Laminin colocalization with AChR aggregates is disrupted on dystroglycan- myotubes. Wild-type (R1) and dystroglycan- myotubes (3C12) were treated overnight with
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Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that
Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells ...
Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrininduced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches
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The first signs of synapse function The decision to form a synapse The sticky synapse Converting growth cones to presynaptic terminals Receptor clustering and postsynaptic differentiation at the NMJ Agrin is a transynaptic clustering signal at the NMJ Receptor clustering signals in the CNS Scaffold proteins and receptor aggregation in the CNS Innervation increases receptor expression and insertion Synaptic activity regulates receptor density Maturation of transmission and receptor isoform transitions Maturation of transmitter reuptake Short-term plasticity Appearance of synaptic inhibition Is inhibition really inhibitory during development?. Summary 9. Refinement of synaptic connections The early pattern of connections Functional synapses are eliminated Many axonal arborizations are eliminated or refined The Sensory Environment Influences Synaptic Connections Activity Influences Synapse Elimination at the NMJ Synapse refinement is reflected in sensory coding properties Activity contributes to ...
Benjamin A. Samuels and Li-Huei Tsai Postsynaptic clusters of acetylcholine receptors (AChR) form at the developing neuromuscular junction (NMJ) prior to apposition by the presynaptic nerve terminal. Subsequently, clusters that are innervated by the nerve terminal become stable, while clusters that are not innervated disperse. It is believed that an important positive signal for stabilization is agrin, but until recently, a signal for dispersion had not been discovered. A recent paper from the lab of Kuo-Fen Lee provides evidence that the Cdk5 kinase plays an essential role in this process (1).. Cdk5 is a small serine/threonine kinase that is primarily active in neurons. However, a report from the lab of Nancy Ip in 2001 first established a potentially important role for Cdk5 in the postsynaptic myotubes of the NMJ by showing it promoted neuregulin-induced transcription of the AChR (2). An abstract from Nancy Ips lab in 2003 also suggested that agrin-induced AChR clusters are significantly ...
Prior studies on dystroglycan support dystroglycan as an agrin-binding protein. More specifically, data suggests that the carbohydrate residues on dystroglycan may be involved in binding to agrin. However, genetic and functional studies on dystroglycans role in agrin-mediated acetylcholine receptor (AChR) clustering remain unclear. Evidence points toward an indirect role for dystroglycan, a role which is important for the aggregation and stabilization of micro-clusters. The question of what regions of dystroglycan are functionally important for its role in agrin-induced AChR clustering is still unanswered. In an attempt to answer this question, we created beta-dystroglycan deletion and point mutants, and expressed these mutants in C2 muscle cells. Next, we assayed transfected myotubes for their responsiveness to agrin by measuring the number of AChR clusters per myotube segment. Last, we characterized a previously unknown protein-lipid interaction for dystroglycan. Our results demonstrate that ...
Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8(+) T cells and regulatory T cells, respectively, bind receptor complexes that share beta- and gamma-signaling subunits. Receptor specificity is provided by unique, nonsignaling alpha-subunits. Whereas IL-2 receptor-alpha (IL-2Ralpha) is expressed together in cis with the beta- and gamma-subunits on T cells and B cells, IL-15Ralpha is expressed in trans on antigen-presenting cells. Here we present a 1.85-Å crystal structure of the human IL-15-IL-15Ralpha complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15-IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15-IL-15Ralpha and IL-2-IL-2Ralpha complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context ...
Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal ...
Adult mammalian Renshaw cells express large and complex postsynaptic gephyrin/glycine receptor clusters on their surface. Larger gephyrin clusters correlate with more
Everything you ever wanted to know about gear - its use and application, limitations, strengths, weaknesses. How to pack for a trip (see the Gear Lists forum), or how to do it yourself (see the Make Your Own Gear forum). Need to know how to use a spork as a tent stake? See the Multiple Use Gear forum. You get the idea. Its all about gear ...
The discovery that the high affinity IgE receptor (Fc epsilon RI) is expressed on APCs of patients with atopic diseases raised the possibility that the functional importance of Fc epsilon RI in the pathogenesis of atopy may extend beyond its role in type I allergic reactions. Here we show that, following removal of in vivo-bound IgE by lactic acid treatment, targeting of allergens to monocytes by Ag-specific IgE critically depends on Fc epsilon RI expression. Even more importantly, lactic acid-treated, monocyte-enriched PBMCs present allergen to T cells 100- to 1000-fold more effectively if the allergen has been targeted to Fc epsilon RI on these cells via allergen-specific IgE. This mechanism may critically lower the atopic individuals threshold to mount allergen-specific T cell responses capable of promoting IgE production and delayed-type hypersensitivity reactions. ...
Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide an excellent platform for sensitive protein recognition. Using nanomaterials as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, the quantitative analysis of the binding affinity of these glyconanomaterials has been lacking. In this Article, we report a new method to measure the binding affinity of glyconanoparticle (GNP)-protein interactions based on a fluorescent competition binding assay, which yielded the apparent dissociation constant (K-d) of GNPs with the interacting protein. Au nanoparticles conjugated with underivatized mono-, oligo-, and polysaccharides were synthesized using our recently developed photocoupling chemistry. The affinities of these GNPs with lectins were measured and were several orders of magnitude higher than the corresponding free ligands with lectins. The effect of ligand display on the binding affinity of GNPs was, furthermore, ...
