DOCSLIB.ORG

Keratin 12-Deficient Mice Have Fragile Corneal Epithelia

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Keratin 12-Deficient Mice Have Fragile Corneal Epithelia Keratin 12-Deficient Mice Have Fragile Corneal Epithelia Winston W.—Y. Kao,* Chia-YangLiu,* Richard L. Converse,* Atsushi Shiraishi* Candace W.-C. Kao,* Masamichi Ishizaki* Thomas Doetschman,^ and John Duffy-f Purpose. Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentia- tion. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. Methods. One allele of murine Krtl.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. Results. The heterozygous mice (+/—) one allele of the Krtl.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (—/—) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (—/—) does not express keratin 12 as judged by immunohistochemis- try, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Nordiern hybrid- ization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of —/— mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in — /— mice de- creases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of — /— mice. Conclusions. The presence of cornea-specific K3-K12 keratin pail's is essential for the maintenance of corneal epithelium integrity. Invest Ophthalmol Vis Sci. 1996;37:2572-2584. IVeratins are a group of water-insoluble proteins that tive charge, immunoreactivity, and sequence homolo- form 10 nm intermediate filaments in epithelial gies to types I and II wool keratins, respectively.5'6 In cells.1"'1 Approximately 30 different keratin molecules vivo, a basic keratin usually is coexpressed and have been identified.1 They can be divided into acidic "paired" with a particular acidic keratin to form a and basic-neutral subfamilies according to their rela- heterodimer.6'7 The expression of various keratin pairs is tissue specific, differentiation dependent, and devel- opmentally regulated.2'58'9 From the Departments of*Ophthalmology and ^Molecular Genetics, University of The presence of specific keratin pairs is essential Cincinnati, Ohio. Supported by National Institutes of Health grants EY10556 and HL41496 and by for the maintenance of epithelium integrity. For ex- Ohio Lions Eye Research Foundation. ample, mutations in human K14-K510"12 and K10- Submitted for publication April 16, 1996; revised July 9, 1996; accepted July 25, Kl1314 genes have been linked to the human skin dis- 1996. Proprietary interest category: N. eases, epidermolysis bullosa simplex and epidermoly- Reprint requests: Winston W.-Y. Kao, Department of Ophthalmology, University of sis hyperkeratosis, respectively. Similar clinical mani- Cincinnati, Eden and Bethesda Avenues, P.O. Box 670527, Cincinnati, OH 45267-0527. festation of these diseases have been reproduced in Investigative Ophthalmology & Visual Science, December 1996, Vol. 37, No. 13 2572 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/29/2021 Keratin 12-Deficient Mice With Fragile Epithelia 2573 transgenic mice carrying dominant negative muta- Histologic Examination and tions of these keratin genes.15'16 In transgenic mice, Immunohistochemical Staining the dominant expression of mutated epidermal kera- The excised corneas were fixed in 4% paraformalde- tin 14 results in clinical features similar to those of hyde at 4°C overnight and embedded in paraffin as human epidermolysis bullosa simplex.1516 Recently, a previously described.20'22 Five-micrometer-thick sec- null mutation of K8 keratin by gene targeting has been tions were mounted on Super Frost slides (Fisher Sci- reported.17 Many of the homozygous mutant mice are entific, Pittsburgh, PA). Histologic examination was embryonic lethal at 10 days of gestation, when the K8- performed after Harris hematoxylin and eosin stain- K18 pair normally would be expressed during em- ing. Immunohistochemical staining was performed us- bryogenesis.17 These results demonstrate that keratin ing epitope-specific anti-keratin 1222 and anti-keratin intermediate filaments are vital for the integrity of 14 antibodies23 (a generous gift of Dr. D. R. Roop, epithelium. Baylor College of Medicine, Houston, TX) as pre- Expression of the K3-K12 keratin pair has been 20 22 viously described. ' found in human, bovine, guinea pig, rabbit, mouse, and chicken corneas and is regarded as a marker for In Situ Hybridization corneal-type epithelial differentiation. 