DOCSLIB.ORG

Regulation of Microvesicle Particle Release in Keratinocytes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Microvesicle Particle Release in Keratinocytes Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2018 Regulation of Microvesicle Particle Release in Keratinocytes Azeezat Afolake Awoyemi Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Awoyemi, Azeezat Afolake, "Regulation of Microvesicle Particle Release in Keratinocytes" (2018). Browse all Theses and Dissertations. 1999. https://corescholar.libraries.wright.edu/etd_all/1999 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. REGULATION OF MICROVESICLE PARTICLE RELEASE IN KERATINOCYTES A thesis submitted in partial fulfilment of the Requirements for the degree of Master of Science By AZEEZAT AFOLAKE AWOYEMI B.S., University of Lagos, 2015 2018 Wright State University i All rights reserved. This work may not be reproduced in whole or in part by photocopy or other means, without permission of the author. COPYRIGHT BY AZEEZAT AFOLAKE AWOYEMI 2018 ii WRIGHT STATE UNIVERSITY GRADUATE SCHOOL JULY 23, 2018 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Azeezat Afolake Awoyemi ENTITLED Regulation of Microvesicle Particle release in keratinocytes. BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. Jeffrey B. Travers, M.D., Ph.D. Thesis Director Jeffrey B. Travers, M.D., Ph.D. Chair, Department of Pharmacology and Toxicology Committee on Final Examination Jeffrey B Travers, M.D., Ph.D. Ravi Sahu, Ph.D. Ji Chen-Bihl, M.D., Ph.D. ________________________________ Barry Milligan, Ph.D. Interim Dean of the Graduate School iii ABSTRACT Awoyemi, Azeezat Afolake. M.S. Department of Pharmacology and Toxicology, Wright State University, 2018. Regulation of Microvesicle Particle release in keratinocytes. Microvesicle particles (MVPs) are produced from cellular membranes and are thought to mediate cell-cell communication, including in response to stressors such as UVB radiation and thermal burn injury. Previous studies have shown that stress-induced MVP release requires the platelet-activating factor (PAF) receptor in human keratinocytes and that pharmacological inhibition of acid sphingomyelinase (ASM) blocked this release. To validate a genetic role for ASM in MVP release, we used CRISPR-Cas9 gene silencing in human keratinocytes and primary fibroblasts derived from ASM-knockout mice. Though MVP release was partially blocked in ASM- deficient mouse fibroblasts, the inability to fully knockdown ASM in HaCaT cells resulted in no significant impact on MVP production. We also found that ethanol, which is known to augment PAF production, stimulated MVP release in human keratinocytes and in mice in vivo. These studies therefore provide additional insights into the role of ASM and ethanol in MVP release. iv TABLE OF CONTENTS CHAPTER 1: INTRODUCTION .................................................................................. 1 1.1 Statement of Problem .................................................................................... 1 1.2 Significance ................................................................................................... 1 1.3 Statement of Purpose ..................................................................................... 2 1.4 Hypothesis ..................................................................................................... 2 1.5 Research Objectives ..................................................................................... 3 1.6 Abbreviations ............................................................................................... 4 1.7 Assumptions ................................................................................................. 5 CHAPTER 2: LITERATURE REVIEW ................................................................ 6 2.1 Microvesicle Particles .................................................................................. 6 2.2 Keratinocyte Damage .................................................................................. 7 2.3 PAF-Receptor .............................................................................................. 7 2.3.1 Role of PAF and PAF-R in Keratinocyte Damage ...................................... 8 2.4 Acid Sphingomyelinase............................................................................... 9 v 2.4.1 ASM and MVP ......................................................................................... 10 2.5 Ethanol ....................................................................................................... 11 2.5.1 Ethanol and thermal burn injury ............................................................ 12 2.6 Translocation of ASM ............................................................................... 13 2.7 CRISPR CAS9 ........................................................................................... 13 CHAPTER 3: Materials and Methods .................................................................. 15 3.1 Introduction ............................................................................................... 15 3.2 Cells and Cell culture ............................................................................... 15 3.2.1 Cell growth media and storage condition ................................................ 15 3.2.2 Cell passage ............................................................................................. 16 3.2.3 Cell count ................................................................................................. 