Colonization history of Scots pine in Eastern Europe and North Asia based on mitochondrial DNA variation

During Quaternary glaciations, the ranges of Northern Eurasia forest species periodically experienced contraction followed by subsequent re-colonizations in the interglacial intervals. However, unlike the broadleaf trees of temperate forests, taiga species seem not to have retreated fully to southern regions in unfavorable periods and possibly survived at mid-latitudes in multiple refugia. Here, we report a study of genetic variation of three mitochondrial DNA markers in 90 populations of Scots pine (Pinus sylvestris) located from Eastern Europe to Eastern Siberia. The geographic distribution of seven mitotypes demonstrated the split between western and eastern populations approximately along the 38th meridian. Genetic diversity in the western part was significantly higher than in the eastern one. Five mitotypes were western- and one eastern-specific. One mitotype was common in both regions, but in the eastern part it occurred only in the South Urals and adjacent areas. The geographic structure in the mitotype distribution supports a hypothesis of post-glacial re-colonization of the studied territory from the European and Ural refugia.     

Effective Synthesis of Fluorescently Labeled Morpholino Nucleoside Triphosphate Derivatives

Morpholino nucleoside triphosphates (A, U, G, C, T) bearing the active functional amino group tethered to morpholine residue and their fluorescently labeled derivatives were synthesized. All compounds were characterized by ¹H, ¹³C, and ³¹P NMR, and mass spectrometry. A possibility of using fluorescently labeled morpholino nucleoside triphosphates as chain terminators in DNA sequencing is discussed.     

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.     

Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.     

Vaccine production in Neurospora crassa

We have chosen to use the filamentous fungus Neurospora crassa to produce subunit vaccines. Here we describe the production and purification of Influenza hemagglutinin and neuraminidase antigens in N. crassa. The N. crassa system used by Neugenesis offers many advantages over other systems for production of recombinant protein. In contrast to mammalian cell culture, N. crassa can be grown in a rapid and economic manner, generating large amounts of recombinant protein in simple, defined medium. Vaccines, therefore, can be produced more rapidly and at lower cost than conventional cell culture or egg-based systems. This has important applications to tailoring the seasonal vaccine supply and responding to new pandemics.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

Inhibition of Cation Channels in Human Erythrocytes by Spermine

In erythrocytes, spermine concentration decreases gradually with age, which is paralleled by increases of cytosolic Ca2+ concentration, with subsequent cell shrinkage and cell membrane scrambling. Cytosolic Ca2+ was estimated from fluo-3 fluorescence, cell volume from forward scatter, cell membrane scrambling from annexin V binding and cation channel activity with whole-cell patch-clamp in human erythrocytes. Extracellular spermine exerted a dual effect on erythrocyte survival. At 200 μM spermine blunted the increase of intracellular Ca2+, cell shrinkage and annexin V binding following 48 h exposure of cells at +37 °C. In contrast, short exposure (10-30 min) of cells to 2 mM spermine was accompanied by increased cytosolic Ca2+ and annexin binding. Intracellular addition of spermine at subphysiological concentration (0.2 μM) significantly decreased the conductance of monovalent cations (Na+, K+, NMDG+) and of Ca2+. Moreover, spermine (0.2 μM) blunted the stimulation of voltage-independent cation channels by Cl? removal. Spermine (0.2 and 200 μM) added to the extracellular bath solution similarly inhibited the cation conductance in Cl?-containing bath solution. The effect of 0.2 μM spermine, but not the effect of 200 μM, was rapidly reversible. Acute addition (250 μM) of a naphthyl acetyl derivative of spermine (200 μM) again significantly decreased basal cation conductance in NaCl bath solution and inhibited voltage-independent cation channels. Spermine is a powerful regulator of erythrocyte cation channel cytosolic Ca2+ activity and, thus, cell survival.     

Mass Spectrometry Defines the Stoichiometry of Ribosomal Stalk Complexes across the Phylogenetic Tree

