

Tuberculosis caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains is a growing problem in many countries. The availability of the complete nucleotide sequences of several MTB genomes allows to use the comparative genomics as a tool to study the relationships of strains and differences in their evolutionary history including acquisition of drug-resistance. In our work, we sequenced three genomes of Russian MTB strains of different phenotypes – drug susceptible, MDR and XDR. Of them, MDR and XDR strains were collected in Tomsk (Siberia, Russia) during the local TB outbreak in 1998–1999 and belonged to rare KQ and KY families in accordance with IS6110 typing, which are considered endemic for Russia. Based on phylogenetic analysis, our isolates belonged to different genetic families, Beijing, Ural and LAM, which made the direct comparison of their genomes impossible. For this reason we performed their comparison in the broader context of all M. tuberculosis genomes available in GenBank. The list of unique individual non-synonymous SNPs for each sequenced isolate was formed by comparison with all SNPs detected within the same phylogenetic group. For further functional analysis, all proteins with unique SNPs were ascribed to 20 different functional classes based on Clusters of Orthologous Groups (COG). We have confirmed drug resistant status of our isolates that harbored almost all known drug-resistance associated mutations. Unique SNPs of an XDR isolate CTRI-4^XDR, belonging to a Beijing family were compared in more detail with SNPs of additional 14 Russian XDR strains of the same family. Only type specific mutations in genes of repair, replication and recombination system (COG category L) were found common within this group. Probably the other unique SNPs discovered in CTRI-4^XDR may have an important role in adaptation of this microorganism to its surrounding and in escape from antituberculosis drugs treatment.     

Interaction of 23S ribosomal RNA helices 89 and 91 of Escherichia coli contributes to the activity of IF2 but is insignificant for elongation factors functioning

The non-canonical base-pair C2475/G2529 joins helices 89 and 91 of the 23S rRNA in the large subunit of E. coli ribosomes. These nucleotides are located at the "crossroads" between the peptidyl transferase center, the sarcin-ricin loop and the GTPase-associated center. We probed the functional role of nucleotides C2475/G2529 by the mutations C2475G, C2475G/G2529C and deltaA2471/U2479 of 23S rRNA. All these mutations had no influence on the elongation factors activity but had different effects on the cell growth, 23S rRNA conformation and translation initiation. C2475G/G2529C and C2475G mutations led to more or less substantial decrease in IF2.GDPNP binding to the ribosomes, and IF2-assisted initiation complex formation. Ribosome-dependent GTPase activity of IF2 was enhanced by both C2475G/G2529C and C2475G mutations. Mutation deltaA2471/U2479 has no influence on IF2.GDPNP binding to the ribosome, but reduces IF2-dependent formation of initiation complex and the ribosome-dependent GTPase activity. Thus, the contact between helices 89 and 91 is important for efficient IF2 functioning in translation initiation.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.