16 3.3 Treatments ............................................................................................... 16 3.4 MVP Isolation ......................................................................................... 17 3.5 MVP Analysis ......................................................................................... 17 3.6 Mice ......................................................................................................... 18 3.6.1 Treatment ................................................................................................ 18 3.6.2 Blood sample collection ......................................................................... 19 3.6.3 Mice skin punch biopsies ....................................................................... 19 3.6.4 MVP extraction ...................................................................................... 19 3.6.5 MVP analysis ......................................................................................... 20 3.7 CRISPR/CAS 9 Knockout of Acid Sphingomyelinase gene ................. 20 vi 3.7.1 Super competent bacterial transformation ............................................... 20 3.7.2 Enzyme restriction ................................................................................... 21 3.7.3 Cell seeding and Co-transfection ............................................................. 23 3.7.4 Cell passaging and colony selection ....................................................... 23 3.7.5 Analysis ................................................................................................. 23 3.7.5.1 Western blot ........................................................................................... 23 3.7.5.2 Immuno-dot blot assay .......................................................................... 24 3.7.5.3 MVP release .......................................................................................... 26 3.7.5.4 MVP Isolation and Analysis: ................................................................ 26 3.8 Flow cytometry ......................................................................................... 26 3.9 Determining the genomic targeting efficiency of Cas9/gRNA with T7 Endonuclease ........................................................................................................ 27 3.10 ASM translocation................................................................................... 28 3.10.1 Cell seeding ............................................................................................ 28 3.10.2 Treatment ............................................................................................... 28 3.10.3 Immunofluorescence .............................................................................. 28 CHAPTER 4: RESULTS ..................................................................................... 30 4.0 In vitro studies ............................................................................................ 30 4.1 Dose response effect of ethanol mediated MVP release in HaCaT cells ... 30 4.2 EtOH induced MVP release in NTERTs and Normal Human Fibroblasts 31 4.3 MVP release from HaCaT cells on exposure to EtOH and TBI ................ 33 vii 4.3.1 Effect of imipramine on MVP-induced EtOH and TBI release in HaCaT cells ……………………………………………………………………………...35 4.4 Effect of EtOH and UVB on MVP release ............................................... 36 4.5 EtOH mediated release of MVP In vivo .................................................... 37 4.6 ASM translocation ..................................................................................... 39 4.7
Load more

Recommended publications
  • Avicin G Is a Potent Sphingomyelinase Inhibitor and Blocks Oncogenic K- and H-Ras Signaling Christian M
    www.nature.com/scientificreports OPEN Avicin G is a potent sphingomyelinase inhibitor and blocks oncogenic K- and H-Ras signaling Christian M. Garrido1, Karen M. Henkels1, Kristen M. Rehl1, Hong Liang2, Yong Zhou2, Jordan U. Gutterman3 & Kwang-jin Cho1 ✉ K-Ras must interact primarily with the plasma membrane (PM) for its biological activity. Therefore, disrupting K-Ras PM interaction is a tractable approach to block oncogenic K-Ras activity. Here, we found that avicin G, a family of natural plant-derived triterpenoid saponins from Acacia victoriae, mislocalizes K-Ras from the PM and disrupts PM spatial organization of oncogenic K-Ras and H-Ras by depleting phosphatidylserine (PtdSer) and cholesterol contents, respectively, at the inner PM leafet. Avicin G also inhibits oncogenic K- and H-Ras signal output and the growth of K-Ras-addicted pancreatic and non-small cell lung cancer cells. We further identifed that avicin G perturbs lysosomal activity, and disrupts cellular localization and activity of neutral and acid sphingomyelinases (SMases), resulting in elevated cellular sphingomyelin (SM) levels and altered SM distribution. Moreover, we show that neutral SMase inhibitors disrupt the PM localization of K-Ras and PtdSer and oncogenic K-Ras signaling. In sum, this study identifes avicin G as a new potent anti-Ras inhibitor, and suggests that neutral SMase can be a tractable target for developing anti-K-Ras therapeutics. Ras proteins are small GTPases that primarily localize to the inner-leafet of the plasma membrane (PM), switch- ing between an active GTP-bound state and inactive GDP-bound state1. In response to epidermal growth factor stimulation or receptor tyrosine kinase activation, guanine nucleotide exchange factors activate Ras by inducing the release of the guanine nucleotides and binding of GTP1.