1~3'5'718'19 We previously cloned the murine Krtl.12 gene and dem- The plasmid DNA containing a full-length keratin 12 onstrated that its expression is corneal epithelial cell cDNA insert was linearized with EcoR I and Kpn I. T- specific, differentiation dependent, and developmen- 7 and T-3 RNA polymerase were used to synthesize tally regulated.20'21 The cornea-specific nature of kera- antisense and sense riboprobes with digoxigenin using procedures recommended by the manufacturer (Boe- tin 12 gene expression signifies that keratin 12 plays hringer-Mannheim, Indianapolis, IN). Tissue sec- a unique role in maintaining normal corneal epithe- tions (5 fim) were incubated with riboprobes (1 //g/ lium functions. No hereditary human corneal epithe- ml using procedures recommended by Boehringer— lial disorder has been linked direcdy to a mutation of Mannheim). The nonspecific binding of riboprobes the keratin 12 gene, although such a human genetic was removed by RNase (20 //g/ml) digestion and a defect may exist. Nevertheless, the exact function of stringent wash in 0.2 X SSC at 65°C.20'22 Tissue sections keratin 12 remains unknown. To elucidate the func- were incubated with anti-digoxigenin antibody alka- tion of keratin 12, we have created knockout mice line phosphatase conjugate at 4°C overnight. The hy- lacking the Krtl.12 gene by gene targeting techniques. bridization signal was visualized with 5-nitroblue tetra- The homozygous mutant mice (Krtl.12, —/ — ) are zolium chloride as recommended by Boehringer— characterized by fragile corneal epithelium. Mannheim. MATERIALS AND METHODS Western Blot Analysis Animal Experiments To isolate keratins, frozen tissues were first homoge- Animal experiments were performed in compliance nized in an extraction solution containing 0.1% Tri- with the ARVO Statement for the Use of Animals in ton X-100 and a mixture of protease inhibitors in Tris- Ophthalmic and Vision Research. Adult mice were saline (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5) with anesthetized by intraperitoneal injections of 70 mg/ a Tissuemizer (Tekmar, Cincinnati, OH) as previously 22 24 kg sodium pentobarbital. Under a stereomicroscope, described. ' The homogenate was centrifuged at a partial epithelial defect was created in both eyes by 12,000 rpm for 15 minutes. The supernatant was dis- scraping the corneal surface with a number 69 Beaver carded, and the pellet was rehomogenized in extrac- blade (Becton-Dickinson, Franklin Lakes, NJ) or tion buffer containing 9 M urea in Tris-saline to ex- brushing with a wet Microsponge (Alcon, Dallas, TX). tract the keratin. After centrifugation, equal volumes Partial defects occupied approximately 60% to 70% of 2 X sodium dodecyl sulfate-polyacrylamide gel of the total corneal epithelium. Neomycin ointment electrophoresis sample buffer was added to the super- was applied on the eyes immediately after surgery. natant. The samples were boiled for 10 minutes and Eyes were examined using a stereomicroscope (Olym- were then subjected to Western immunoblot analysis pus, Melville, NY) every other day beginning on the in 7% or 8% acrylamide using epitope-specific anti- first day after wounding to evaluate reepithelialization keratin 12 antibodies, anti-K12n, and anti-K12c as pre- and to detect any signs of infection.22 The animals viously described.22'24 were killed in a CO2 chamber, and the corneas were removed. Corneas either were embedded in paraffin Northern Blot Analysis for histology, immunohistochemistry, or in situ hy- Total RNAs were isolated from mouse corneas with bridization, or they were prepared for Western blot TRI reagent (MRC, Cincinnati, OH), using proce- analysis as described below. dures recommended
Load more

Recommended publications
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • 140503 IPF Signatures Supplement Withfigs Thorax
    Supplementary material for Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis Daryle J. DePianto1*, Sanjay Chandriani1⌘*, Alexander R. Abbas1, Guiquan Jia1, Elsa N. N’Diaye1, Patrick Caplazi1, Steven E. Kauder1, Sabyasachi Biswas1, Satyajit K. Karnik1#, Connie Ha1, Zora Modrusan1, Michael A. Matthay2, Jasleen Kukreja3, Harold R. Collard2, Jackson G. Egen1, Paul J. Wolters2§, and Joseph R. Arron1§ 1Genentech Research and Early Development, South San Francisco, CA 2Department of Medicine, University of California, San Francisco, CA 3Department of Surgery, University of California, San Francisco, CA ⌘Current address: Novartis Institutes for Biomedical Research, Emeryville, CA. #Current address: Gilead Sciences, Foster City, CA. *DJD and SC contributed equally to this manuscript §PJW and JRA co-directed this project Address correspondence to Paul J. Wolters, MD University of California, San Francisco Department of Medicine Box 0111 San Francisco, CA 94143-0111 [email protected] or Joseph R. Arron, MD, PhD Genentech, Inc. MS 231C 1 DNA Way South San Francisco, CA 94080 [email protected] 1 METHODS Human lung tissue samples Tissues were obtained at UCSF from clinical samples from IPF patients at the time of biopsy or lung transplantation. All patients were seen at UCSF and the diagnosis of IPF was established through multidisciplinary review of clinical, radiological, and pathological data according to criteria established by the consensus classification of the American Thoracic Society (ATS) and European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and the Latin American Thoracic Association (ALAT) (ref. 5 in main text). Non-diseased normal lung tissues were procured from lungs not used by the Northern California Transplant Donor Network.
