Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

On the ATP-Dependent Activation of the Radical Enzyme (R)-2-Hydroxyisocaproyl-CoA Dehydratase

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 ? resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.     

Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry

Introduction

Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA.

Methods

Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis.

Results

CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis.

Conclusions

In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.     

The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.

The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an Deltaost3Deltaost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a approximately 240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.     

Capitalizing on multi-element interactions through bal-anced nutrition——A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Peripartum Cardiomyopathy Presenting With Ventricular Tachycardia: A Rare Presentation

A 25-year-old previously asymptomatic pregnant woman at 36 weeks'' gestation was noticed to have repetitive monomorphic ventricular tachycardia. A dilated left ventricle with moderately reduced systolic function was found on echocardiographic examination. This is a very rare presentation of peripartum cardiomyopathy (PPCMP) presenting with repetitive monomorphic ventricular tachycardia.     

Pollinator adaptation and the evolution of floral nectar sugar composition

A long‐standing debate concerns whether nectar sugar composition evolves as an adaptation to pollinator dietary requirements or whether it is ‘phylogenetically constrained’. Here, we use a modelling approach to evaluate the hypothesis that nectar sucrose proportion (NSP) is an adaptation to pollinators. We analyse ~ 2100 species of asterids, spanning several plant families and pollinator groups (PGs), and show that the hypothesis of adaptation cannot be rejected: NSP evolves towards two optimal values, high NSP for specialist‐pollinated and low NSP for generalist‐pollinated plants. However, the inferred adaptive process is weak, suggesting that adaptation to PG only provides a partial explanation for how nectar evolves. Additional factors are therefore needed to fully explain nectar evolution, and we suggest that future studies might incorporate floral shape and size and the abiotic environment into the analytical framework. Further, we show that NSP and PG evolution are correlated – in a manner dictated by pollinator behaviour. This contrasts with the view that a plant necessarily has to adapt its nectar composition to ensure pollination but rather suggests that pollinators adapt their foraging behaviour or dietary requirements to the nectar sugar composition presented by the plants. Finally, we document unexpectedly sucrose‐poor nectar in some specialized nectarivorous bird‐pollinated plants from the Old World, which might represent an overlooked form of pollinator deception. Thus, our broad study provides several new insights into how nectar evolves and we conclude by discussing why maintaining the conceptual dichotomy between adaptation and constraint might be unhelpful for advancing this field.