DOCSLIB.ORG

The Role of Lumican in the Formation of Bio-Glass: Transparent Cornea

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of Lumican in the Formation of Bio-Glass: Transparent Cornea UNIVERSITY OF CINCINNATI DATE: January 31, 2003 I, Eric Curtis Carlson , hereby submit this as part of the requirements for the degree of: Doctorate of Philosophy (Ph.D.) in: Cell and Molecular Biology It is entitled: The Role of Lumican in Bio-glass Formation: Transparent Cornea Approved by: Winston Kao, Ph.D. Gary Dean, Ph.D. James Funderburgh, Ph.D. Wallace Ip, Ph.D. Lawrence Sherman, Ph.D. The Role of Lumican in the Formation of Bio-glass: Transparent Cornea A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctorate of Philosophy (Ph.D.) in the Graduate Program of Cell and Molecular Biology of the College of Medicine 2002 by Eric C. Carlson B.A. Biology, Thiel College, 1997 Committee Chair: Winston W.-Y. Kao, Ph.D. Gary Dean, Ph.D. James Funderburgh, Ph.D. Wallace Ip, Ph.D. Lawrence Sherman, Ph.D. Abstract Corneal opacity leaves 1.5 million people visually impaired worldwide. One potential key player in the formation and maintenance of corneal transparency is a keratan sulfate proteoglycan lumican. The role of lumican in the formation and maintenance of bio-glass, transparent cornea, was examined using three different experimental systems. First, a wound healing system was used to understand the expression of lumican by corneal cells during stromal matrix restructuring and remodeling. Three different types of wounds were generated on mouse corneas: partial epithelial debridement, total epithelial debridement, and alkali burn wounds. Lumican expression was analyzed over a 12 week time course. Second, cultured corneal fibroblasts were used in an ex vivo expression system to determine the impact of lumican on collagen fibrillogenesis. Cultured corneal fibroblasts were stably transfected with either a wild- type lumican (lumWT) or 41cysteine to serine mutated lumican (lumC/S). Under proper conditions stable transformants formed a three-dimensional extracellular matrix over a 4 or 6 week time period. Finally, DNA microinjection into the corneal stroma was used as an in vivo expression system. Our results demonstrate the following: (1) the amount of lumican expression by corneal stroma cells in a wound healing situation decreases during stromal remodeling and is also dependent on the severity of the wound. This finding indicates that the gene expression pattern of keratocytes is lost during the wound healing response. (2) Mutation of 41Cys to serine is adequate to impair the role of lumican in ex vivo collagen fibrillogenesis. The actual interaction of lumican with collagen is disrupted by this single amino acid mutation. The actual impact is possibly due to a structural change caused by this mutation. (3) Plasmid DNA ii microinjection into the corneal stroma is an effective tool to drive protein expression and is adequate to rescue the thin corneal stroma phenotype of lumican null mice. DNA microinjection into the corneal stroma is capable of being an efficient and safe way to serve as a gene therapy strategy in treating ocular surface diseases. This technique is simple, non-invasive, and effective in expressing a therapeutic protein of interest. iii iv This dissertation is dedicated to my wife, Becky, and my daughter, Courtney. v Acknowledgements I would like to thank the Departments of Cell Biology, Neurobiology, and Anatomy and Ophthalmology for the opportunity to pursue my doctorate degree. A special thank you goes to my advisor, Dr. Winston W-Y Kao, for providing guidance and resources during my thesis research, and still allowing me to think independently. I would like to thank my thesis committee: Dr. Gary Dean, Dr. James Funderburgh, Dr. Wally Ip, and Dr. Larry Sherman for their time, comments, and