    [Show full text]
  • 75 2. INTRODUCTION Triple-Negative Breast Cancer (TNBC)
    [Frontiers in Bioscience, Scholar, 11, 75-88, March 1, 2019] The persisting puzzle of racial disparity in triple negative breast cancer: looking through a new lens Chakravarthy Garlapati1, Shriya Joshi1, Bikram Sahoo1, Shobhna Kapoor2, Ritu Aneja1 1Department of Biology, Georgia State University, Atlanta, GA, USA, 2Department of Chemistry, Indian Institute of Technology Bombay, Powai, India TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Dissecting the TNBC racially disparate burden 3.1. Does race influence TNBC onset and progression? 3.2. Tumor microenvironment in TNBC and racial disparity 3.3. Differential gene signatures and pathways in racially distinct TNBC 3.4. Our Perspective: Looking racial disparity through a new lens 4. Conclusion 5. Acknowledgement 6. References 1. ABSTRACT 2. INTRODUCTION Triple-negative breast cancer (TNBC) Triple-negative breast cancer (TNBC), is characterized by the absence of estrogen a subtype of breast cancer (BC), accounts for and progesterone receptors and absence 15-20% of all BC diagnoses in the US. It has of amplification of human epidermal growth been recognized that women of African descent factor receptor (HER2). This disease has no are twice as likely to develop TNBC than approved treatment with a poor prognosis women of European descent (1). As the name particularly in African-American (AA) as foretells, TNBCs lack estrogen, progesterone, compared to European-American (EA) and human epidermal growth factor receptors. patients. Gene ontology analysis showed Unfortunately, TNBCs are defined by what they specific gene pathways that are differentially “lack” rather than what they “have” and thus this regulated and gene signatures that are negative nomenclature provides no actionable differentially expressed in AA as compared to information on “druggable” targets.
    [Show full text]
  • A Novel Keratin 12 Mutation in a German Kindred with Meesmann's
    Br J Ophthalmol 2000;84:527–530 527 A novel keratin 12 mutation in a German kindred Br J Ophthalmol: first published as 10.1136/bjo.84.5.527 on 1 May 2000. Downloaded from with Meesmann’s corneal dystrophy L D Corden, O Swensson, B Swensson, R Rochels, B Wannke, H J Thiel, W H I McLean Abstract inherited disorder aVecting the corneal epithe- Aim—To study a kindred with Mees- lium of both eyes.1–3 It manifests in early child- mann’s corneal dystrophy (MCD) to de- hood and is characterised by myriads of termine if a mutation within the cornea intraepithelial microcysts of variable distribu- specific K3 or K12 genes is responsible for tion and density. The microcysts are best seen the disease phenotype. by retroillumination and slit lamp Methods—Slit lamp examination of the examination.45 Further clinical signs of the cornea in four members of the kindred disease are recurrent punctate erosions with lacrimation and photophobia. Symptoms in- Epithelial Genetics was carried out to confirm the diagnosis Group, Human of MCD. The region encoding the helix clude blurred vision and foreign body sensa- Genetics Unit, initiation motif (HIM) of the K12 polypep- tion. Histologically, the corneal epithelium Department of tide was polymerase chain reaction (PCR) appears irregularly thickened and contains Molecular and Cellular amplified from genomic DNA derived numerous vacuolated suprabasal cells.6–8 In Pathology, Ninewells addition, there is intraepithelial cyst formation Medical School, from aVected individuals in the kindred. Dundee DD1 9SY PCR products generated were subjected to with accumulated periodic acid SchiV (PAS) L D Corden direct automated sequencing.