Initial attempts to end-label polymers bearing highly polar substituents . (a) Jurkat cell labeled with a fluorescent anti-L-selectin antibody, FITC-DREG-56. added to a solution of triethylene glycol (1; 8.9 ml, 66.6 mmol) in 50% aq NaOH (5.3 ml), .. Sialyl Lewis x liposomes as a multivalent ligand and inhibitor of E-selectin. ...
Does anybody know a source for monoclonal antibodies against high affinity IgE receptors (Fc-epsilon-RI)? Ive heard that Roche produce an antibody. Anybody know anything? -- Gordon ...
p>The checksum is a form of redundancy check that is calculated from the sequence. It is useful for tracking sequence updates.,/p> ,p>It should be noted that while, in theory, two different sequences could have the same checksum value, the likelihood that this would happen is extremely low.,/p> ,p>However UniProtKB may contain entries with identical sequences in case of multiple genes (paralogs).,/p> ,p>The checksum is computed as the sequence 64-bit Cyclic Redundancy Check value (CRC64) using the generator polynomial: x,sup>64,/sup> + x,sup>4,/sup> + x,sup>3,/sup> + x + 1. The algorithm is described in the ISO 3309 standard. ,/p> ,p class="publication">Press W.H., Flannery B.P., Teukolsky S.A. and Vetterling W.T.,br /> ,strong>Cyclic redundancy and other checksums,/strong>,br /> ,a href="http://www.nrbook.com/b/bookcpdf.php">Numerical recipes in C 2nd ed., pp896-902, Cambridge University Press (1993),/a>),/p> Checksum:i ...
cyclo(L-262): inhibits the interaction between immunoglobulin E (IgE) and its high affinity receptor, Fc epsilon RI; structure given in first source
Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):11082-7. Epub 2004 Jul 19. IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells. Vαmosi G, Bodnαr A, Vereb G, Jenei A, Goldman CK, Langowski J, Tσth K, Mαtyus L, Szφllφsi J, Waldmann TA, Damjanovich S ...
Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):11082-7. Epub 2004 Jul 19. IL-2 and IL-15 receptor alpha-subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells. Vαmosi G, Bodnαr A, Vereb G, Jenei A, Goldman CK, Langowski J, Tσth K, Mαtyus L, Szφllφsi J, Waldmann TA, Damjanovich S ...
Signaling between nerve and muscle occurs at neuromuscular junctions (NMJs), which consist of specialized and precisely apposed pre- and postsynaptic structures separated by a synaptic cleft (Sanes and Lichtman 1999). Agrin, a heparan sulfate proteoglycan of ∼400-600 kD, is essential for the induction and organization of the postsynaptic structures (McMahan 1990; Gautam et al. 1996; Cohen et al. 1997; Jones et al. 1997; Meier et al. 1997; Rimer et al. 1997). Agrin is synthesized by motor neurons, transported to axon terminals, and released into the synaptic cleft of NMJs, where it binds to the basal lamina (Magill-Solc and McMahan 1988, Magill-Solc and McMahan 1990; Cohen and Godfrey 1992; Reist et al. 1992). Motor neurons express a mixture of agrin isoforms, alternatively spliced at two sites (A and B in chick, y and z in rat) in their COOH-terminal halves (Ruegg et al. 1992; Rupp et al. 1992; Hoch et al. 1993). The neural isoform of agrin containing inserts of four and eight amino acids at ...
The aim of this study was to investigate the influence of the β chain of the high affinity IgE receptor on the development of the atopic phenotype, as postulated by Cookson et al.8 9 11 In order to make the comparison more valuable, we have chosen the same phenotypes for the study as the group from Oxford, we used the same microsatellite probes for linkage analysis, and checked for the same polymorphisms. We studied two populations, one recruited through a population based study and the other through our outpatient department. Thus, the much higher number of clinically affected children in the second population (45.8% versus 5.4%) was expected. Assuming that atopy represents a genetically heterogeneous disease, it might well be associated with a different set of genes involved or a different degree of involvement of each gene in our populations.. We could not find definite evidence for involvement of the the β chain of the high affinity IgE receptor on enhanced IgE responsiveness in a cognate ...
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MUSK兔多克隆抗体(ab58697)可与重组片段样本反应并经WB, ELISA实验严格验证。中国75%以上现货,所有产品均提供质保服务,可通过电话、电邮或微信获得本地专属技术支持。
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CHAPTER 1 Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that aggregates formed by trivalent antigens are more complicated than those of divalent antigens. CHAPTER 2 Membrane-active antibiotics include antimicrobial peptides (AMPs) and a class of amphiphilic steroids termed ceragenins. Recent studies of membrane-active antibiotics show that cationic, facially amphiphilic molecules could disrupt bacterial membranes
At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, beta-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyn-deficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and beta-dystroglycan, and for the agrin-induced increase in association ...
About Steve Burden. Dr. Burdens laboratory has made significant contributions to our current understanding of the signaling pathways for forming and maintaining neuromuscular synapse. His experiments led to the discovery of the neural signal, Agrin, a secreted, extracellular matrix protein that is necessary to form and stabilize neuromuscular synapses. His laboratory went on to identify Lrp4 as the muscle receptor for Agrin and to discover MuSK, the kinase that associates with Lrp4 and transduces the Agrin signal to stimulate postsynaptic differentiation. Further, his laboratory showed that Lrp4 acts bi-directionally, as Lrp4 not only serves as a muscle receptor for Agrin but also signals in a retrograde manner to motor neurons to control presynaptic differentiation. These studies have led to a good understanding of the signaling events that control synapse formation and provide a basis for understanding how dysfunction of this pathway causes neuromuscular disease and insights into the design ...