words of wisdom. I would also like to thank my collaborators Dr. David Birk (Thomas Jefferson University) and Dr. Jim Funderburgh (University of Pittsburgh) for their technical expertise. I would like to thank the members of the Kao Lab, especially Dr. Chia-Yang Liu, for all their help. A sincere appreciation is expressed to Drs. Robert Brackenbury and Linda Parysek for providing an educational guidance, a hunting sanctuary, but especially for being good friends. Finally, I would like to thank my wife Becky. The pursuit of my doctorate was frustrating at times, but her support, love, and encouragement helped me through it. I would also like to thank her for my future hunting partner, Courtney. vi Table of Contents Title Page i Abstract ii Blank Page iv Dedication v Acknowledgements vi Table of Contents 1 List of Tables and Figures 3 List of Symbols 10 Introduction 13 Chapter One: Corneal Wound Healing 20 Abstract 21 Introduction 23 Materials and Methods 26 Results 31 Discussion 35 - 1 - Figures 39 Chapter Two: Role of Lumican in Collagen Fibrillogenesis 51 Abstract 52 Introduction 54 Materials and Methods 58 Results 63 Discussion 69 Figures 73 Conclusions and Future Directions 87 References 92 Appendix: In vivo Lumican overexpression 104 Role of 41Cys in the N-terminal 125 Domain of Lumican in Ex Vivo Collagen Fibrillogenesis by Cultured Corneal Stromal Cells Biochemical Journal 2002 0593. - 2 - List of Tables and Figures Chapter One Figure 1 Fluorescein staining of partial epithelial debridement, total epithelial debridement, and alkali burn wounds using light microscopy. Figure 2 Hematoxylin and eosin staining of paraffin sections of partial epithelial debridement, total epithelial debridement, and alkali burn wounds at 2 hours, 1 week, and 2 weeks post-injury. Figure 3 Whole mount light microscopy depicting corneal opacity and neovascularization of total epithelial debridement and partial epithelial debridement wounds. - 3 - Figure 4 Paraffin section of total epithelial debridement wounded cornea stained with methyl green to show presence of goblet cells in corneal epithelium. Figure 5 Keratin 12 immunostaining of partial epithelial debridement and total epithelial debridement wounds. Figure 6 Keratin 12 in situ hybridization of partial and total epithelial debridement wounds. Figure 7 Keratocan in situ hybridization of partial epithelial debridement, total epithelial debridement, and alkali burn wounds at 6 weeks post-injury. Figure 8 Keratocan in situ hybridization of partial epithelial debridement, total epithelial debridement, and alkali burn wounds at 12 weeks post-injury. - 4 - Figure 9 Histogram of data generated from Real-Time RT-PCR using lumican probes in the three wound types. Chapter Two Figure 11 Schematic Diagram of Lumican minigenes cloned into pSecTag2A vector with corresponding DNA and amino acid sequences. Figure 12 1% agarose gel stained with Ethidium Bromide of stable transformant PCR genotyping Figure 13 Growth curve of stable transformant over a 7 day period - 5 - Figure 14 Western blot analysis of stable transformant conditioned media probed with anti-lumican antibody. Figure 15 Light microscopy of 6 week 3-D cell culture semi-thin section. Figure 16 Transmission electron micrograph of lumWT and lumC/S 3-D cell culture extracellular matrix collagen fibrils at 4 weeks and 6 weeks. Figure 17 Histogram of transmission electron microscopy measurement of collagen fibrils in the lumWT and lumC/S cultures at 4 weeks and 6 weeks. Figure 18 Confocal micrograph of propidium iodide staining and immunofluorescence of anti-lumican and anti-collagen I in lumWT and lumC/S 3-D cultures - 6 - Figure 19 Confocal micrograph of anti-lumican, anti-collagen I immunofluorescent staining, and an overlay. Figure 20 Histogram of pixel values calculated from anti-lumican and anti-collagen I