    [Show full text]
  • Role of Acid Sphingomyelinase and IL-6 As Mediators Of
    Thorax Online First, published on July 27, 2016 as 10.1136/thoraxjnl-2015-208067 Respiratory research ORIGINAL ARTICLE Thorax: first published as 10.1136/thoraxjnl-2015-208067 on 27 July 2016. Downloaded from Role of acid sphingomyelinase and IL-6 as mediators of endotoxin-induced pulmonary vascular dysfunction Rachele Pandolfi,1,2,3 Bianca Barreira,1,2,3 Enrique Moreno,1,2,3 Victor Lara-Acedo,2 Daniel Morales-Cano,1,2,3 Andrea Martínez-Ramas,1,2,3 Beatriz de Olaiz Navarro,4 Raquel Herrero,1,5 José Ángel Lorente,1,5,6 Ángel Cogolludo,1,2,3 Francisco Pérez-Vizcaíno,1,2,3 Laura Moreno1,2,3 ▸ Additional material is ABSTRACT published online only. To view Background Pulmonary hypertension (PH) is frequently Key messages please visit the journal online (http://dx.doi.org/10.1136/ observed in patients with acute respiratory distress thoraxjnl-2015-208067). syndrome (ARDS) and it is associated with an increased risk of mortality. Both acid sphingomyelinase (aSMase) 1Ciber Enfermedades What is the key question? Respiratorias (CIBERES), activity and interleukin 6 (IL-6) levels are increased in ▸ Pulmonary hypertension and right ventricular Madrid, Spain patients with sepsis and correlate with worst outcomes, dysfunction are prominent prognostic features 2 Department of Pharmacology, but their role in pulmonary vascular dysfunction of acute respiratory distress syndrome (ARDS) School of Medicine, pathogenesis has not yet been elucidated. Therefore, the which, given the unclear pathophysiology and Universidad Complutense de Madrid, Madrid, Spain aim of this study was to determine the potential the lack of approved pharmacological therapies, 3Gregorio Marañón Biomedical contribution of aSMase and IL-6 in the pulmonary demand the identification of new therapeutic Research Institution (IiSGM), vascular dysfunction induced by lipopolysaccharide (LPS).
    [Show full text]
  • Abcam Enzymatic Activity Assay Kits
    Less haste, more speed 用更快的速度，從容的完成實驗 我們擁有品項齊全的抗體、相關免疫實驗試劑，以及數百種偵測酵素活性的試劑 Signal transduction Metabolism 套組，協助研究者進行訊號傳遞 ( )、代謝 ( )、神經 Neuroscience Gene regulation Epigenetics 科學 ( )、基因調控 ( )、表觀遺傳 ( )、 Cancer Cardiovascular Oxidative stress 癌症 ( )、心血管 ( )、氧化壓力 ( ) 等相關研 瀏覽相關產品目錄 Cell culture Tissue lysate 究。 涵蓋樣本來源諸如：細胞培養 ( )、組織裂解物 ( ) 或體 Body fluid 液 ( ) 等 。 我們總是追求卓越，完整呈現給您最佳的抗體與分析試劑盒， 以協助您快速取得所需的實驗結果。 Abcam 各項特惠活動進行中，詳情請洽 台灣代理 ― 伯森生技。 酵素測定成功秘訣 Activity assay kits 活性測定試劑套組 ( ) Target/ Protein Detection method Cat. no. Target/ Protein Detection method Cat. no. Acetylcholinesterase Colorimetric ab138871 Aldo-Keto Reductase Colorimetric ab211112 Acetylcholinesterase Fluorescent ab138872 Aldolase Colorimetric ab196994 Acetylcholinesterase Colorimetric/ ab138873 Alkaline Phosphatase Colorimetric ab83369 Fluorometric Alkaline Phosphatase Fluorescent ab83371 Acetyltransferase Fluorescent ab204536 Alkaline Phosphatase Fluorescent ab138887 Acid Phosphatase Colorimetric ab83367 Alkaline Phosphatase Luminescent ab233466 Acid Phosphatase Fluorescent ab83370 Alkaline Sphingomyelinase Colorimetric ab241039 Acid Sphingomyelinase Colorimetric ab252889 Alpha Galactosidase Fluorescent ab239716 Acidic Sphingomyelinase Fluorescent ab190554 Alpha-Glucosidase Colorimetric ab174093 Aconitase Colorimetric ab83459 Alpha-Ketoglutarate Dehydrogenase Colorimetric ab185440 Aconitase Colorimetric ab109712 Amylase Colorimetric ab102523 Adenosine Deaminase Fluorescent ab204695 Arginase Colorimetric ab180877 Adenosine Deaminase Colorimetric ab211093 Asparaginase Colorimetric/
