DOCSLIB.ORG

Down-Regulation of Nedd4l Is Associated with the Aggressive Progression and Worse Prognosis of Malignant Glioma

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Down-Regulation of Nedd4l Is Associated with the Aggressive Progression and Worse Prognosis of Malignant Glioma Jpn J Clin Oncol 2012;42(3)196–201 doi:10.1093/jjco/hyr195 Advance Access Publication 4 January 2012 Down-regulation of Nedd4L is Associated with the Aggressive Progression and Worse Prognosis of Malignant Glioma Shiming He1,†, Jianping Deng1,†, Gang Li1, Boliang Wang2, Yizhan Cao2 and Yanyang Tu2,* 1Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University and 2Department of Emergency, Tangdu Hospital, Fourth Military Medical University, Xi’an, China Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 *For reprints and all correspondence: Yanyang Tu, Department of Emergency, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China. E-mail: [email protected] †These authors contributed equally to this work. Received September 6, 2011; accepted December 10, 2011 Objective: Human neural precursor cell-expressed developmentally down-regulated 4 like (Nedd4L), a ubiquitin protein ligase, is expressed by various cancer cells and might have an oncogenic property. Its expression pattern in glioma tissues is unknown. Therefore, the aim of this study was to investigate whether Nedd4L is present in glioma and to evaluate the correlation of Nedd4L expression with the progression and prognosis of the disease. Methods: Immunohistochemistry and western blot were used to investigate the expression of Nedd4L protein in 128 patients with gliomas. Results: Immunohistochemistry showed a strong-to-weak range of Nedd4L staining with increasing pathologic grade of glioma (P , 0.001), which was in line with the results from western blot analysis. In addition, a non-parametric analysis revealed that the attenuated Nedd4L expression was significantly correlated with a large tumor diameter (P ¼ 0.02), low Karnofsky performance score (P ¼ 0.008), frequent intra-tumor necrosis (P ¼ 0.01) and worse overall survival (P ¼ 0.009). Furthermore, multivariate analysis showed that Nedd4L expression (P ¼ 0.02) and intra-tumor necrosis (P ¼ 0.03) were two important independent prognostic factors identified by the Cox proportional hazards model. Conclusions: Our results provide convincing evidence for the first time that the expression of Nedd4L is down-regulated in human gliomas. The glioma patients with lower Nedd4L expression have a worse prognosis. Key words: neural precursor cell-expressed developmentally down-regulated 4 like – glioma – immunohistochemistry – western blot analysis – expression INTRODUCTION surgical resection, radiation therapy and chemotherapy (2). Glioma, as the most common primary brain tumors in adult Numerous studies have showed that age, performance status, humans, continues to be the cause of a disproportionate level histologic grade and tumor necrosis are important prognostic of morbidity and mortality across a wide range of indivi- factors for gliomas. However, these clinical parameters do duals. The key features of this disease are local invasive not fully account for the observed variation in survival rates growth and strong angiogenesis. Gliomas are histologically and the prognosis of both high- and low-grade tumors classified into four grades (Grades I–IV), according to the remains heterogeneous (3). Therefore, additional indicators World Health Organization (WHO) guidelines (1). Grade IV are needed to more accurately determine the prognosis of glioma, glioblastoma, has the worst prognosis even after patients with gliomas. # The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Jpn J Clin Oncol 2012;42(3) 197 The neural precursor cell-expressed developmentally radiotherapy prior to surgery. The clinicopathological fea- down-regulated 4-like (Nedd4L) gene encodes a ubiquitin tures and the treatment strategies of all the patients are indi- ligase that targets the epithelial sodium channel (ENaC) for catedinTable1. Paraffin and snap-frozen sections of degradation (4). It participates in the regulation of plasma non-neoplastic brain tissues from 10 patients with intractable volume and blood pressure by controlling expression of epilepsy were also included as controls. A 