immunofluorescent staining and superimposed images. Figure 21 Light stereomicroscopy photograph of corneal stroma DNA injection using 33 gauge needle Figure 22 Fluorescent microscopy picture of EGFP fluorescence emitted from cornea of lumican knock-out (-/-) mouse microinjected with CMV-EGFP. Figure 23 Paraffin sections of lumican knock-out (-/-) cornea following DNA microinjection and anti-lumican immunostaining. - 7 - Figure 24 Schematic of keratocan promoter driven lumican wild- type and lumican C/S constructs for transgenic mouse generation. Figure 25 Schematic of LumX minigenes cloned into keratocan promoter. Figure 26 pKeraLumWT construct for blastocyst injection digested with FspI and SacII to release transgene from the vector backbone. Figure 27 pKeraLumC/S construct for blastocyst injection digested with FspI and SacII to release transgene from the vector backbone. Figure 28 1% agarose gel stained with Ethidium Bromide of transgenic mouse founders’ genomic DNA PCR genotyping. - 8 - Figure 29 RT-PCR of KeraLumWT transgenic founders for lines 5, 25, and 38. Figure 30 A Western blot of corneal extracts using anti-lumican antibody to determine lumican expression level in transgenic mouse cornea. Figure 31 Real-time RT-PCR of transgenic mouse cornea from KeraLumWT line 25 Figure 32 Western blot analysis of corneal extracts using anti- keratocan antibody to determine the expression level in transgenic mouse cornea. Figure 33 PCR genotyping of KeraLumC/S transgenic mice founders on 1% agarose gel stained with Ethidium Bromide. - 9 - LIST OF SYMBOLS 3-D Three dimensional α Anti- ANOVA Analysis of variance bp Base pair Cys Cysteine DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid DNase Deoxyribonuclease dNTPs Deoxynucleotidetriphosphate DTT Dithiothreitol ECL Enhanced chemiluminescence ECM Extracellular matrix EDTA Disodium Ethylenediaminetetraacetate EGFP Enhanced green fluorescent protein fg femtogram g Gram GAPDH
Load more

Recommended publications
  • Generation and Characterization of a Novel Mouse Line, Keratocan-Rtta (Kerart), for Corneal Stroma and Tendon Research
    Cornea Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research Yujin Zhang,1 Winston W.-Y. Kao,2 Yasuhito Hayashi,3 Lingling Zhang,1 Mindy Call,2 Fei Dong,2 Yong Yuan,2 Jianhua Zhang,2 Yen-Chiao Wang,1 Okada Yuka,1,4 Atsushi Shiraishi,3 and Chia-Yang Liu1 1School of Optometry, Indiana University, Bloomington, Indiana, United States 2Edith J. Crawley Vision Research Center/Department of Ophthalmology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States 3Department of Ophthalmology, School of Medicine, Ehime University, Ehime, Japan 4Department of Ophthalmology, School of Medicine, Wakayama Medical University, Wakayama, Japan RT Correspondence: Yujin Zhang, Indi- PURPOSE. We created a novel inducible mouse line Keratocan-rtTA (Kera ) that allows ana University School of Optometry, specific genetic modification in corneal keratocytes and tenocytes during development and in 800 Atwater Avenue, Bloomington, adults. IN 47405, USA; [email protected]. METHODS. A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after Chia-Yang Liu, Indiana University the termination codon of the mouse Kera allele via gene targeting techniques. The resulting RT RT School of Optometry, 800 Atwater Kera mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain Kera /TH2B-EGFP Avenue, Bloomington, IN 47405, compound transgenic mice, in which cells expressing Kera are labeled with green USA; fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of [email protected]. RT RT GFP and endogenous Kera were examined in Kera /TH2B-EGFP. Moreover, Kera was bred Submitted: July 21, 2017 with tet-O-TGF-a to generate a double transgenic mouse, KeraRT/tet-O-TGF-a, to overexpress Accepted: August 16, 2017 TGF-a in corneal keratocytes upon Dox induction.