    [Show full text]
  • Proteomic Expression Profile in Human Temporomandibular Joint
    diagnostics Article Proteomic Expression Profile in Human Temporomandibular Joint Dysfunction Andrea Duarte Doetzer 1,*, Roberto Hirochi Herai 1 , Marília Afonso Rabelo Buzalaf 2 and Paula Cristina Trevilatto 1 1 Graduate Program in Health Sciences, School of Medicine, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba 80215-901, Brazil; [email protected] (R.H.H.); [email protected] (P.C.T.) 2 Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Bauru 17012-901, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-41-991-864-747 Abstract: Temporomandibular joint dysfunction (TMD) is a multifactorial condition that impairs human’s health and quality of life. Its etiology is still a challenge due to its complex development and the great number of different conditions it comprises. One of the most common forms of TMD is anterior disc displacement without reduction (DDWoR) and other TMDs with distinct origins are condylar hyperplasia (CH) and mandibular dislocation (MD). Thus, the aim of this study is to identify the protein expression profile of synovial fluid and the temporomandibular joint disc of patients diagnosed with DDWoR, CH and MD. Synovial fluid and a fraction of the temporomandibular joint disc were collected from nine patients diagnosed with DDWoR (n = 3), CH (n = 4) and MD (n = 2). Samples were subjected to label-free nLC-MS/MS for proteomic data extraction, and then bioinformatics analysis were conducted for protein identification and functional annotation. The three Citation: Doetzer, A.D.; Herai, R.H.; TMD conditions showed different protein expression profiles, and novel proteins were identified Buzalaf, M.A.R.; Trevilatto, P.C.
    [Show full text]
  • Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs S
    University of Kentucky UKnowledge Neuroscience Faculty Publications Neuroscience 6-2018 Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs S. Randal Voss University of Kentucky, [email protected] David Murrugarra University of Kentucky, [email protected] Tyler B. Jensen Northeastern University James R Monaghan Northeastern University Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/neurobio_facpub Part of the Anatomy Commons, Biochemistry Commons, Comparative and Evolutionary Physiology Commons, Neuroscience and Neurobiology Commons, and the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Voss, S. Randal; Murrugarra, David; Jensen, Tyler B.; and Monaghan, James R, "Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs" (2018). Neuroscience Faculty Publications. 54. https://uknowledge.uky.edu/neurobio_facpub/54 This Article is brought to you for free and open access by the Neuroscience at UKnowledge. It has been accepted for inclusion in Neuroscience Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Transcriptional Correlates of Proximal-Distal Identify and Regeneration Timing in Axolotl Limbs Notes/Citation Information Published in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, v. 208, p. 53-63. © 2017 Elsevier Inc. All rights reserved. This manuscript version is made available under the CC‐BY‐NC‐ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/. The document available for download is the author's post-peer-review final draft of the ra ticle.
    [Show full text]
  • S Epithelial Corneal Dystrophy Phenotype Due to A
    Eye (2013) 27, 367–373 & 2013 Macmillan Publishers Limited All rights reserved 0950-222X/13 www.nature.com/eye 1 2 2 2 Severe Meesmann’s H Hassan , C Thaung , ND Ebenezer , G Larkin , CLINICAL STUDY AJ Hardcastle2 and SJ Tuft1;2 epithelial corneal dystrophy phenotype due to a missense mutation in the helix- initiation motif of keratin 12 Abstract Purpose To describe a severe phenotype of protein. The clinical effects are markedly Meesmann’s epithelial corneal dystrophy more severe than the phenotype usually (MECD) and to determine the underlying associated with the Arg135Thr mutation molecular cause. within this motif, most frequently seen in Methods We identified a 30-member family European patients with MECD. affected by MECD and examined 11 of the 14 Eye (2013) 27, 367–373; doi:10.1038/eye.2012.261; affected individuals. Excised corneal tissue published online 7 December 2012 from one affected individual was examined histologically. We used PCR and direct Keywords: corneal epithelium; Meesmann sequencing to identify mutation of the corneal dystrophy; human KRT12 protein coding regions of the KRT3 and KRT12 genes. Introduction Results Cases had an unusually severe phenotype with large numbers of Meesmann’s epithelial corneal dystrophy 1Corneal Service, Moorfields intraepithelial cysts present from infancy and (MECD; OMIM #122100) is an autosomal Eye Hospital NHS they developed subepithelial fibrosis in the dominant inherited change of the corneal Foundation Trust, London, second to third decade. In some individuals, epithelium of the eye characterized by the UK the cornea became superficially vascularized, bilaterally symmetric development of numerous 2 a change accompanied by the loss of intraepithelial cysts.1,2 The cysts are best Department of Ocular clinically obvious epithelial cysts.
