Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

Background

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.

Results

We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.

Conclusions

The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.     

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.     

Association of social jetlag experienced by young northerners with their appetite after having breakfast

Young residents of the European North of Russia with normal body weight (n = 66) were divided into three groups, SJL ≤ 1 h, 1 < SJL ≤ 2 h, and SJL > 2 h, using the Munich ChronoType Questionnaire. Participants consumed a breakfast consisting of pizza ad libitum and rated their appetite sensations using visual analog scales. Participants in three SJL groups consumed the same amount of the ad libitum meal and reported similar levels of satiety and fullness right after food intake (intra-meal satiation). However, participants with SJL > 2 h were found to feel significantly hungrier in comparison with participants with SJL ≤ 1 h. In addition, participants with SJL > 2 h reported a diminished satisfaction with food consumption (inter-meal satiety) during 120 min after test breakfast. Thus, the study demonstrates that SJL appears to be associated with disturbance of appetite regulation in young northerners with normal body weight.     

Diversity of the major capsid genes (g23) of T4-like bacteriophages in the eutrophic Lake Kotokel in East Siberia, Russia

Numerous studies revealed high diversity of T4-like bacteriophages in various environments, but so far, little is known about T4-like virus diversity in freshwater bodies, particularly in eutrophic lakes. The present study was aimed at elucidating molecular diversity of T4-like bacteriophages in eutrophic Lake Kotokel located near Lake Baikal by partial sequencing of the major capsid genes (g23) of T4-like bacteriophages. The majority of g23 fragments from Lake Kotokel were most similar to those from freshwater lakes and paddy fields. Despite the proximity and direct water connection between Lake Kotokel and Lake Baikal, g23 sequence assemblages from two lakes were different. UniFrac analysis showed that uncultured T4-like viruses from Lake Kotokel tended to cluster with those from the distant lake of the same trophic status. This fact suggested that the trophic conditions affected the formation of viral populations, particularly of T4-like viruses, in freshwater environments.     

Regeneration processes of a boreal forest in Kamchatka with special reference to the contribution of sprouting to population maintenance

We studied regeneration patterns of three tree species Picea ajanensis, Betula platyphylla and Populus tremula from 1998 to 2000 in the Central Depression of the Kamchatka Peninsula. We paid special attention to the contribution of sprouting to their regeneration. P. ajanensis was the only species that regenerated by seedling. In a 40 × 40 m study plot, the density of P. ajanensis saplings < 2.0 cm in diameter at basal area (DBH) was 1132, and this was the highest among the three species studied. The number of saplings 2 cm in DBH declined sharply with size class. The spatial distribution of P. ajanensis saplings (< 2 cm in DBH) showed a significant positive correlation with that of adult trees and a negative correlation with that of gaps. These trends were not changed after re-measurement in 2000, although nearly half of the juveniles had died or been injured during the two years. These results suggest that small Picea saplings prefer habitats under the canopy of adult trees rather than in gaps for establishment. Most small individuals of B. platyphylla were produced from sprouts. The number of saplings in the smallest size class (< 2 cm in DBH) was much less than that of P. ajanensis, although the number of larger individuals did not decrease remarkably. The spatial distribution of B. platyphylla saplings showed a positive correlation with that of adult trunks and a negative correlation with that of canopy trees of P. ajanensis. These results suggest an effective contribution of sprouts to the regeneration of B. platyphylla. P. tremula was the only species that could invade big gaps and produce many root suckers efficiently. There were 181 suckers of P. tremula in the smallest size class (< 2 cm in DBH) in the study plot, although the number of saplings 2 cm in DBH declined abruptly. The spatial distribution of saplings of this species showed a slight positive correlation with that of gaps, and negative correlation with that of adult trees of B. platyphylla, P. ajanensis, and P. tremula. The root suckering strategy of P. tremula might be adaptive under severe conditions in high-latitude regions. Our data suggest, however, that it does not necessarily contribute to regeneration in mature forests. The three component species in this forest did not seem to utilize canopy gaps for regeneration; we suggest that gap dynamics do not work in this forest. The sparse canopy, which is a typical character of forests in high-latitude regions, might be a consequence of high mortalities of seedlings and root suckers inside gaps.     

Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like cells to myocardial scar formation

Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A_2B adenosine receptors on mouse cardiac Sca1⁺CD31⁻ mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A_2B receptor signaling regulates the transition of Sca1⁺CD31⁻ cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFβ1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1⁺CD31⁻ cells. Stimulation of A_2B receptors attenuated TGFβ1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A_2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1⁺CD31⁻ cells remained significantly higher in wild type compared with A_2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1⁺CD31⁻ cells to the accumulation of αSMA-expressing cells after infarction and implicated A_2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9410-y) contains supplementary material, which is available to authorized users.     

Expression pattern of Filamin-240 in Drosophila blood cells

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.     

Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.