Showing papers by "Jon Lindstrom published in 2008"

Roles of Accessory Subunits in α4β2* Nicotinic Receptors

Abstract: Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. α5 and β3 subunits can function only as accessory subunits. We show that both α5 and β3 efficiently assemble in human α4β2* AChRs expressed in permanently transfected human embryonic kidney (HEK) cell lines. Only (α4β2)2α5, not (α4β2)2β3 AChRs, have been detected in brain. The α4β2α5 line expressed 40% more AChRs than the parent α4β2 line and was equally sensitive to up-regulation by nicotine. The α4β2β3 line expressed 25-fold more AChRs than the parental line and could not be further up-regulated by nicotine. Relative sensitivity to activation by ACh depends on the accessory subunit, β2 conferring the greatest sensitivity, α5 less, and β3 and α4 much less. Accessory subunits form binding sites for positive allosteric modulators, as illustrated by the observation that α5 conferred high sensitivity to galanthamine. In the presence of α5 or β3, stable, partially degraded, dead end intermediates accumulated within the cells. These may have the form α5α4β2α5. The efficiency with which α5 and β3 assemble with α4 and β2 and the necessity of avoiding formation of potentially toxic intermediates may explain why α5 and β3 seem to be transcribed at low levels in brain. Autosomal dominant nocturnal frontal lobe epilepsy can be caused by the α4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with α5, β3, or α6 to replace α4 as accessory subunit.

The European Perspective of Type 2 Diabetes Prevention: Diabetes in Europe - Prevention Using Lifestyle, Physical Activity and Nutritional Intervention (DE-PLAN) Project

Abstract: Background The prevalence of type 2 diabetes (T2D) has dramatically increased in Europe, and the age-at-diagnosis has become younger. Action is needed now to develop targeted prevention management program for T2D. The DE-PLAN ("Diabetes in Europe - Prevention using Lifestyle, Physical Activity and Nutritional intervention") project, led by the University of Helsinki is currently addressing this major public health concern in Europe. Methods The DE-PLAN project aims at developing and testing models of efficient identification and intervention of individuals at high risk of type 2 diabetes in the community. It conducts a lifestyle modification intervention in people at high risk for T2D. Furthermore, it tests the feasibility and cost-effectiveness of the translation of the current research evidence about preventive intervention program into clinical settings within existing health care systems in 17 European countries. Results This 3-year project spanning has commenced mid-2005. By now, 25 institutions from 17 countries are involved. Conclusion The development of efficient screening strategies for type 2 diabetes risk as well as the development of core intervention strategies for the primary prevention of type 2 diabetes should significantly enhance the ability of health care professionals to respond swiftly to the drastic increase of T2D and its burden to the society.

Activated cholinergic signaling provides a target in squamous cell lung carcinoma

Abstract: The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non-small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of alpha5 and beta3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC.

Myasthenia Gravis and the Tops and Bottoms of AChRs Antigenic Structure of the MIR and Specific Immunosuppression of EAMG Using AChR Cytoplasmic Domains

Abstract: The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of α1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR α3 subunits, but MG patient antibodies do not. By making chimeras of α1 subunits with α7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of α1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in α1/α7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human α1, β1, γ, δ, and e subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG.

The genetic variation in the tenomodulin gene is associated with serum total and LDL cholesterol in a body size-dependent manner.

Abstract: We have reported that the sequence variation in the tenomodulin (TNMD) gene is associated with the risk of type 2 diabetes (T2DM), central obesity and serum levels of systemic immune mediators in the Finnish Diabetes Prevention Study (DPS), which is a longitudinal lifestyle intervention study on 522 middle-aged persons with impaired glucose tolerance (IGT). The aim of this study was to investigate whether the association with T2DM, observed in the DPS could be replicated in a larger, cross-sectional population-based random sample of 5298 men (3020 with normoglycaemia, 984 with impaired fasting glucose, 436 with IGT and 811 with T2DM) from the region of Kuopio, eastern Finland. To further explore the putative mechanisms linking TNMD to T2DM and metabolic syndrome, we studied the associations of TNMD sequence variation with lipid abnormalities characteristic to metabolic syndrome. The association with T2DM risk was not replicated, but significant associations were found with serum low-density lipoprotein and total cholesterol in a body mass index-dependent manner. These associations were also observed in the men of DPS, whereas in women these associations were not significant. These results from two independent study populations suggest that the genetic variation in TNMD could modulate cholesterol metabolism in obese men.