    [Show full text]
  • Structural Study of the Acid Sphingomyelinase Protein Family
    Structural Study of the Acid Sphingomyelinase Protein Family Alexei Gorelik Department of Biochemistry McGill University, Montreal August 2017 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Alexei Gorelik, 2017 Abstract The acid sphingomyelinase (ASMase) converts the lipid sphingomyelin (SM) to ceramide. This protein participates in lysosomal lipid metabolism and plays an additional role in signal transduction at the cell surface by cleaving the abundant SM to ceramide, thus modulating membrane properties. These functions are enabled by the enzyme’s lipid- and membrane- interacting saposin domain. ASMase is part of a small family along with the poorly characterized ASMase-like phosphodiesterases 3A and 3B (SMPDL3A,B). SMPDL3A does not hydrolyze SM but degrades extracellular nucleotides, and is potentially involved in purinergic signaling. SMPDL3B is a regulator of the innate immune response and podocyte function, and displays a partially defined lipid- and membrane-modifying activity. I carried out structural studies to gain insight into substrate recognition and molecular functions of the ASMase family of proteins. Crystal structures of SMPDL3A uncovered the helical fold of a novel C-terminal subdomain, a slightly distinct catalytic mechanism, and a nucleotide-binding mode without specific contacts to their nucleoside moiety. The ASMase investigation revealed a conformational flexibility of its saposin domain: this module can switch from a detached, closed conformation to an open form which establishes a hydrophobic interface to the catalytic domain. This open configuration represents the active form of the enzyme, likely allowing lipid access to the active site. The SMPDL3B structure showed a narrow, boot-shaped substrate binding site that accommodates the head group of SM.
    [Show full text]
  • Acid Sphingomyelinase Regulates the Localization and Trafficking of Palmitoylated Proteins
    Chemistry and Biochemistry Faculty Publications Chemistry and Biochemistry 5-29-2019 Acid Sphingomyelinase Regulates the Localization and Trafficking of Palmitoylated Proteins Xiahui Xiong University of Nevada, Las Vegas, [email protected] Chia-Fang Lee Protea Biosciences Wenjing Li University of Nevada, Las Vegas, [email protected] Jiekai Yu University of Nevada, Las Vegas, [email protected] Linyu Zhu University of Nevada, Las Vegas SeeFollow next this page and for additional additional works authors at: https:/ /digitalscholarship.unlv.edu/chem_fac_articles Part of the Biochemistry, Biophysics, and Structural Biology Commons Repository Citation Xiong, X., Lee, C., Li, W., Yu, J., Zhu, L., Kim, Y., Zhang, H., Sun, H. (2019). Acid Sphingomyelinase Regulates the Localization and Trafficking of Palmitoylated Proteins. Biology Open 1-56. Company of Biologists. http://dx.doi.org/10.1242/bio.040311 This Article is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Article in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Article has been accepted for inclusion in Chemistry and Biochemistry Faculty Publications by an authorized administrator of Digital Scholarship@UNLV. For
    [Show full text]
  • 67-Kda Laminin Receptor Increases Cgmp to Induce Cancer- Selective Apoptosis
