STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.     

Towards physiologically meaningful water‐use efficiency estimates from eddy covariance data

Intrinsic water‐use efficiency (iWUE) characterizes the physiological control on the simultaneous exchange of water and carbon dioxide in terrestrial ecosystems. Knowledge of iWUE is commonly gained from leaf‐level gas exchange measurements, which are inevitably restricted in their spatial and temporal coverage. Flux measurements based on the eddy covariance (EC) technique can overcome these limitations, as they provide continuous and long‐term records of carbon and water fluxes at the ecosystem scale. However, vegetation gas exchange parameters derived from EC data are subject to scale‐dependent and method‐specific uncertainties that compromise their ecophysiological interpretation as well as their comparability among ecosystems and across spatial scales. Here, we use estimates of canopy conductance and gross primary productivity (GPP) derived from EC data to calculate a measure of iWUE (G₁, “stomatal slope”) at the ecosystem level at six sites comprising tropical, Mediterranean, temperate, and boreal forests. We assess the following six mechanisms potentially causing discrepancies between leaf and ecosystem‐level estimates of G₁: (i) non‐transpirational water fluxes; (ii) aerodynamic conductance; (iii) meteorological deviations between measurement height and canopy surface; (iv) energy balance non‐closure; (v) uncertainties in net ecosystem exchange partitioning; and (vi) physiological within‐canopy gradients. Our results demonstrate that an unclosed energy balance caused the largest uncertainties, in particular if it was associated with erroneous latent heat flux estimates. The effect of aerodynamic conductance on G₁ was sufficiently captured with a simple representation. G₁ was found to be less sensitive to meteorological deviations between canopy surface and measurement height and, given that data are appropriately filtered, to non‐transpirational water fluxes. Uncertainties in the derived GPP and physiological within‐canopy gradients and their implications for parameter estimates at leaf and ecosystem level are discussed. Our results highlight the importance of adequately considering the sources of uncertainty outlined here when EC‐derived water‐use efficiency is interpreted in an ecophysiological context.     

Bericht über einige pflanzengesellschaften islands und deren bedeutung für die landwirtschaft

Zusammenfassung Die von Gletschern und Vulkanen geprägte Oberfläche Islands wird nur an wenigen Stellen intensiv landwirtschaftlich genutzt. Neben hofnahen, eingezäunten Wirtschaftsgrünländerein überwiegen hofferne Magerrasen, wie die Mattenvegetation, die Heiden u.a. und die von wenigen Arten dünn besiedelten Lava- und Erosionsflächen. Viele derzeitigen Ödlandflächen können als potentielles Grünland angesehen werden; d.h. auf Island könnte die Viehhaltung innerhalb kurzer Zeit gewaltig erhöht werden. Dazu ist jedoch eine Umwandlung vieler rezenter Pflanzengesellschaften in leistungsfähigere Ersatzgesellschaften notwendig. Die vorliegend beschriebenen Untersuchungen und Beobachtungen zeigen, dass für eine Umwandlung vieler Ödlandgesellschaften in Wirtschaftsgrünland die allgemein geläufigen Voraussetzungen Gültigkeit haben. Den stärksten Einfluss auf die Artenzusammensetzung hat intensive Nutzung. Das führt schliesslich so weit, dasssich Trittrasen auf Island von Trittrasen in Mitteleuropa überhaupt nicht unterscheiden. Eine starke Ertragssteigerung ist durch Einsatz der Stickstoffdüngung zu erwarten. Durch Einsatz von Handelsdünger und durch Intensierung der Nutzung könnte der derzeitige Viehbestand auf viel kleinerer Fläche ernährt werden. Die Schafhaltung könnte dadurch grundlegend geändert werden, d.h. alle zur Zeit stark erodierten und auch alle erosionsgefährdeten Flächen könnten von Schafen freigehalten werden. Ansaat von brauchbaren Arten auf bisher fast vegetationslosen Flächen und richtige Nutzung aller ausreichend mit Pflanzen besiedelten Flächen müssten zu einer weitgehenden Begrünung Islands führen.     

Borrelia burgdorferi potently activates bone marrow-derived conventional dendritic cells for production of IL-23 required for IL-17 release by T cells

Lyme borreliosis is characterized by cellular inflammatory responses at multiple body sites. Recently, an association of interleukin-17 (IL-17) and Lyme arthritis was suggested. In this context, it is of special interest that the heterodimeric cytokine IL-23 can act on T cells and initiate the up-regulation of effector cytokines such as IL-17. To determine the role of this specific cytokine cascade for the induction of subsequently induced proinflammatory events we developed an in vitro system to investigate the IL-23-inducing capacity of Borrelia burgdorferi and the potential of the spirochete for inducing the IL-23/IL-17 axis. We used cells derived from mice deficient for IL-23 or IL-12 only or deficient for both IL-12 and IL-23 to define precisely the function of these cytokines. Experiments with bone marrow-derived dendritic cells (BMDC) identified these cells as sources for IL-23 but not for IL-12 after B. burgdorferi exposure. Subsequent investigations with T cell-depleted splenocyte fractions revealed a tight IL-23/IL-17 axis in response to the spirochetes. Monoclonal antibodies that block IL-23 showed further that BMDC-derived IL-23 was required for production of IL-17 in this experimental model. These in vitro data describing a spirochete-induced release of IL-23 may help to define IL-17-dependent inflammatory responses in the disease.     