5-year follow-up ENaC which is a heteromultimeric protein complex. The was performed, and all patients had a complete follow-up ubiquitination of ENaC is catalyzed by Nedd4L, initiating until death. The overall survival time was calculated from ubiquitin-mediated endocytosis and lysosomal targeting of the date of the initial surgical operation to death. Patients, ENaC for its removal from the luminal cell membrane in who died of diseases not directly related to their gliomas, or renal collecting ducts (5). Recent studies have demonstrated due to unexpected events, were excluded from this study. In that Nedd4L targets a broad range of molecules along with addition, 20 glioma specimens [5 pilocytic astrocytomas ENaC and is thus thought to be involved in various biologic- (WHO I), 3 diffuse astrocytomas (WHO II), 3 anaplasia Downloaded from https://academic.oup.com/jjco/article/42/3/196/1015725 by guest on 28 September 2021 al properties other than regulating ion channels (6). Human astrocytomas (WHO III) and 9 primary glioblastomas (WHO Nedd4L is a homologue of the mouse Nedd4-2, which was IV)] were snap frozen in liquid nitrogen and stored at identified as a novel protein which could inhibit transforming 2808C following surgery for analysis by western blot growth factor b (TGF-b) signaling in TGF-b receptors and analysis. Smadslevels(7). It has been reported that TGF-b signaling is involved in a variety of biological processes, including regulation of cell growth, angiogenesis, immune response IMMUNOHISTOCHEMISTRY ASSAY and modulation of extracellular matrix (8). In gliomas, previ- Formalin-fixed, paraffin-embedded, sectioned tissues (4 mm ous studies have indicated that TGF-b signaling plays an thick) were immunostained using the Labelled Streptavidin important role in the pathobiology of invasion of the tumor Biotin 2 System (BioGenex; San Ramon, CA, USA). (9). Therefore, investigation into the TGF-b system in Following peroxidase blocking with 0.3% H2O2/methanol glioma is needed in order to learn whether there are candi- for 30 min, specimens were blocked with phosphate-buffered date prognostic markers in this system can be manipulated saline (PBS) containing 5% normal horse serum (Vector for survival prediction of gliomas. Nedd4L has been Laboratories Inc., Burlingame, CA, USA). All incubations detected in various cancer cells and might have an oncogenic with a monoclonal antibody against human Nedd4L property. However, its expression pattern in glioma tissues is (HPA024618, Sigma-Aldrich, St Louis, MO, USA) at 1:10 unknown. Therefore, the aim of this study was to investigate dilution were carried out overnight at 48C. Then, the whether Nedd4L is present in glioma and to evaluate the correlation of Nedd4L expression with the progression and prognosis of the disease. Table 1. Clinicopathological features of 128 patients with gliomas Features WHO I WHO II WHO III WHO IV PATIENTS AND METHODS Case no. 18 14 38 58 PATIENTS AND TISSUE SAMPLES Mean age (years) 38.6 45.9 53.1 54.2 This study was approved by the Research Ethics Committee Gender of Tangdu Hospital, Fourth Military Medical University, Male 12 6 25 33 China. Written informed consent was obtained from all of Female 6 8 13 25 the patients. All specimens were handled and made anonymous according to the ethical and legal standards. KPS A total of 128 formalin-fixed, paraffin-embedded speci- .80 15 11 9 15 mens of gliomas resected between 2000 and 2005 were ,80 3 3 29 43 retrieved from the archives of the Pathology Department of Surgery Tangdu Hospital, Fourth Military Medical University, China. Gross total resection 18 14 28 38 All the slides were re-evaluated according to the WHO clas- Partial resection 0 0 9 15 sifications (1) by two pathologists, with differences resolved by careful discussion. A total of 76 males and 52 females Biopsy 0 0 1 5 (1.46:1) were enrolled in this study, and the median age was Adjuvant treatment 52 years (range, 12–71). Thirty-two of the 128 gliomas were Radiotherapy 0 0 30 12 classified as low-grade gliomas [18 pilocytic astrocytomas Chemotherapy 0 1 0 6 (WHO I) and 14 diffuse astrocytomas (WHO II)] and 96 Radiotherapy and 00 5 28 were classified as high-grade gliomas [38 anaplasia astrocy- chemotherapy combination tomas (WHO III) and 58 primary glioblastomas (WHO IV)]. None of the patients had received chemotherapy or KPS, Karnofsky performance score. 