HybF, a zinc-containing protein involved in NiFe hydrogenase maturation

HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a K(D) of 1.87 microM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.     

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.     

Ultra‐Conserved Elements and morphology reciprocally illuminate conflicting phylogenetic hypotheses in Chalcididae (Hymenoptera,Chalcidoidea)

Recent technical advances combined with novel computational approaches have promised the acceleration of our understanding of the tree of life. However, when it comes to hyperdiverse and poorly known groups of invertebrates, studies are still scarce. As published phylogenies will be rarely challenged by future taxonomists, careful attention must be paid to potential analytical bias. We present the first molecular phylogenetic hypothesis for the family Chalcididae, a group of parasitoid wasps, with a representative sampling (144 ingroups and seven outgroups) that covers all described subfamilies and tribes, and 82% of the known genera. Analyses of 538 Ultra‐Conserved Elements (UCEs) with supermatrix (RAx ML and IQTREE) and gene tree reconciliation approaches (ASTRAL, ASTRID) resulted in highly supported topologies in overall agreement with morphology but reveal conflicting topologies for some of the deepest nodes. To resolve these conflicts, we explored the phylogenetic tree space with clustering and gene genealogy interrogation methods, analyzed marker and taxon properties that could bias inferences and performed a thorough morphological analysis (130 characters encoded for 40 taxa representative of the diversity). This joint analysis reveals that UCEs enable attainment of resolution between ancestry and convergent/divergent evolution when morphology is not informative enough, but also shows that a systematic exploration of bias with different analytical methods and a careful analysis of morphological features is required to prevent publication of artifactual results. We highlight a GC content bias for maximum‐likelihood approaches, an artifactual mid‐point rooting of the ASTRAL tree and a deleterious effect of high percentage of missing data (>85% missing UCEs) on gene tree reconciliation methods. Based on the results we propose a new classification of the family into eight subfamilies and ten tribes that lay the foundation for future studies on the evolutionary history of Chalcididae.     

Social inequalities and mortality in europe - results from a large multi-national cohort

Background

Socio-economic inequalities in mortality are observed at the country level in both North America and Europe. The purpose of this work is to investigate the contribution of specific risk factors to social inequalities in cause-specific mortality using a large multi-country cohort of Europeans.

Methods

A total of 3,456,689 person/years follow-up of the European Prospective Investigation into Cancer and Nutrition (EPIC) was analysed. Educational level of subjects coming from 9 European countries was recorded as proxy for socio-economic status (SES). Cox proportional hazard model''s with a step-wise inclusion of explanatory variables were used to explore the association between SES and mortality; a Relative Index of Inequality (RII) was calculated as measure of relative inequality.

Results

Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest (HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78). The risk reduction was attenuated by 7% in men and 3% in women by the introduction of smoking and to a lesser extent (2% in men and 3% in women) by introducing body mass index and additional explanatory variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women). Social inequalities were highly statistically significant for all causes of death examined in men. In women, social inequalities were less strong, but statistically significant for all causes of death except for cancer-related mortality and injuries.

Discussion

In this European study, substantial social inequalities in mortality among European men and women which cannot be fully explained away by accounting for known common risk factors for chronic diseases are reported.     

Variable δ15N Diet-Tissue Discrimination Factors among Sharks: Implications for Trophic Position,Diet and Food Web Models

The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ¹⁵N diet-tissue discrimination factors (∆¹⁵N). As ∆¹⁵N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆¹⁵N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆¹⁵N values for large and/or endangered species, our objective was to conduct an assessment of a range of reported ∆¹⁵N values that can hypothetically serve as surrogates for describing the predator-prey relationships of four shark species that feed on prey from different trophic levels (i.e., different mean δ¹⁵N dietary values). Overall, the most suitable species-specific ∆¹⁵N values decreased with increasing dietary-δ¹⁵N values based on stable isotope Bayesian ellipse overlap estimates of shark and the principal prey functional groups contributing to the diet determined from stomach content analyses. Thus, a single ∆¹⁵N value was not supported for this speciose group of marine predatory fishes. For example, the ∆¹⁵N value of 3.7‰ provided the highest percent overlap between prey and predator isotope ellipses for the bonnethead shark (mean diet δ¹⁵N = 9‰) whereas a ∆¹⁵N value < 2.3‰ provided the highest percent overlap between prey and predator isotope ellipses for the white shark (mean diet δ¹⁵N = 15‰). These data corroborate the previously reported inverse ∆¹⁵N-dietary δ¹⁵N relationship when both isotope ellipses of principal prey functional groups and the broader identified diet of each species were considered supporting the adoption of different ∆¹⁵N values that reflect the predators’ δ¹⁵N-dietary value. These findings are critical for refining the application of stable isotope modeling approaches as inferences regarding a species’ ecological role in their community will be influenced with consequences for conservation and management actions.     

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression

The glycosylphosphatidylinositol (GPI) - anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts. J. Cell. Physiol. 180:225–235, 1999. © 1999 Wiley-Liss, Inc.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.