Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn −/− myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR β and δ subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation

Interaction of solid organic acids with carbon nanotube field effect transistors

A series of solid organic acids were used to p-dope carbon nanotubes. The extent of doping is shown to be dependent on the pKa value of the acids. Highly fluorinated carboxylic acids and sulfonic acids are very effective in shifting the threshold voltage and making carbon nanotube field effect transistors to be more p-type devices. Weaker acids like phosphonic or hydroxamic acids had less effect. The doping of the devices was accompanied by a reduction of the hysteresis in the transfer characteristics. In-solution doping survives standard fabrication processes and renders p-doped carbon nanotube field effect transistors with good transport characteristics.Comment: 5 pages, 4 figures, 1 tabl

Incorporation of atmospheric 15NO2-nitrogen into free amino acids by Norway spruce Picea abies (L.) Karst.

During spring and autumn 1991, potted 6-yearold spruce trees (Picea abies (L.) Karst.) were fumigated with 60 nl1–1 15NO2 for 4 days under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total 15N content and incorporation of 15N into the -amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. Nitrate reductase activity was significantly higher in the needles of fumigated trees compared to control trees exposed to filtered air. With an average of 9.1% 15N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO2 to the N nutrition of the needles. 15N-labelled amino acids were also detected in the bark and the roots, indicating export from the needles

Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations

Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, rapsyn. Most patients have mutations within the rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in rapsyn−/− myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of rapsyn with AChR and prevented agrin-induced AChR clusters in rapsyn−/− myotubes. In TE671 cells, R91L reduced the ability of rapsyn to self-associate, and K373del-rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type rapsyn, L361R-rapsyn showed reduced expression/stability, and both N88K-rapsyn and L361R-rapsyn showed significantly reduced co-localization with AChR. N88K-rapsyn and L361R-rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severit

Dynamic interactions of the asialoglycoprotein receptor subunits with coated pits. Enhanced interactions of H2 following association with H1

Lateral mobility studies comparing native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, can measure membrane protein-coated pit interactions in intact cells (Fire, E., Zwart, D., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). We applied this approach to study the interactions of the H1 and H2 human asialoglycoprotein receptor subunits with coated pits. The lateral mobilities of singly expressed and coexpressed H1 and H2B (the H2 species that reaches the cell surface) were measured by fluorescence photobleaching recovery. They were compared with mutant proteins, H1(5A) (Tyr-5 replaced by Ala) and H2(5A) (Phe-5 replaced by Ala). While the mobile fractions of H1, H2B, and their mutants were similar, the lateral diffusion rate (measured by D, the lateral diffusion coefficient) was significantly slower for H1, whether expressed alone or with H2B. Coexpression with H1 reduced D of H2B to that of H1. Disruption of the clathrin lattices by hypertonic medium elevated D of H1, H1(5A), H2B, and H2(5A) to the same final level, without affecting their mobile fractions. Cytosol acidification, which retains altered clathrin lattices attached to the membrane and prevents coated vesicle formation, immobilized part of the H1 molecules, reflecting stable entrapment in "frozen" coated pits. H1(5A), H2B, and H2(5A) were not affected; however, coexpression of H2B with H1 conferred the sensitivity to cytosol acidification on H2B. Our results suggest that H1 lateral mobility is inhibited by dynamic interactions with coated pits in which Tyr-5 is involved. H2B resembles H1(5A) rather than H1, and its interactions with coated pits are weaker; efficient interaction of H2B with coated pits depends on complex formation with H1

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases

The Global Atmospheric Pollution Forum (GAPF) emission inventory preparation tool and its application to Côte d’Ivoire

