Composition,diversity and distribution of microbenthos across the intertidal zones of Ryazhkov Island (the White Sea)

The composition and distribution of the main unicellular eukaryotic groups (diatom algae, ciliates, dinoflagellates (DF), other phototrophic (PF) and heterotrophic flagellates (HF)) were investigated in sandy sediments at five stations allocated across the tidal sheltered beach of the White Sea. Overall, 75 diatoms, 98 ciliates, 16 DF, 3 PF and 34 HF species were identified; some are new records for the White Sea. Common species for each group are illustrated. Diatoms and ciliates showed high alpha-diversity (species richness per sample), whereas flagellates were characterized by high beta-diversity (species turnover across the intertidal flat). Each group demonstrated its own spatial pattern that was best matched with its own subset of abiotic variables, reflecting group-specific responses to environmental gradients. Species richness increased from the upper intertidal zone seaward for ciliates but decreased for HF, whereas autotrophs showed a relatively uniform pattern with a slight peak at the mid-intertidal zone. Across the littoral zone, all groups showed distinct compositional changes; however, the position of the boundary between “upper” and “lower” intertidal communities varied among groups. Most of the species found at Ryazhkov Island are known from many other regions worldwide, indicating a wide geographic distribution of microbial eukaryotic species.     

The biomolecule ubiquinone exerts a variety of biological functions

The chemistry of ubiquinone allows reversible addition of single electrons and protons. This unique property is used in nature for aerobic energy gain, for unilateral proton accumulation, for the generation of reactive oxygen species involved in physiological signaling and a variety of pathophysiological events. Since several years ubiquinone is also considered to play a major role in the control of lipid peroxidation, since this lipophilic biomolecule was recognized to recycle alpha-tocopherol radicals back to the chain-breaking form, vitamin E. Ubiquinone is therefore a biomolecule which has increasingly focused the interest of many research groups due to its alternative pro- and antioxidant activity. We have intensively investigated the role of ubiquinone as prooxidant in mitochondria and will present experimental evidences on conditions required for this function, we will also show that lysosomal ubiquinone has a double function as proton translocator and radical source under certain metabolic conditions. Furthermore, we have addressed the antioxidant role of ubiquinone and found that the efficiency of this activity is widely dependent on the type of biomembrane where ubiquinone exerts its chain-breaking activity.     

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.     

Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.     

Human factors and pathways essential for mediating epigenetic gene silencing

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.