198 Down-regulation of Nedd4L in glioma specimens were briefly washed in PBS and incubated at Nedd4L as an optical densitometry ratio that was scored as room temperature with the anti-rabbit antibody and avidin– the densitometry of Nedd4L relative to the densitometry of biotin peroxidase (Vector Laboratories Inc.). The specimens b-actin. were then washed in PBS and color-developed by diamino- benzidine solution (Dako Corporation, Carpinteria, CA, STATISTICAL ANALYSIS USA). After washing with water, specimens were counter- stained with Meyer’s hematoxylin (Sigma Chemical Co., St All computations were carried out using the software of Louis, MO, USA). Non-neoplastic brain tissues were used as SPSS version 13.0 for Windows (SPSS Inc., IL, USA). Data control tissues and non-immune IgG was also used as a were expressed as means + standard deviation. The amount negative control antibody for immunohistochemical staining. of Nedd4L in positively expressed tissue samples was pre- Assessment
Load more

Recommended publications
  • Screening and Identification of Key Biomarkers in Clear Cell Renal Cell Carcinoma Based on Bioinformatics Analysis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Screening and identification of key biomarkers in clear cell renal cell carcinoma based on bioinformatics analysis Basavaraj Vastrad1, Chanabasayya Vastrad*2 , Iranna Kotturshetti 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. 3. Department of Ayurveda, Rajiv Gandhi Education Society`s Ayurvedic Medical College, Ron, Karnataka 562209, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.21.423889; this version posted December 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignancy of the urinary system. The pathogenesis and effective diagnosis of ccRCC have become popular topics for research in the previous decade. In the current study, an integrated bioinformatics analysis was performed to identify core genes associated in ccRCC. An expression dataset (GSE105261) was downloaded from the Gene Expression Omnibus database, and included 26 ccRCC and 9 normal kideny samples. Assessment of the microarray dataset led to the recognition of differentially expressed genes (DEGs), which was subsequently used for pathway and gene ontology (GO) enrichment analysis.
    [Show full text]
  • NEDD4L Regulates the Proliferation and Metastasis of Non-Small-Cell Lung Cancer by Mediating CPNE1 Ubiquitination
    NEDD4L Regulates the Proliferation and Metastasis of Non-small-cell Lung Cancer by Mediating CPNE1 Ubiquitination Ruochen Zhang Institute of Resporatory Diseases, soochow University Yuanyuan Zeng Institute of Respiratory Diseases, Soochow University. Weijie Zhang Soochow University Yue Li Soochow University Jieqi Zhou Soochow University Yang Zhang Soochow University Anqi Wang Soochow University Yantian Lv Soochow University Jianjie Zhu First Aliated Hospital of Soochow University Zeyi Liu ( [email protected] ) First Aliated Hospital of Soochow University https://orcid.org/0000-0003-2528-6909 Jian-an Huang First Aliated Hospital of Soochow University Research Keywords: NSCLC, CPNE1, NEDD4L, ubiquitination, degradation Posted Date: February 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-250598/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Abstract High expression of CPNE1 is positively correlated with the occurrence, TNM stage, lymph node metastasis, and distant metastasis of non-small-cell lung cancer (NSCLC), suggesting that CPNE1 may be an effective target for the treatment of NSCLC. No direct role of post-translational modication of CPNE1 in NSCLC has been reported. This study conrms that CPNE1 is degraded by two pathways: the ubiquitin-proteasome pathway and the autophagy- lysosome pathway. CPNE1 binds with the ubiquitin molecule via its K157 residue. Moreover, we determine that the ubiquitin ligase NEDD4L can mediate the ubiquitination of CPNE1 and promote its degradation. In addition, we nd that NEDD4L knockout promotes the proliferation and metastasis of NSCLC cells by regulating CPNE1. This study aims to further investigate the mechanism of CPNE1 ubiquitination in the occurrence and development of NSCLC and provide a new potential target for NSCLC treatment.
