Point mutation of an RGD sequence in the human P2Y2 receptor to a QGD sequence conserves Go-mediated signal transduction

Abstract only availableThe P2Y2 nucleotide receptor is a Go/q coupled receptor that is activated equipotently by extracellular nucleotides such as ATP or UTP and is upregulated in a variety of tissues in response to injury or stress. The biological effects of extracellular nucleotides are mediated through activation of P1 and P2 purinergic receptors. P1 receptors are responsive to adenine and P2 receptors are activated by a variety of nucleotides including ATP and UTP. The P2 receptors are subdivided into two distinct categories, the ionotropic ligand-gated channel (P2X) receptors and G-protein coupled P2Y-receptors, with seven transmembrane domains. Previous studies have shown that the human P2Y2 nucleotide receptor contains an arginine-glycine-aspartic acid (RGD), integrin-binding domain. This domain is located in the first extracellular loop of the receptor and binds specifically to the v3/5 group of integrins. The P2Y2 receptor interacts with the v integrins by the RGD domain to activate Go and induce cell migration. The human and murine P2Y2R's have the RGD integrin-binding domain, whereas the rat homologue has a QGD domain. However, this change in the arginine position in the RGD integrin-binding domain is considered to be a conservative substitution that maintains integrin binding. In order to confirm this assumption we changed the RGD domain of the human P2Y2 receptor into the QGD domain by in vitro mutagenesis. The wild-type and the QGD mutant P2Y2 receptors were transiently transfected into P2 receptor null, 1321 N1 astrocytoma cell line. Preliminary data suggest that the QGD mutant can stimulate PLC dependent intracellular Ca2+ mobilization and also activate cofillin and extracellular signal-regulated kinases (Erk) with equal efficacy and agonist potency as the wild type receptor. Further tests need to be done to verify that integrin bindin and signaling by the Rac and Rho pathways remain unaffected in the QGD mutant to induce integrin dependent cell migration in response to UTP and ATP.NSF-REU Program in Biological Sciences & Biochemistr

Effects of pro-inflamatory cytokines on polarized rat parotid Par-C10 monolayers [abstract]

Abstract only availableSjögren's syndrome (SS), an autoimmune disorder, is distinguished by inflammation and salivary gland cell death, leading to xerostomia (dry mouth). The G protein-coupled P2Y2 receptor (P2Y2R) is up-regulated in response to damage or stress in salivary epithelium. Pro-inflammatory cytokines associated with SS can be produced by infiltrating lymphocytes or salivary epithelium. Correlations have been found between lymphocytic infiltration and increased production of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-Éø (TNFα) and interferon-γ (IFNγ) and decreased function of exocrine glands in SS. Recent data has shown that P2Y2R activation enhances the activity of metalloproteases that release TNFα. OBJECTIVES: To study the effects of cytokines on polarized salivary epithelium. METHODS: Polarized rat parotid (Par-C10) monolayers were used to perform these studies. Cytokines released by UTP-induced P2Y2R activation were identified by ELISA. To evaluate the role of cytokines associated with SS on epithelial integrity, epithelial resistance was determined and correlated with the expression and distribution of tight junction (TJ) proteins by immunofluorescence and Western analysis, respectively. RESULTS: Activation of P2Y2Rs in Par-C10 monolayers induced the release of TNFα. The cytokines TNFα and IFNγ, but not IL-6 or IL1β, decreased the resistance of Par-C10 cells. However, the expression/distribution of the TJ protein ZO-1 was unaffected. CONCLUSIONS: The data support a hypothesis that P2Y2R expression and activation in salivary gland cells contribute to epithelial dysfunction in SS by generating pro-inflammatory cytokines that regulate ion transport and epithelial integrity in salivary glands. Future studies will determine the role of cytokines on the expression and distribution of other TJ molecules including occludin, claudins and junctional adhesion molecules. These studies may lead to better therapeutic strategies for minimizing autoimmune-associated dysfunction of salivary gland that contributes to xerostomia in SS patient.Life Sciences Undergraduate Research Opportunity Progra

P2Y receptors in the mammalian nervous system: pharmacology, ligands and therapeutic potential