    67-kDa laminin receptor increases cGMP to induce cancer- selective apoptosis Motofumi Kumazoe, … , Koji Yamada, Hirofumi Tachibana J Clin Invest. 2013;123(2):787-799. https://doi.org/10.1172/JCI64768. Research Article The 67-kDa laminin receptor (67LR) is a laminin-binding protein overexpressed in various types of cancer, including bile duct carcinoma, colorectal carcinoma, cervical cancer, and breast carcinoma. 67LR plays a vital role in growth and metastasis of tumor cells and resistance to chemotherapy. Here, we show that 67LR functions as a cancer-specific death receptor. In this cell death receptor pathway, cGMP initiated cancer-specific cell death by activating the PKCδ/acid sphingomyelinase (PKCδ/ASM) pathway. Furthermore, upregulation of cGMP was a rate-determining process of 67LR- dependent cell death induced by the green tea polyphenol (–)-epigallocatechin-3-O-gallate (EGCG), a natural ligand of 67LR. We found that phosphodiesterase 5 (PDE5), a negative regulator of cGMP, was abnormally expressed in multiple cancers and attenuated 67LR-mediated cell death. Vardenafil, a PDE5 inhibitor that is used to treat erectile dysfunction, significantly potentiated the EGCG-activated 67LR-dependent apoptosis without affecting normal cells and prolonged the survival time in a mouse xenograft model. These results suggest that PDE5 inhibitors could be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death. Find the latest version: https://jci.me/64768/pdf Related Commentary, page 556 Research article 67-kDa laminin receptor increases cGMP to induce cancer-selective apoptosis Motofumi Kumazoe,1 Kaori Sugihara,1 Shuntaro Tsukamoto,1 Yuhui Huang,1 Yukari Tsurudome,1 Takashi Suzuki,1 Yumi Suemasu,1 Naoki Ueda,1 Shuya Yamashita,1 Yoonhee Kim,1 Koji Yamada,1 and Hirofumi Tachibana1,2 1Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, and 2Food Functional Design Research Center, Kyushu University, Fukuoka, Japan.
    [Show full text]
  • Phosphatidylcholine-Specific Phospholipase C
    The EMBO Journal vol.14 no.23 pp.5859-5868, 1995 Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal Maria Grazia Cifone1, Paola Roncaioli1, 1994; Krammer et al., 1994; Nagata and Golstein, 1995). Ruggero De Maria2, Grazia Camarda2, TNF-Rl and Fas/APO-1 belong to a family of receptors, Angela Santoni3, Giovina Ruberti4 and which includes the low-affinity nerve growth factor recep- tor, CD40, CD30, CD27, OX40, tumour necrosis factor Roberto Testi2'5 receptor p75, involved in mediating a variety of cell 'Department of Experimental Medicine, University of L'Aquila, growth modulation or cell differentiation effects. While 67100 L'Aquila, 2Department of Experimental Medicine and all members of the family typically display two or more Biochemical Sciences, University of Rome 'Tor Vergata', via di Tor cysteine-rich subdomains in the extracellular portion, TNF- Vergata 135, 100133 Rome, 3Department of Experimental Medicine 1 of limited homology and Pathology, University of Rome 'La Sapienza', 00161 Rome and RI and Fas/APO- share also a region 4Department of Immunobiology, Institute of Cell Biology, CNR, close to the intracellular C-terminus, which is essential 00137 Rome, Italy for the ability of the receptors to generate the apoptotic 5Corresponding author signal and which has been named 'death domain' (Itoh and Nagata, 1993; Tartaglia et al., 1993). This region The early signals generated following cross-linking of might be involved in binding putative cytosolic effectors, Fas/APO-1, a transmembrane receptor whose engage- also expressing death domain-like regions, recently identi- ment by ligand results in apoptosis induction, were fied by yeast two-hybrid system searches (Boldin et al., investigated in human HuT78 lymphoma cells.