    [Show full text]
  • NEDD4 2 (NEDD4L) (NM 001144964) Human Tagged ORF Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC227866 NEDD4 2 (NEDD4L) (NM_001144964) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: NEDD4 2 (NEDD4L) (NM_001144964) Human Tagged ORF Clone Tag: Myc-DDK Symbol: NEDD4L Synonyms: hNEDD4-2; NEDD4-2; NEDD4.2; PVNH7; RSP5 Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 6 NEDD4 2 (NEDD4L) (NM_001144964) Human Tagged ORF Clone – RC227866 ORF Nucleotide >RC227866 representing NM_001144964 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGGAGCGACCCTATACATTTAAGGACTTTCTCCTCAGACCAAGAAGTCATAAGTCTCGAGTTAAGGGAT TTTTGCGATTGAAAATGGCCTATATGCCAAAAAATGGAGGTCAAGATGAAGAAAACAGTGACCAGAGGGA TGACATGGAGCATGGATGGGAAGTTGTTGACTCAAATGACTCGGCTTCTCAGCACCAAGAGGAACTTCCT CCTCCTCCTCTGCCTCCCGGGTGGGAAGAAAAAGTGGACAATTTAGGCCGAACTTACTATGTCAACCACA ACAACCGGACCACTCAGTGGCACAGACCAAGCCTGATGGACGTGTCCTCGGAGTCGGACAATAACATCAG ACAGATCAACCAGGAGGCAGCACACCGGCGCTTCCGCTCCCGCAGGCACATCAGCGAAGACTTGGAGCCC GAGCCCTCGGAGGGCGGGGATGTCCCCGAGCCTTGGGAGACCATTTCAGAGGAAGTGAATATCGCTGGAG ACTCTCTCGGTCTGGCTCTGCCCCCACCACCGGCCTCCCCAGGATCTCGGACCAGCCCTCAGGAGCTGTC
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • Inherited Renal Tubulopathies—Challenges and Controversies
    G C A T T A C G G C A T genes Review Inherited Renal Tubulopathies—Challenges and Controversies Daniela Iancu 1,* and Emma Ashton 2 1 UCL-Centre for Nephrology, Royal Free Campus, University College London, Rowland Hill Street, London NW3 2PF, UK 2 Rare & Inherited Disease Laboratory, London North Genomic Laboratory Hub, Great Ormond Street Hospital for Children National Health Service Foundation Trust, Levels 4-6 Barclay House 37, Queen Square, London WC1N 3BH, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-2381204172; Fax: +44-020-74726476 Received: 11 February 2020; Accepted: 29 February 2020; Published: 5 March 2020 Abstract: Electrolyte homeostasis is maintained by the kidney through a complex transport function mostly performed by specialized proteins distributed along the renal tubules. Pathogenic variants in the genes encoding these proteins impair this function and have consequences on the whole organism. Establishing a genetic diagnosis in patients with renal tubular dysfunction is a challenging task given the genetic and phenotypic heterogeneity, functional characteristics of the genes involved and the number of yet unknown causes. Part of these difficulties can be overcome by gathering large patient cohorts and applying high-throughput sequencing techniques combined with experimental work to prove functional impact. This approach has led to the identification of a number of genes but also generated controversies about proper interpretation of variants. In this article, we will highlight these challenges and controversies. Keywords: inherited tubulopathies; next generation sequencing; genetic heterogeneity; variant classification. 1. Introduction Mutations in genes that encode transporter proteins in the renal tubule alter kidney capacity to maintain homeostasis and cause diseases recognized under the generic name of inherited tubulopathies.