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases

Up-regulation of the P2Y2 receptor by cytokines in neuronal cells

Abstract only availableAlzheimer's Disease (AD) is characterized by inflammation and neurodegeneration in the brain due to the presence of extracellular amyloid beta (A β) plaques and neurofibrillary tangles. Microglial and astrocyte cells associated with these plaques and tangles have been shown to release cytokines in AD patients, which have a proinflammatory effect on the brain. The P2Y2 receptor (P2Y2R) is a receptor protein that is up-regulated in response to damage or stress in a variety of tissues, including blood vessels and salivary gland epithelium. Recently our laboratory has shown that activation of the P2Y2R enhances α -secretase-dependent amyloid precursor protein (APP) processing. APP is proteolytically processed by β - and γ -secretases to release neurodegenerative A β. Alternatively, APP can be cleaved within the A β domain by α -secretase releasing the non-amyloidogenic product, sAPP α, which has been shown to have neuroprotective properties. Primary neurons have low P2Y2R expression, however, it has been demonstrated that cytokines up-regulate P2Y2R in smooth muscle cells. Therefore, this study will explore if cytokines up-regulate P2Y2R expression in primary rat neurons and in SH-SY5Y human neuroblastoma cells. Primary rat neurons and SH-SY5Y human neuroblastoma cells were plated on glass cover slips 24 or 48 hours with individual treatment, or a combination of, human interleukin-1 β (IL1- β), tumor necrosis factor α (TNF α), and interferon γ (IF γ). P2Y2R activity was measured by increases in intracellular calcium concentration ([Ca2+]i ) in response to the P2Y2R agonist UTP. Results support the hypothesis that P2Y2R is up-regulated by cytokines in neuronal cells. Furthermore, real-time PCR results indicate a two-fold increase in P2Y2R mRNA after cytokine treatment. Therefore, activation of the up-regulated P2Y2R in stressed neurons generates a neuroprotective (sAPP α) rather than neurodegenerative (A β) peptide. These results could have a substantial impact on the understanding and treatment of neurological disorders such as AD.Life Sciences Undergraduate Research Opportunity Progra

Phosphorylation of EGFR, ERK 1/2 and downstream transcription factors after P2Y2 receptor activation in a human submandibular gland cell line

Abstract only availableP2 nucleotide receptors mediate a variety of biological responses and are activated by the extracellular nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate (UTP), uridine diphosphate (UDP). The P2Y2 nucleotide receptor is a seven transmembrane spanning domain receptor activated by the nucleotides ATP and UTP, and is up-regulated in a variety of tissues in response to injury or stress. For example, the P2Y2 receptors are not normally expressed in salivary glands, but upon disruption of tissue homeostasis, the P2Y2 receptors are up-regulated. Sjogren's disease is an autoimmune disorder that affects salivary and lacrimal glands resulting in a decreased ability to produce saliva and tears. Previous work by our lab has shown that the P2Y2 receptor is up-regulated in submandibular glands of a Sjogren's syndrome mouse model, suggesting that it may be up-regulated in human Sjogren's syndrome. The goal of this project is to analyze the function of P2Y2 receptors in salivary gland tissues. HSG cells, which endogenously express P2Y2 receptors and are derived from a human submandibular gland tumor, were utilized as a cell model to analyze downstream signaling pathways in response to UTP. Our results show that UTP, the P2Y2 receptor selective agonist, causes phosphorylation of the epidermal growth factor receptor (EGFR), extracellular regulated kinases (ERK 1/2) and the downstream transcription factors p90RSK, and ELK, suggesting that P2Y2 receptors may play a role in gene transcription in salivary gland tissues.NSF-REU Biology & Biochemistr

P2Y2 nucleotide receptors mediate inflammatory responses in mouse salivary gland cells

Abstract only availableSjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by destruction of salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). Although the mechanisms involved have not been adequately elucidated, the diminished function of exocrine glands in SS is often associated with lymphocytic infiltration of the tissue. Aberrant expression of specific adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) is also observed in salivary gland with SS, which enable salivary epithelium to interact directly with infiltrating lymphocytes. P2Y2 nucleotide receptor (P2Y2R) is G protein-couple receptor that is activated by extracellular ATP and UTP. P2Y2R expression and activity is up-regulated in response to damage or stress in a variety of tissues, including submandibular glands (SMGs), where it mediates a complex set of cellular responses to injury of disease. Additionally, P2Y2R activation up-regulates VCAM-1 expression in dispersed rat SMG cell culture and human submandibular gland (HSG) cells. Our objective is to investigate weather P2Y2R up-regulation correlates with increased expression of adhesion molecules in SMGs from a mouse model for SS (C57BL/6.NOD-Aec1Aec2) as compared with normal mouse strain (C57BL/6). P2Y2R expression was measured by RT-PCR and adhesion molecules expression was determined by Western blot analysis. Salivary flow was preformed by cannulation of individual glands. We could see that P2Y2R expression and ICAM-1 expression were both up-regulated in the SMGs from a mouse model for SS as compared with normal mouse strain. And salivary flow was decreased in salivary glands from a mouse model for SS. These results suggest that P2Y2R mediate inflammatory responses related to secretory dysfunction in the mouse model for SS. Our ultimate goal would be to translate all this information to the human salivary gland in order to understand SS and to develop new therapies for salivary dysfunction in SS.Gyeongsang National Universit