The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution.Ribosomes universally translate the genetic code into proteins. They consist of two asymmetric subunits between which mRNA is decoded and amino acids are added to a growing peptide chain. On the large subunit there is a noticeable protrusion, observable by electron microscopy, known as the stalk complex (also denoted as L8) (1). This complex is involved in the binding and orientation of translation factors and exists with variable composition throughout all three domains of life. In bacteria we and others have shown previously that it is composed of either two or three dimers of the protein L12 (termed L7 when N-acetylated) attached to a single copy of the scaffolding protein L10 (2, 3). These assemblies of stalk proteins, either L10(L7/L12)₄ or L10(L7/L12)₆, are referred to as pentameric or heptameric stalk complexes hereafter. In eukaryotes there is an identical arrangement for the stalk complex but of unrelated proteins with no sequence homology to L10/L12. In this case P0 is the L10 equivalent scaffolding protein, and two different but related proteins (P1 and P2) take the place of L12 (nomenclature according to Ref. 4). In plants P3 occurs in addition to P1 and P2 (5). The P-proteins are named after their propensity for phosphorylation when attached to the ribosome. In yeast, but not in higher eukaryotes, P1 and P2 have both evolved into two α and β proteins (6). In archaea the stalk complex constituents, although named L10 and L12, share sequence homology with the P-proteins (7). L12 and its P1/P2 counterparts are the only ribosomal proteins that have acidic pI values, do not interact directly with rRNA, and are present in multiple copies on the ribosome.We have shown previously that by applying a combination of MS and tandem MS approaches to intact MDa particles such as ribosomes we can obtain precise information, especially regarding the overall stoichiometry and composition of different stalk complexes (2, 8–11). This is due to the fact that the stalk complex is readily observable in mass spectra of intact ribosomes (Fig. 1). This is in contrast to the situation in most crystallographic investigations where the dynamics and heterogeneity of the stalk prevent its high resolution structure determination. Because it dissociates readily in the mass spectrometer as an intact, oligomeric species we can exploit this property, and previously we have shown that for the mesophilic bacteria Bacillus subtilis and Escherichia coli the stalk complex is unequivocally a pentamer comprising L10 and four copies of L7/L12 (2). For ribosomes of the extreme thermophilic bacteria Thermus thermophilus and Thermatoga maritima we demonstrated that the stalk complex was in fact a heptamer, rather than the anticipated pentamer, comprising one L10 and six copies of L12 (2). This led us to speculate that heptameric stalk complexes were likely to be present on ribosomes from species growing at higher temperatures. Although classification of the species has changed since their discovery, the generally accepted consensus at present separates them into four distinct categories based on their optimal growth temperatures (OGTs)¹: thermophiles (>55 °C), moderate thermophiles (>65 °C), extreme thermophiles (>75 °C), and hyperthermophiles (>85 °C) (12).Open in a separate windowFig. 1.Mass spectrum of intact ribosomes from T. thermophilus. The mass spectrum of intact ribosomes from T. Thermophilus in 1 m ammonium acetate showing well resolved charge states for the 30 S subunit at 14,000–18,000 m/z and 70 S subunit at 25,000–28,000 m/z is shown. The 50 S subunit lacks charge state resolution probably because of the heterogeneity associated with the partial loss of the stalk complex, which appears as a series of well resolved charge states at 4000–6000 m/z. Individual ribosomal proteins and tRNA are observed at the lower m/z regions of the spectrum.Here we report on investigations of ribosome stalk compositions from a range of species from all three domains of life, including examples with different OGTs. For bacteria we extended our earlier findings by including in our analysis the thermophile Bacillus stearothermophilus (OGT 55 °C) and compared it with the extreme thermophile Thermus aquaticus (OGT of 70–75 °C). For eukaryotes we compared ribosomes from three animals (brine shrimp, silkworm, and rabbit) and a thermophilic red alga, Galdieria sulphuraria, the eukaryote with the highest known OGT of 56 °C. Within the archaea we targeted the mesophilic methanogens Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri with OGTs of 35, 35–40, and 37 °C, respectively. Methanogens, capable of producing methane, are the most common and widely dispersed of the archaea. M. maripaludis is a model species among the methanogenic archaea. M. barkeri, unlike most methanogens, which only use carbon dioxide, is able to ferment a variety of carbon sources (13). Surprisingly stalk complexes of different stoichiometries were present simultaneously on ribosomes from these mesophilic archaea. Our MS study of ribosomes isolated from M. vannielii and M. barkeri harvested at different stages of growth showed that the ratio of pentameric versus heptameric stalks changes during cell growth with pentameric species being predominant at the early stages and during the lag phase and the proportion of heptameric complexes increasing toward the latter stages of cell growth. Overall therefore in this study we widened our previous MS investigations into ribosomal stalk complexes and targeted species with different OGTs within bacterial, eukaryotic, and archaeal domains.     

X-ray structural studies of the fungal laccase from Cerrena maxima

Laccases are members of the blue multi-copper oxidase family. These enzymes oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and following the transfer of four electrons is reduced to two molecules of water. The X-ray structure of a laccase from Cerrena maxima has been elucidated at 1.9 A resolution using synchrotron data and the molecular replacement technique. The final refinement coefficients are Rcryst = 16.8% and Rfree = 23.0%, with root mean square deviations on bond lengths and bond angles of 0.015 A and 1.51 degrees , respectively. The type 1 copper centre has an isoleucine residue at the axial position and the "resting" state of the trinuclear centre comprises a single oxygen (OH) moiety asymmetrically disposed between the two type 3 copper ions and a water molecule attached to the type 2 ion. Several carbohydrate binding sites have been identified and the glycan chains appear to promote the formation of well-ordered crystals. Two tyrosine residues near the protein surface have been found in a nitrated state.