Synthesis and SAR comparison of regioisomeric aryl naphthyridines as potent mGlu5 receptor antagonists

We describe three novel regioisomeric series of aryl naphthyridine analogs, which are potent antagonists of the Class III GPCR mGlu5 receptor. The synthesis and in vitro and in vivo pharmacological activities of these analogs are discussed.     

Endonuclease G regulates budding yeast life and death

Endonuclease G (EndoG) is located in mitochondria yet translocates into the nucleus of apoptotic cells during human degenerative diseases. Nonetheless, a direct involvement of EndoG in cell-death execution remains equivocal, and the mechanism for mitochondrio-nuclear translocation is not known. Here, we show that the yeast homolog of EndoG (Nuc1p) can efficiently trigger apoptotic cell death when excluded from mitochondria. Nuc1p induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factor. Instead, the permeability transition pore, karyopherin Kap123p, and histone H2B interact with Nuc1p and are required for cell death upon Nuc1p overexpression, suggesting a pathway in which mitochondrial pore opening, nuclear import, and chromatin association are successively involved in EndoG-mediated death. Deletion of NUC1 diminishes apoptotic death when mitochondrial respiration is increased but enhances necrotic death when oxidative phosphorylation is repressed, pointing to dual--lethal and vital--roles for EndoG.     

Effects of mesophyll conductance on vegetation responses to elevated CO2 concentrations in a land surface model

Mesophyll conductance (g_m) is known to affect plant photosynthesis. However, g_m is rarely explicitly considered in land surface models (LSMs), with the consequence that its role in ecosystem and large‐scale carbon and water fluxes is poorly understood. In particular, the different magnitudes of g_m across plant functional types (PFTs) are expected to cause spatially divergent vegetation responses to elevated CO₂ concentrations. Here, an extensive literature compilation of g_m across major vegetation types is used to parameterize an empirical model of g_m in the LSM JSBACH and to adjust photosynthetic parameters based on simulated A_n ? C_i curves. We demonstrate that an explicit representation of g_m changes the response of photosynthesis to environmental factors, which cannot be entirely compensated by adjusting photosynthetic parameters. These altered responses lead to changes in the photosynthetic sensitivity to atmospheric CO₂ concentrations which depend both on the magnitude of g_m and the climatic conditions, particularly temperature. We then conducted simulations under ambient and elevated (ambient + 200 μmol/mol) CO₂ concentrations for contrasting ecosystems and for historical and anticipated future climate conditions (representative concentration pathways; RCPs) globally. The g_m‐explicit simulations using the RCP8.5 scenario resulted in significantly higher increases in gross primary productivity (GPP) in high latitudes (+10% to + 25%), intermediate increases in temperate regions (+5% to + 15%), and slightly lower to moderately higher responses in tropical regions (?2% to +5%), which summed up to moderate GPP increases globally. Similar patterns were found for transpiration, but with a lower magnitude. Our results suggest that the effect of an explicit representation of g_m is most important for simulated carbon and water fluxes in the boreal zone, where a cold climate coincides with evergreen vegetation.     

Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition

The serine protease granzyme B (GrB) of cytotoxic lymphocytes efficiently induces apoptosis by direct activation of caspases and cleavage of central caspase substrates. We employed human GrB as an effector function in chimeric fusion proteins that also contain the EGFR ligand TGFalpha or an ErbB2-specific single-chain antibody fragment (scFv) for selective targeting to tumor cells. GrB-TGFalpha (GrB-T) and GrB-scFv(FRP5) (GrB-5) molecules expressed in the yeast Pichia pastoris were bifunctional, cleaving synthetic and natural GrB substrates, and binding specifically to cells expressing EGFR or ErbB2 target receptors. Upon cell binding the chimeric molecules were internalized into intracellular vesicles, but could be released into the cytosol by the endosomolytic reagent chloroquine. Treatment with picomolar to nanomolar concentrations of GrB-5 and GrB-T resulted in selective and rapid tumor cell killing, accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases.     

Label-Free Detection of Neuronal Differentiation in Cell Populations Using High-Throughput Live-Cell Imaging of PC12 Cells

Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.