    [Show full text]
  • Integrated Bioinformatics Analysis of the NEDD4 Family Reveals a Prognostic Value of NEDD4L in Clear-Cell Renal Cell Cancer
    Integrated bioinformatics analysis of the NEDD4 family reveals a prognostic value of NEDD4L in clear-cell renal cell cancer Hui Zhao1,2,* Junjun Zhang3,* Xiaoliang Fu4 Dongdong Mao1 Xuesen Qi1 Shuai Liang1 Gang Meng1 Zewen Song3 Ru Yang5 Zhenni Guo6 Binghua Tong6 Meiqing Sun6 Baile Zuo7 Guoyin Li6,8 1 Department of Urology, Affiliated Hospital of Weifang Medical University, Weifang, China 2 Department of Urology, China Rehabilitation Research Centre, Rehabilitation School of Capital Medical University, Beijing, China 3 Department of Oncology, The Third Xiangya Hospital of Central South University, Changsha, China 4 Department of Urology, The Second Affiliated Hospital of Air Force Medical University, Xian, China 5 Henan Key Laboratory of Neurorestoratology, The First Affliated Hospital of Xinxiang Medical University, Weihui, China 6 College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, China 7 Tumor Molecular Immunology and Immunotherapy Laboratory, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China 8 Academy of Medical Science, Zhengzhou University, Zhengzhou, China * These authors contributed equally to this work. ABSTRACT The members of the Nedd4-like E3 family participate in various biological processes. However, their role in clear cell renal cell carcinoma (ccRCC) is not clear. This study systematically analyzed the Nedd4-like E3 family members in ccRCC data sets from multiple publicly available databases. NEDD4L was identified as the only NEDD4 family member differentially expressed in ccRCC compared with normal samples. Bioinformatics tools were used to characterize the function of NEDD4L in ccRCC. It indicated that NEDD4L might regulate cellular energy metabolism by co-expression Submitted 3 February 2021 analysis, and subsequent gene ontology (GO) and Kyoto Encyclopedia of Genes and Accepted 7 July 2021 Genomes (KEGG) analysis.
    [Show full text]
  • Systems-Level Identification of PKA-Dependent Signaling In
    Systems-level identification of PKA-dependent PNAS PLUS signaling in epithelial cells Kiyoshi Isobea, Hyun Jun Junga, Chin-Rang Yanga,J’Neka Claxtona, Pablo Sandovala, Maurice B. Burga, Viswanathan Raghurama, and Mark A. Kneppera,1 aEpithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1603 Edited by Peter Agre, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, and approved August 29, 2017 (received for review June 1, 2017) Gproteinstimulatoryα-subunit (Gαs)-coupled heptahelical receptors targets are as yet unidentified. Some of the known PKA targets regulate cell processes largely through activation of protein kinase A are other protein kinases and phosphatases, meaning that PKA (PKA). To identify signaling processes downstream of PKA, we de- activation is likely to result in indirect changes in protein phos- leted both PKA catalytic subunits using CRISPR-Cas9, followed by a phorylation manifest as a signaling network, the details of which “multiomic” analysis in mouse kidney epithelial cells expressing the remain unresolved. To identify both direct and indirect targets of Gαs-coupled V2 vasopressin receptor. RNA-seq (sequencing)–based PKA in mammalian cells, we used CRISPR-Cas9 genome editing transcriptomics and SILAC (stable isotope labeling of amino acids in to introduce frame-shifting indel mutations in both PKA catalytic cell culture)-based quantitative proteomics revealed a complete loss subunit genes (Prkaca and Prkacb), thereby eliminating PKA-Cα of expression of the water-channel gene Aqp2 in PKA knockout cells. and PKA-Cβ proteins. This was followed by use of quantitative SILAC-based quantitative phosphoproteomics identified 229 PKA (SILAC-based) phosphoproteomics to identify phosphorylation phosphorylation sites.