    [Show full text]
  • Consensus Recommendation for a Diagnostic Guideline for Acid Sphingomyelinase Deficiency
    Official journal of the American College of Medical Genetics and Genomics SPECIAL ARTICLE Open Consensus recommendation for a diagnostic guideline for acid sphingomyelinase deficiency Margaret M. McGovern, MD, PhD1, Carlo Dionisi-Vici, MD2, Roberto Giugliani, MD, PhD3, Paul Hwu, MD, PhD4, Olivier Lidove, MD5, Zoltan Lukacs, PhD6, Karl Eugen Mengel, MD7, Pramod K. Mistry, MD, PhD8, Edward H. Schuchman, PhD9 and Melissa P. Wasserstein, MD10 Background: Acid sphingomyelinase deficiency (ASMD) is a rare, base and share personal experience in order to develop a guideline progressive, and often fatal lysosomal storage disease. The underlying for diagnosis of the various ASMD phenotypes. metabolic defect is deficiency of the enzyme acid sphingomyelinase that results in progressive accumulation of sphingomyelin in target Conclusions: Although care of ASMD patients is typically provided tissues. ASMD manifests as a spectrum of severity ranging from rap- by metabolic disease specialists, the guideline is directed at a wide idly progressive severe neurovisceral disease that is uniformly fatal to range of providers because it is important for primary care providers more slowly progressive chronic neurovisceral and chronic visceral (e.g., pediatricians and internists) and specialists (e.g., pulmonolo- forms. Disease management is aimed at symptom control and regular gists, hepatologists, and hematologists) to be able to identify ASMD. assessments for multisystem involvement. Genet Med advance online publication 13 April 2017 Purpose and methods: An
    [Show full text]
  • Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism
    International Journal of Molecular Sciences Review Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism Bernadette Breiden 1 and Konrad Sandhoff 2,* 1 Independent Researcher, 50181 Bedburg, Germany; [email protected] 2 Membrane Biology and Lipid Biochemistry Unit, LIMES Institute, University of Bonn, 53121 Bonn, Germany * Correspondence: [email protected]; Tel.: +49-228-73-5346 Abstract: Here, we present the main features of human acid sphingomyelinase (ASM), its biosyn- thesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal lu- minal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrol- yses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumu- lation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical Citation: Breiden, B.; Sandhoff, K. characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact Acid Sphingomyelinase, a Lysosomal on physiological processes will be discussed.
    [Show full text]
  • K735 -100Lysophosphatidylcholine Assay Kit (Colorimetric/Fluorometric)
    FOR RESEARCH USE ONLY! Lysophosphatidylcholine Assay Kit (Colorimetric/Fluorometric) Rev 07/19 (Catalog # K735-100; 100 assays; Store at -20°C) I. Introduction: Lysophosphatidylcholine (LPC), also referred to as lysolecithin, is a phospholipid intermediate whose concentrations have been correlated with such diseases as cancer, artherosclerosis and diabetes. Generally produced through the action of phospholipases on phosphatidylcholine, LPC consists of a glycerol backbone with a phosphocholine at one hydroxyl group and a single acyl chain attached to either the 1- or 2- position hydroxyl of the glycerol moiety. LPC is typically at concentrations in the high micromolar range in human serum and plasma, and is capable of activating second messengers such as Ca2+ and cyclic AMP. It is through these pathways that LPC has been found to affect such biological events as the pro-inflammatory response and intestinal uptake. BioVision’s Lysophosphatidylcholine Assay Kit utilizes LPC-specific enzymes to generate an intermediate that then reacts with a probe, yielding a signal that can be quantified either colorimetrically or fluorometrically, and is proportional to the amount of LPC present in the sample. When used as described, the assay is capable of detecting as little as 10 pmole of lysophosphatidylcholine. Enzyme Mix Developer/Probe LPC Intermediate Colorimetric (570 nm)/Fluorescence (λex/em = 535 nm/587 nm) II. Applications: Measurement of LPC content of various tissue/cell extracts Determination of LPC concentration in biological fluids III. Sample Type: Tissue and cell lysates Biological fluids (e.g. serum, plasma) IV. Kit Contents: Components K735-100 Cap Code Part Number LPC Assay Buffer 25 ml WM K735-100-1 LPC Enzyme Mix 1 vial Purple K735-100-2 LPC Developer 1 vial Green K735-100-3 LPC Probe (in DMSO) 200 µl Red K735-100-4 LPC Standard (0.5 µmol) 1 vial Yellow K735-100-5 2X Lipid Resuspension Buffer 2 x 1 ml Amber K735-100-6 V.