Conditional knockout of the Menkes disease copper transporter demonstrates its critical role in embryogenesis

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 7 (2012): e43039, doi:10.1371/journal.pone.0043039.The transition metal, copper (Cu), is an enzymatic cofactor required for a wide range of biochemical processes. Its essentiality is demonstrated by Menkes disease, an X-linked copper deficiency disorder characterized by defects in nervous-, cardiovascular- and skeletal systems, and is caused by mutations in the ATP7A copper transporter. Certain ATP7A mutations also cause X-linked Spinal Muscular Atrophy type 3 (SMAX3), which is characterized by neuromuscular defects absent an underlying systemic copper deficiency. While an understanding of these ATP7A-related disorders would clearly benefit from an animal model that permits tissue-specific deletion of the ATP7A gene, no such model currently exists. In this study, we generated a floxed mouse model allowing the conditional deletion of the Atp7a gene using Cre recombinase. Global deletion of Atp7a resulted in morphological and vascular defects in hemizygous male embryos and death in utero. Heterozygous deletion in females resulted in a 50% reduction in live births and a high postnatal lethality. These studies demonstrate the essential role of the Atp7a gene in mouse embryonic development and establish a powerful model for understanding the tissue-specific roles of ATP7A in copper metabolism and disease.This work was supported by National Institutes of Health Grants DK59893 and DK093386 to M.J.P., and DK44464 to J.D.G

Diffractive Guiding of Waves by a Periodic Array of Slits

We show that in order to guide waves, it is sufficient to periodically truncate their edges. The modes supported by this type of wave guide propagate freely between the slits, and the propagation pattern repeats itself. We experimentally demonstrate this general wave phenomenon for two types of waves: (i) plasmonic waves propagating on a metal-air interface that are periodically blocked by nanometric metallic walls, and (ii) surface gravity water waves whose evolution is recorded, the packet is truncated, and generated again to show repeated patterns. This guiding concept is applicable for a wide variety of waves.Comment: 5 pages, 4 figure

Intraoperative visualisation and treatment of salivary glands in Sjögren's syndrome by contrast-enhanced ultrasound sialendoscopy (CEUSS):protocol for a phase I single-centre, single-arm, exploratory study

INTRODUCTION: We established a promising sialendoscopic treatment for in vivo enhancement of salivation in salivary glands affected by Sjögren's syndrome (SS). In this technique, the ducts of the salivary glands are irrigated with saline and steroids. This allows for dilatation of ductal strictures and removal of debris. Unfortunately, it is not possible to assess the delivery and penetration of saline or medications in the ductal system and parenchyma. To address this problem, we will conduct contrast-enhanced ultrasound sialendoscopy (CEUSS) using sulphur hexafluoride microbubbles. To the best of our knowledge, microbubbles have never been used for the treatment of salivary glands in SS. It is, therefore, imperative to test this application for its safety and feasibility. METHODS AND ANALYSIS: A single-arm phase I study will be performed in 10 SS patients. Under local anaesthesia, ultrasound (US) guided infusion of the parotid and submandibular glands with microbubbles will be performed. Continuous US imaging will be used to visualise the glands, including the location of strictures and occlusions. Main outcomes will be the evaluation of safety and technical feasibility of the experimental treatment. Secondary outcomes will consist of determinations of unstimulated whole mouth saliva flow, stimulated whole mouth saliva flow, stimulated parotid saliva flow, clinical oral dryness, reported pain, xerostomia, disease activity, salivary cytokine profiles and clinical SS symptoms. Finally, salivary gland topographical alterations will be evaluated by US. ETHICS AND DISSEMINATION: Ethical approval for this study was obtained from the Medical Ethics Committee of the Amsterdam University Medical Centre, Amsterdam, The Netherlands (NL68283.029.19). data will be presented at national and international conferences and published in a peer-reviewed journal. The study will be implemented and reported in line with the Standard Protocol Items: Recommendations for Interventional Trials' statement. TRIAL REGISTRATION NUMBERS: The Netherlands Trial Register: NL7731, MREC Trial Register: NL68283.029.19; Pre-results