    [Show full text]
  • NEDD4L Monoclonal Antibody (M04), Clone 1D2
    NEDD4L monoclonal antibody (M04), clone 1D2 Catalog # : H00023327-M04 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a partial recombinant Western Blot (Transfected lysate) Description: NEDD4L. Immunogen: NEDD4L (AAH32597, 1 a.a. ~ 100 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: MATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLY VADENRELALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLT enlarge Host: Mouse Western Blot (Recombinant protein) Reactivity: Human Immunoprecipitation Isotype: IgG2a Kappa Quality Control Antibody Reactive Against Recombinant Protein. Testing: enlarge Sandwich ELISA (Recombinant protein) enlarge Western Blot detection against Immunogen (36.63 KDa) . ELISA Storage Buffer: In 1x PBS, pH 7.4 Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: MSDS: Download Datasheet: Download Applications Western Blot (Transfected lysate) Page 1 of 3 2019/2/4 Western Blot analysis of NEDD4L expression in transfected 293T cell line by NEDD4L monoclonal antibody (M04), clone 1D2. Lane 1: NEDD4L transfected lysate (Predicted MW: 104.9 KDa). Lane 2: Non-transfected lysate. Protocol Download Western Blot (Recombinant protein) Protocol Download Immunoprecipitation Immunoprecipitation of NEDD4L transfected lysate using anti-NEDD4L monoclonal antibody and Protein A Magnetic Bead (U0007), and immunoblotted with NEDD4L monoclonal antibody. Protocol Download Sandwich ELISA (Recombinant protein) Detection
    [Show full text]
  • Chromosome 18 Gene Dosage Map 2.0
    Human Genetics (2018) 137:961–970 https://doi.org/10.1007/s00439-018-1960-6 ORIGINAL INVESTIGATION Chromosome 18 gene dosage map 2.0 Jannine D. Cody1,2 · Patricia Heard1 · David Rupert1 · Minire Hasi‑Zogaj1 · Annice Hill1 · Courtney Sebold1 · Daniel E. Hale1,3 Received: 9 August 2018 / Accepted: 14 November 2018 / Published online: 17 November 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract In 2009, we described the first generation of the chromosome 18 gene dosage maps. This tool included the annotation of each gene as well as each phenotype associated region. The goal of these annotated genetic maps is to provide clinicians with a tool to appreciate the potential clinical impact of a chromosome 18 deletion or duplication. These maps are continually updated with the most recent and relevant data regarding chromosome 18. Over the course of the past decade, there have also been advances in our understanding of the molecular mechanisms underpinning genetic disease. Therefore, we have updated the maps to more accurately reflect this knowledge. Our Gene Dosage Map 2.0 has expanded from the gene and phenotype maps to also include a pair of maps specific to hemizygosity and suprazygosity. Moreover, we have revamped our classification from mechanistic definitions (e.g., haplosufficient, haploinsufficient) to clinically oriented classifications (e.g., risk factor, conditional, low penetrance, causal). This creates a map with gradient of classifications that more accurately represents the spectrum between the two poles of pathogenic and benign. While the data included in this manuscript are specific to chro- mosome 18, they may serve as a clinically relevant model that can be applied to the rest of the genome.
    [Show full text]
  • The Role of Nedd4l in the Regulation of Muscle Stem Cell Function
    The Role of Nedd4L in the Regulation of Muscle Stem Cell Function Claudia Yvette Dominici Department of Human Genetics, McGill University, Montreal December 2016 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Claudia Dominici, 2016 Abstract Muscle wasting diseases exist as a spectrum of diseases in which muscle function is impaired. Adult stem cells are the drivers of regeneration in damaged tissue. In patients with muscle degenerative diseases, the balance between the processes of muscle stem cell (MuSC) self-renewal and differentiation is perturbed; thus creating an environment that is not conducive to tissue homeostasis. Therefore, in order to assist in developing effective cell-based therapies for muscle wasting diseases, we must investigate the molecular mechanisms that are crucial for maintaining the critical balance that promotes normal MuSC function. E3 ubiquitin ligases target proteins for degradation through the proteasome, and they are known to be critical regulators of cell function. Interestingly, our data indicate that in muscle stem cells, Nedd4L (Neural Precursor Cell Expressed, Developmentally Down-Regulated 4-Like) is the only E3 ubiquitin ligase that is highly up regulated during a specific window following MuSC activation. Given this unique time frame, we hypothesize that Nedd4L is involved in a specific set of cellular functions that determine whether an activated satellite cell will self-renew or differentiate. In order to elucidate the function of Nedd4L in the regulation of MuSCs, I have utilized a series of in vitro and in vivo analyses. C2C12 mouse myoblasts were used to generate stable cell lines overexpressing Nedd4L and a mutated Nedd4L to assess the effect of Nedd4L on their proliferation and differentiation.