Low- and middle-income countries (LMICs) often lack the necessary tools, guidance, and capacity for compiling an emission inventory (EI) for air pollutants. A reliable EI is an important prerequisite for the identification of key emissions sources, as an input to modelling atmospheric transport and impacts of air pollutants, and the identification of appropriate mitigation policies. The publicly-available Global Atmospheric Pollution Forum Emission Inventory (GAPF-EI) tool meets the need of LMICs for a user-friendly tool allowing in-country practitioners to compile their own EIs. The species covered are SO2, NOX, CO, NMVOC, CH4, NH3, PM10, PM 2.5, black carbon, organic carbon and CO2. Output from the tool can therefore support the development of integrated air quality and climate change mitigation strategies. This tool incorporates default emission factors and inventory methods conforming with internationally recognised approaches. The GAPF-EI tool enables emissions to be estimated for technologies or practices that are often of little or no relevance to developed countries, but may represent key sources in LMICs. This paper describes the GAPF-EI tool and its application to Côte d’Ivoire where emissions from traditional biomass cookstoves, vegetation fires, traditional charcoal manufacture, road transport (including dust from unpaved roads) and open burning of municipal solid waste were found to be particularly important components of the inventory. The application of the GAPF-EI approach to Côte d’Ivoire has demonstrated its utility in addressing sources of particular relevance to LMICs in addition to providing a user-friendly, transparent and flexible EI preparation tool

The History of Science and Current Perspectives in Dialogue

Research on urban space has a long tradition in classical studies. Current research of ancient urban spaces continues on in this tradition. It is essential to determine the position of research history in order to define the prospects of current urban studies in a more dif- ferentiated way: only those who are conversant with the history-of-science determination of their respective fields are able, intuitively and critically, to put innovative approaches and methods to the test. Based on the projects of the research group C-6, the various threads of history-of-science developments will be outlined and the current approaches defined in the context of constant re- orientation and new orientation of classical urban studie

The ESCAPE project : Energy-efficient Scalable Algorithms for Weather Prediction at Exascale

In the simulation of complex multi-scale flows arising in weather and climate modelling, one of the biggest challenges is to satisfy strict service requirements in terms of time to solution and to satisfy budgetary constraints in terms of energy to solution, without compromising the accuracy and stability of the application. These simulations require algorithms that minimise the energy footprint along with the time required to produce a solution, maintain the physically required level of accuracy, are numerically stable, and are resilient in case of hardware failure. The European Centre for Medium-Range Weather Forecasts (ECMWF) led the ESCAPE (Energy-efficient Scalable Algorithms for Weather Prediction at Exascale) project, funded by Horizon 2020 (H2020) under the FET-HPC (Future and Emerging Technologies in High Performance Computing) initiative. The goal of ESCAPE was to develop a sustainable strategy to evolve weather and climate prediction models to next-generation computing technologies. The project partners incorporate the expertise of leading European regional forecasting consortia, university research, experienced high-performance computing centres, and hardware vendors. This paper presents an overview of the ESCAPE strategy: (i) identify domain-specific key algorithmic motifs in weather prediction and climate models (which we term Weather & Climate Dwarfs), (ii) categorise them in terms of computational and communication patterns while (iii) adapting them to different hardware architectures with alternative programming models, (iv) analyse the challenges in optimising, and (v) find alternative algorithms for the same scheme. The participating weather prediction models are the following: IFS (Integrated Forecasting System); ALARO, a combination of AROME (Application de la Recherche a l'Operationnel a Meso-Echelle) and ALADIN (Aire Limitee Adaptation Dynamique Developpement International); and COSMO-EULAG, a combination of COSMO (Consortium for Small-scale Modeling) and EULAG (Eulerian and semi-Lagrangian fluid solver). For many of the weather and climate dwarfs ESCAPE provides prototype implementations on different hardware architectures (mainly Intel Skylake CPUs, NVIDIA GPUs, Intel Xeon Phi, Optalysys optical processor) with different programming models. The spectral transform dwarf represents a detailed example of the co-design cycle of an ESCAPE dwarf. The dwarf concept has proven to be extremely useful for the rapid prototyping of alternative algorithms and their interaction with hardware; e.g. the use of a domain-specific language (DSL). Manual adaptations have led to substantial accelerations of key algorithms in numerical weather prediction (NWP) but are not a general recipe for the performance portability of complex NWP models. Existing DSLs are found to require further evolution but are promising tools for achieving the latter. Measurements of energy and time to solution suggest that a future focus needs to be on exploiting the simultaneous use of all available resources in hybrid CPU-GPU arrangements