    [Show full text]
  • Genomics of Inherited Bone Marrow Failure and Myelodysplasia Michael
    Genomics of inherited bone marrow failure and myelodysplasia Michael Yu Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Mary-Claire King, Chair Akiko Shimamura Marshall Horwitz Program Authorized to Offer Degree: Molecular and Cellular Biology 1 ©Copyright 2015 Michael Yu Zhang 2 University of Washington ABSTRACT Genomics of inherited bone marrow failure and myelodysplasia Michael Yu Zhang Chair of the Supervisory Committee: Professor Mary-Claire King Department of Medicine (Medical Genetics) and Genome Sciences Bone marrow failure and myelodysplastic syndromes (BMF/MDS) are disorders of impaired blood cell production with increased leukemia risk. BMF/MDS may be acquired or inherited, a distinction critical for treatment selection. Currently, diagnosis of these inherited syndromes is based on clinical history, family history, and laboratory studies, which directs the ordering of genetic tests on a gene-by-gene basis. However, despite extensive clinical workup and serial genetic testing, many cases remain unexplained. We sought to define the genetic etiology and pathophysiology of unclassified bone marrow failure and myelodysplastic syndromes. First, to determine the extent to which patients remained undiagnosed due to atypical or cryptic presentations of known inherited BMF/MDS, we developed a massively-parallel, next- generation DNA sequencing assay to simultaneously screen for mutations in 85 BMF/MDS genes. Querying 71 pediatric and adult patients with unclassified BMF/MDS using this assay revealed 8 (11%) patients with constitutional, pathogenic mutations in GATA2 , RUNX1 , DKC1 , or LIG4 . All eight patients lacked classic features or laboratory findings for their syndromes.
    [Show full text]
  • UCLA Electronic Theses and Dissertations
    UCLA UCLA Electronic Theses and Dissertations Title Proteomic Analysis of Cancer Cell Metabolism Permalink https://escholarship.org/uc/item/8t36w919 Author Chai, Yang Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Proteomic Analysis of Cancer Cell Metabolism A thesis submitted in partial satisfaction of the requirements of the degree Master of Science in Oral Biology by Yang Chai 2013 ABSTRACT OF THESIS Proteomic Analysis of Cancer Cell Metabolism by Yang Chai Master of Science in Oral Biology University of California, Los Angeles, 2013 Professor Shen Hu, Chair Tumor cells can adopt alternative metabolic pathways during oncogenesis. This is an event characterized by an enhanced utilization of glucose for rapid synthesis of macromolecules such as nucleotides, lipids and proteins. This phenomenon was also known as the ‘Warburg effect’, distinguished by a shift from oxidative phosphorylation to increased aerobic glycolysis in many types of cancer cells. Increased aerobic glycolysis was also indicated with enhanced lactate production and glutamine consumption, and has been suggested to confer growth advantage for proliferating cells during oncogenic transformation. Development of a tracer-based ii methodology to determine de novo protein synthesis by tracing metabolic pathways from nutrient utilization may certainly enhance current understanding of nutrient gene interaction in cancer cells. We hypothesized that the metabolic phenotype of cancer cells as characterized by nutrient utilization for protein synthesis is significantly altered during oncogenesis, and 13C stable isotope tracers may incorporate 13C into non-essential amino acids of protein peptides during de novo protein synthesis to reflect the underlying mechanisms in cancer cell metabolism.