    [Show full text]
  • Decreased Activity of Blood Acid Sphingomyelinase in the Course of Multiple Myeloma
    International Journal of Molecular Sciences Article Decreased Activity of Blood Acid Sphingomyelinase in the Course of Multiple Myeloma Marzena W ˛atek 1,2,* , Ewelina Piktel 3, Joanna Barankiewicz 1, Ewa Sierlecka 4, Sylwia Ko´sciołek-Zgódka 4, Anna Chabowska 5, Łukasz Suprewicz 3, Przemysław Wolak 2 , Bonita Durna´s 2, Robert Bucki 2,3 and Ewa Lech-Mara ´nda 1,6 1 Institute of Hematology and Transfusion Medicine, Indiry Gandhi 14, 02-776 Warsaw, Poland; [email protected] (J.B.); [email protected] (E.L.-M.) 2 Department of Microbiology and Immunology, The Faculty of Medicine and Health Sciences of the Jan Kochanowski University in Kielce, Stefana Zeromskiego˙ 5, 25-001 Kielce, Poland; [email protected] (P.W.); [email protected] (B.D.); [email protected] (R.B.) 3 Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Mickiewicza 2c, 15-222 Bialystok, Poland; [email protected] (E.P.); [email protected] (Ł.S.) 4 Holy Cross Cancer Center, Artwinskiego 4, 25-734 Kielce, Poland; [email protected] (E.S.); [email protected] (S.K.-Z.) 5 Regional Blood Transfusion Center in Bialystok, 15-950 Bialystok, Poland; [email protected] 6 Centre of Postgraduate Medical Education, Marymoncka 99/103, 01-813 Warsaw, Poland * Correspondence: [email protected]; Tel.: +48-41-349-69-09; +48-41-349-69-16 Received: 8 November 2019; Accepted: 27 November 2019; Published: 30 November 2019 Abstract: Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway.
    [Show full text]
  • Secretory Sphingomyelinase in Health and Disease
    Biol. Chem. 2015; 396(6-7): 707–736 Review Open Access Johannes Kornhuber*, Cosima Rhein, Christian P. Müller and Christiane Mühle Secretory sphingomyelinase in health and disease Abstract: Acid sphingomyelinase (ASM), a key enzyme may be both a promising clinical chemistry marker and a in sphingolipid metabolism, hydrolyzes sphingomy- therapeutic target. elin to ceramide and phosphorylcholine. In mammals, the expression of a single gene, SMPD1, results in two Keywords: ceramide; inflammation; lipids; secretory forms of the enzyme that differ in several characteristics. sphingomyelinase; sphingomyelin; sphingomyelinase. Lysosomal ASM (L-ASM) is located within the lysosome, 2+ requires no additional Zn ions for activation and is gly- DOI 10.1515/hsz-2015-0109 cosylated mainly with high-mannose oligosaccharides. Received January 20, 2015; accepted February 16, 2015; previously By contrast, the secretory ASM (S-ASM) is located extra- published online March 24, 2015 cellularly, requires Zn2+ ions for activation, has a complex glycosylation pattern and has a longer in vivo half-life. In this review, we summarize current knowledge regarding the physiology and pathophysiology of S-ASM, includ- Introduction ing its sources and distribution, molecular and cellular Acid sphingomyelinase (ASM, EC 3.1.4.12) plays a major mechanisms of generation and regulation and relevant role in sphingolipid metabolism because it catalyzes the in vitro and in vivo studies. Polymorphisms or mutations hydrolysis of sphingomyelin (SM) to ceramide and phos- of SMPD1 lead to decreased S-ASM activity, as detected in phorylcholine. Ceramide and related products, such as patients with Niemann-Pick disease B. Thus, lower serum/ spingosine-1-phosphate, are important lipid signaling plasma activities of S-ASM are trait markers.
    [Show full text]