Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.     

Comparison of Bacterioneuston and Bacterioplankton Dynamics during a Phytoplankton Bloom in a Fjord Mesocosm

The bacterioneuston is the community of Bacteria present in surface microlayers, the thin surface film that forms the interface between aquatic environments and the atmosphere. In this study we compared bacterial cell abundances and bacterial community structures of the bacterioneuston and the bacterioplankton (from the subsurface water column) during a phytoplankton bloom mesocosm experiment. Bacterial cell abundance, determined by flow cytometry, followed a typical bacterioplankton response to a phytoplankton bloom, with Synechococcus and high-nucleic acid content (HNA) bacterial cell numbers initially falling, probably due to selective protist grazing. Subsequently HNA and low-nucleic acid content bacterial cells increased in abundance, but Synechococcus cells did not. There was no significant difference between bacterioneuston and bacterioplankton cell abundances during the experiment. Conversely, distinct and consistent differences between the bacterioneuston and the bacterioplankton community structures were observed. This was monitored simultaneously by Bacteria 16S rRNA gene terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis. The conserved patterns of community structure observed in all of the mesocosms indicate that the bacterioneuston is distinctive and nonrandom.Determining and understanding both spatial and temporal patterns in bacterioplankton community structure are a core aim of marine microbial ecology (15). Distributions of bacterioplankton over space and time can be correlated to environmental parameters, and subsequent links can therefore be made to ecosystem function. A broad range of spatial studies made on macro- (34), meso- (20), and microscales (27) have shown clear patterns in distribution of the bacterioplankton.The sea surface microlayer is part of the air-sea interface and is generally considered to be the top 1 mm or less of the ocean (26). Surface microlayers have a fundamental role in regulating transport processes between the ocean and the atmosphere (26) and are often referred to as the neuston (28, 31). For more than 25 years it has been hypothesized that the sea surface microlayer is a hydrated gelatinous layer (40) that contains surface-active organic compounds such as carbohydrates, proteins, lipids, and humic substances in relatively high concentrations (17, 45, 48). Recently, gel-like transparent expolymer particles (TEP) have been shown to be enriched in the surface microlayer, supporting the concept of a gelatinous interfacial layer (46).Bacteria present in surface microlayers or the neuston are regarded as the bacterioneuston. There are relatively few studies which have directly compared the community structure of the bacterioneuston with that of the cognate subsurface (bacterioplankton) in the marine environment. Analysis of Bacteria 16S rRNA gene clone libraries constructed using DNA isolated from surface microlayer and subsurface water (<1 m) samples from the North Sea revealed that the bacterioneuston was dominated by two operational taxonomic units which accounted for 81% of clones analyzed (13). Community structure profiling using denaturing gradient gel electrophoresis (DGGE) of the bacterioneuston at three sites around Oahu Island in the Pacific Ocean showed that the bacterioneuston forms consistent and distinct community structures. Conversely, the archaeal community structure of the same samples using Archaea 16S rRNA gene DGGE analysis did not show the same surface microlayer-specific response, indicating that bacteria and archaea respond to their environment in fundamentally different ways in the neuston (7).Other studies have, however, reported no consistent differences between the bacterioneuston and the bacterioplankton. Samples collected from two separate sites in the Mediterranean Sea were analyzed using single-strand conformation polymorphism of Bacteria 16S rRNA genes (1). The authors did not report any significant differences between the surface microlayer and subsurface samples using this community profiling method.Nonmarine studies of the bacterioneuston and Archaea communities in estuarine (10) and freshwater (5, 19) environments have also shown distinct microbial community structures present in the surface microlayer compared to those in subsurface water ≤1 m below.Recurring phytoplankton blooms are a key feature of coastal waters and strongly influence bacterioplankton community structure and succession (4, 14, 38). Phytoplankton blooms stimulate the bacterioplankton by the release of dissolved organic matter (22) or affect bacterioplankton negatively by direct competition for resources (6). Bacterioplankton community structure may also be influenced by grazing flagellates or viral lysis (47).Mesocosm experiments have been used to study plankton ecology for many decades (33). Mesocosms facilitate study of the effects of key environmental parameters, such as temperature, on plankton communities and allow the succession of natural plankton communities that resemble those found in the marine environment (11). The enclosed water mass means that experiments can be designed which manipulate physicochemical parameters to observe biological effects. Furthermore, with replicated mesocosms, the data collected can be analyzed with statistics rigorously. In this study we monitored the dynamics of the bacterioneuston and the bacterioplankton in mesocosms of fjord surface water during an artificially induced phytoplankton bloom and compared bacterial abundances and bacterial community structures in the surface microlayer and subsurface water.     

The anatomical and compositional basis of leaf mass per area

Leaf dry mass per unit leaf area (LMA) is a central trait in ecology, but its anatomical and compositional basis has been unclear. An explicit mathematical and physical framework for quantifying the cell and tissue determinants of LMA will enable tests of their influence on species, communities and ecosystems. We present an approach to explaining LMA from the numbers, dimensions and mass densities of leaf cells and tissues, which provided unprecedented explanatory power for 11 broadleaved woody angiosperm species diverse in LMA (33–262 g m^?2; R² = 0.94; P < 0.001). Across these diverse species, and in a larger comparison of evergreen vs. deciduous angiosperms, high LMA resulted principally from larger cell sizes, greater major vein allocation, greater numbers of mesophyll cell layers and higher cell mass densities. This explicit approach enables relating leaf anatomy and composition to a wide range of processes in physiological, evolutionary, community and macroecology.     

Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1β

Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808–819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1β constructs by measuring ¹⁵N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τ_c-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.     

Parent–environmental interactions shape acoustic signatures in tree swallows: a cross‐fostering experiment

Acoustic signatures are common components of avian vocalizations and are important for the recognition of individuals and groups. The proximate mechanisms by which these signatures develop are poorly understood, however. The development of acoustic signatures in nestling birds is of particular interest, because high rates of extra‐pair paternity or egg dumping can cause nestlings to be unrelated to at least one of the adults that are caring for them. In such cases, nestlings might conceal their genetic origins, by developing acoustic signatures through environmental rather than genetic mechanisms. In a cross‐fostering experiment with tree swallows Tachycineta bicolor, we investigated whether brood signatures of nestlings that were about to fledge were attributable to their genetic/maternal origins or to their rearing environment. We found that the calls of cross‐fostered nestlings did not vary based on their genetic/maternal origin, but did show some variation based on their rearing environment. Control nestlings that were not swapped, however, showed stronger brood signatures than either experimental group, suggesting that acoustic signatures develop through an interaction between rearing environment and genetic/maternal effects.     

Iron-Doping of Copper Oxide Nanoparticles Lowers Their Toxic Potential on C6 Glioma Cells

Neurochemical Research - Copper oxide nanoparticles (CuO-NPs) are well known for their cytotoxicity which in part has been attributed to the release of copper ions from CuO-NPs. As iron-doping has...     

Sex differentiation in seasonal distribution of the starry smooth-hound Mustelus asterias

This mark-recapture study of starry smooth-hound Mustelus asterias tagged during the summer months near the Dutch coast demonstrates a large-scale spatial sex differentiation in their circannual migration patterns and small-scale spatial sex differentiation during summer. Overwintering occurs in the North Sea, English Channel and Bay of Biscay, with significantly more males in the Northern North Sea and more females in the Bay of Biscay. During summer, sheltered sea arms off the Dutch coast were almost exclusively used by adult females. In subsequent summers post-release, both sexes were mostly confined to the Southern North Sea, suggesting philopatry.