Cellular prion protein co‐localizes with nAChR β4 subunit in brain and gastrointestinal tract

Abstract: PrP C , the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrP Sc , in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrP C has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the b4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co-immunoprecipitation assays and double-label immunofluorescence. The interaction between prion protein and native b4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of b4 subunit in the pathogenesis of TSE was studied by in vivo assays. b4 ‐ ⁄ ‐ and wild-type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of b4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrP C is a member of a multiprotein membrane complex participating in the formation and function of a3b4 nAChR.

'Seronegative' myasthenia gravis is no longer seronegative.

Abstract: High-affinity IgG autoantibodies to muscle nicotinic acetylcholine receptors (AChRs) were discovered to cause myasthenia gravis (MG) and its animal model more than 30 years ago (Patrick and Lindstrom, 1973; Lindstrom et al ., 1976 a , b ; Vincent et al ., 2006), and the antigenic structure of muscle AChRs is still being actively investigated (Kalamida et al ., 2007; Lindstrom et al ., 2008). Immune precipitation of AChRs tagged in their acetylcholine-binding sites with 125I-labelled α−bungarotoxin provided a sensitive immunodiagnostic assay for MG (Lindstrom et al ., 1976 b ). However, up to 20% of those who appeared to have autoimmune MG, because they benefited from plasmapheresis or exhibited antibodies bound to their neuromuscular junctions, did not have autoantibodies detectable by the conventional assay (Vincent et al ., 2006). Angela Vincent and her co-workers discovered that about half of the putative seronegative MG patients actually had autoantibodies to muscle-specific …

Effects of Redox Reagents and Arsenical Compounds on [3H]-Cytisine Binding to Immunoisolated Nicotinic Acetylcholine Receptors from Chick Brain Containing α4 β2 Subunits

Abstract: All known nicotinic receptor alpha subunits include a conserved disulfide bond that is essential for function and is a site for labeling via biochemical modification. In an effort to develop a universal ligand for all subtypes of nicotinic receptors, we previously studied the effects of arsenylation with two compounds, p-aminophenyldichloroarsine (APA) and bromoacetyl-p-aminophenylarsenoxide (BAPA) on nicotinic receptors from Torpedo electroplax. Here we apply these reagents to immunoisolated receptors containing alpha 4, beta 2, and possibly other subunits from chick brain that bind [3H]cytisine with high affinity (KD approximately 5 nM). These are distinct from another receptor subtype that also binds [3H]cytisine and [3H]nicotine and can be arsenylated with APA, but instead contains alpha 5, beta 2, and probably other subunits. Reduction of alpha 4 beta 2 receptors with dithiothreitol blocked [3H]cytisine binding and this effect was reversed upon reoxidation by dithiobisnitrobenzoic acid. APA or BAPA prevented the dithiobisnitrobenzoic acid reactivation of dithiothreitol-treated receptors with IC50 values of 15 and 70 nM, respectively. However, the antiarsenical dimercaptopropanesulfonic acid restored function to APA- or BAPA- "arsenylated" receptors (EC50 approximately 100 microM). APA-treated receptors remained blocked for up to 24 h, but treatment with dimercaptopropanesulfonic acid at any time restored [3H]cytisine binding. APA treatment of reduced receptors protected against irreversible alkylation by bromoacetylcholine, indicating that arsenylation occurs at least in part in the agonist binding site. Thus, these reagents have similar effects on different nicotinic receptor subtypes from both muscle and nerves.(ABSTRACT TRUNCATED AT 250 WORDS)