    [Show full text]
  • KRT3 Gene Keratin 3
    KRT3 gene keratin 3 Normal Function The KRT3 gene provides instructions for making a protein called keratin 3. Keratins are a group of tough, fibrous proteins that form the structural framework of epithelial cells, which are cells that line the surfaces and cavities of the body. Keratin 3 is produced in a tissue on the surface of the eye called the corneal epithelium. This tissue forms the outermost layer of the cornea, which is the clear front covering of the eye. The corneal epithelium acts as a barrier to help prevent foreign materials, such as dust and bacteria, from entering the eye. The keratin 3 protein partners with another keratin protein, keratin 12, to form molecules known as intermediate filaments. These filaments assemble into strong networks that provide strength and resilience to the corneal epithelium. Health Conditions Related to Genetic Changes Meesmann corneal dystrophy At least three mutations in the KRT3 gene have been found to cause Meesmann corneal dystrophy, an eye disease characterized by the formation of tiny cysts in the corneal epithelium. All of the identified KRT3 gene mutations associated with Meesmann corneal dystrophy change single protein building blocks (amino acids) in the keratin 3 protein. These changes occur in a region of the protein that is critical for the formation and stability of intermediate filaments. The altered keratin 3 protein interferes with the assembly of intermediate filaments, weakening the structural framework of the corneal epithelium. As a result, this outer layer of the cornea is abnormally fragile and develops the cysts that characterize Meesmann corneal dystrophy. The cysts likely contain clumps of abnormal keratin proteins and other cellular debris.
    [Show full text]
  • Sharon Carr Mphil Thesis
    ADENOVIRUS AND ITS INTERACTION WITH HOST CELL PROTEINS Sharon Carr A Thesis Submitted for the Degree of MPhil at the University of St. Andrews 2007 Full metadata for this item is available in Research@StAndrews:FullText at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/219 This item is protected by original copyright Adenovirus and its interaction with host cell proteins Sharon Carr School of Biomedical Sciences University of St Andrews A thesis submitted for the degree of Master of Philosophy September 2006 1 I, …………………., hereby certify that this thesis, which is approximately 75,000 words in length, has been written by me, that it is the record of work carried out by me and that it has not been submitted in any previous application for a higher degree. date…………… signature of candidate………………………… I was admitted as a research student in September 2002 and as a candidate for the degree of Doctor of Philosophy in September 2002; the higher study for which this is a record was carried out in the University of St Andrews between 2002 and 2005, and at the University of Dundee between 2005 and 2006. date…………… signature of candidate………………………… I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriated for the degree of Master of Philosophy in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. date…………… signature of supervisors………………………… ………………………… 2 In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby.
    [Show full text]
  • Mass Spectrometry Analysis Reveals Dynamic Changes of Protein
    Japanese Journal of Gastroenterology and Hepatology Research Article Mass Spectrometry Analysis Reveals Dynamic Changes of Protein Components in Lipid Rafts from Rat Liver after Partial Hepatectomy Zhu B1*, Li J1*, Yang C1, Jiang M1, Zhou Q1, Wang Y1, 2* and Chen P1, 2* 1Department of Gastroenterology, The National & Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha 410081, P.R. China 2Department of Gastroenterology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha, 410081, P.R. China Received: 08 Apr 2020 1. Abstract Accepted: 27 Apr 2020 Lipid raft, as scaffolding platform for signal transduction, plays important role in the liver re- Published: 30 Apr 2020 generation. But the lipid raft protein expression pattern during liver regeneration has been not * Corresponding author: reported. In this study, lipid raft proteins from the liver of 72 h post partial hepatectomy and Ping Chen, Department of Gastroen- sham-operated group were identified by liquid chromatography-tandem mass spectrometry (LC- terology, The National & Local Joint MS/MS) combined with label-free semi-quantitative analysis. Totally 458 lipid raft proteins were Engineering Laboratory of Animal identified, and most of identified lipid raft proteins were mainly involved in transporter, signal Peptide Drug Development, College transduction, and metabolism. Moreover, label-free quantification analysis suggested that the level of Life Sciences, Hunan Normal Uni- of 46 plasma membrane-related proteins have changed obviously (with ratio≥2) after 72 h hepa- versity, Changsha 410081, P.R. China, tectomy. Several differently expressed proteins, including caveolin-1 and flotillin-1, were validated E-mail: [email protected] by western blotting.
    [Show full text]