    [Show full text]
  • Regulators of Epithelial Sodium Channels in Aldosterone-Sensitive Distal Nephrons (ASDN): Critical Roles of Nedd4l/Nedd4-2 and Salt-Sensitive Hypertension
    International Journal of Molecular Sciences Review Regulators of Epithelial Sodium Channels in Aldosterone-Sensitive Distal Nephrons (ASDN): Critical Roles of Nedd4L/Nedd4-2 and Salt-Sensitive Hypertension Tomoaki Ishigami *, Tabito Kino, Shintaro Minegishi, Naomi Araki, Masanari Umemura , Hisako Ushio, Sae Saigoh and Michiko Sugiyama Department of Medical Science and Cardio-Renal Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Yokohama, Kanazawa-Ku 236-0004, Japan; [email protected] (T.K.); [email protected] (S.M.); [email protected] (N.A.); [email protected] (M.U.); [email protected] (H.U.); [email protected] (S.S.); [email protected] (M.S.) * Correspondence: [email protected]; Tel.: +81-45-787-2635 (ext. 6312) Received: 19 April 2020; Accepted: 27 May 2020; Published: 29 May 2020 Abstract: Ubiquitination is a representative, reversible biological process of the post-translational modification of various proteins with multiple catalytic reaction sequences, including ubiquitin itself, in addition to E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, deubiquitinating enzymes, and proteasome degradation. The ubiquitin–proteasome system is known to play a pivotal role in various molecular life phenomena, including the cell cycle, protein quality, and cell surface expressions of ion-transporters. As such, the failure of this system can lead to cancer, neurodegenerative diseases, cardiovascular diseases, and hypertension. This review article discusses Nedd4-2/NEDD4L, an E3-ubiquitin ligase involved in salt-sensitive hypertension, drawing from detailed genetic dissection analysis and the development of genetically engineered mice model. Based on our analyses, targeting therapeutic regulations of ubiquitination in the fields of cardio-vascular medicine might be a promising strategy in future.
    [Show full text]
  • Identification of Pain Genes Contributing to Ciguatoxin-Induced Pain Using Haplotype-Based Computational Genetic Mapping in Mice
    Identification of pain genes contributing to ciguatoxin-induced pain using haplotype-based computational genetic mapping in mice by Julian Soh Bachelor of Medicine/Bachelor of Surgery Submitted for the degree of Masters of Philosophy at The University of Queensland in 2018 School of Medicine Abstract Pain is a complex process involving numerous underlying genetic contributions still not completely appreciated, which may explain the high degree of interindividual differences seen with nociception. Chronic pain is responsible for a significant socioeconomic burden on the individual as well as society and is affected by several patient specific aspects including physiological, psychological and environmental factors. Interindividual response to current analgesics is exceedingly inconsistent and the functional basis for the unpredictable efficacies of medications has not been elucidated. It is highly probable that variation seen in nociception is due to undiscovered genetic mechanisms, and specific polymorphisms within genes can be potentially singled out by observing deviations in spontaneous pain responses between animals of differing genotypes using the ciguatoxin marine poison. Ciguatoxin is isolated from the Gambierdiscus toxicus dinoflagellate, a specific type of plankton, which is shown to produce spontaneous pain as well as cold allodynia in a dose dependent manner. The objectives of this thesis are centred on the demonstration of the genotypic influence on pain perception, identifying possible genetic sources implicated in nociception, and validating these potential targets with in vivo methods. In Chapter 2, pain behaviours of sixteen mouse strains with recognised genotypes were quantified after administration of the ciguatoxin, which is known to induce spontaneous flinching and licking pain reactions after intraplantar injection.
    [Show full text]