    [Show full text]
  • Comparative Analysis of a Teleost Skeleton Transcriptome Provides Insight Into Its Regulation
    Accepted Manuscript Comparative analysis of a teleost skeleton transcriptome provides insight into its regulation Florbela A. Vieira, M.A.S. Thorne, K. Stueber, M. Darias, R. Reinhardt, M.S. Clark, E. Gisbert, D.M. Power PII: S0016-6480(13)00264-5 DOI: http://dx.doi.org/10.1016/j.ygcen.2013.05.025 Reference: YGCEN 11541 To appear in: General and Comparative Endocrinology Please cite this article as: Vieira, F.A., Thorne, M.A.S., Stueber, K., Darias, M., Reinhardt, R., Clark, M.S., Gisbert, E., Power, D.M., Comparative analysis of a teleost skeleton transcriptome provides insight into its regulation, General and Comparative Endocrinology (2013), doi: http://dx.doi.org/10.1016/j.ygcen.2013.05.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Comparative analysis of a teleost skeleton transcriptome 2 provides insight into its regulation 3 4 Florbela A. Vieira1§, M. A. S. Thorne2, K. Stueber3, M. Darias4,5, R. Reinhardt3, M. 5 S. Clark2, E. Gisbert4 and D. M. Power1 6 7 1Center of Marine Sciences, Universidade do Algarve, Faro, Portugal. 8 2British Antarctic Survey – Natural Environment Research Council, High Cross, 9 Madingley Road, Cambridge, CB3 0ET, UK.
    [Show full text]
  • Supplementary Table 1: Adhesion Genes Data Set
    Supplementary Table 1: Adhesion genes data set PROBE Entrez Gene ID Celera Gene ID Gene_Symbol Gene_Name 160832 1 hCG201364.3 A1BG alpha-1-B glycoprotein 223658 1 hCG201364.3 A1BG alpha-1-B glycoprotein 212988 102 hCG40040.3 ADAM10 ADAM metallopeptidase domain 10 133411 4185 hCG28232.2 ADAM11 ADAM metallopeptidase domain 11 110695 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 195222 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 165344 8751 hCG20021.3 ADAM15 ADAM metallopeptidase domain 15 (metargidin) 189065 6868 null ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme) 108119 8728 hCG15398.4 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 117763 8748 hCG20675.3 ADAM20 ADAM metallopeptidase domain 20 126448 8747 hCG1785634.2 ADAM21 ADAM metallopeptidase domain 21 208981 8747 hCG1785634.2|hCG2042897 ADAM21 ADAM metallopeptidase domain 21 180903 53616 hCG17212.4 ADAM22 ADAM metallopeptidase domain 22 177272 8745 hCG1811623.1 ADAM23 ADAM metallopeptidase domain 23 102384 10863 hCG1818505.1 ADAM28 ADAM metallopeptidase domain 28 119968 11086 hCG1786734.2 ADAM29 ADAM metallopeptidase domain 29 205542 11085 hCG1997196.1 ADAM30 ADAM metallopeptidase domain 30 148417 80332 hCG39255.4 ADAM33 ADAM metallopeptidase domain 33 140492 8756 hCG1789002.2 ADAM7 ADAM metallopeptidase domain 7 122603 101 hCG1816947.1 ADAM8 ADAM metallopeptidase domain 8 183965 8754 hCG1996391 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 129974 27299 hCG15447.3 ADAMDEC1 ADAM-like,
    [Show full text]
  • 3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
    RESEARCH ARTICLE 3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture Chiara E. Ghezzi1, Benedetto Marelli1, Fiorenzo G. Omenetto1, James L. Funderburgh2, David L. Kaplan1* 1 Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America, 2 Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America * [email protected] a1111111111 a1111111111 a1111111111 Abstract a1111111111 a1111111111 The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells OPEN ACCESS (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison Citation: Ghezzi CE, Marelli B, Omenetto FG, to previously developed 2D approaches. Silk film contact guidance was used to control the Funderburgh JL, Kaplan DL (2017) 3D Functional alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were Corneal Stromal Tissue Equivalent Based on then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture. PLoS ONE 12(1): e0169504. systems were compared similar systems generated with human corneal fibroblasts (hCFs). doi:10.1371/journal.pone.0169504 Both cell types were viable and preferentially aligned along the biomaterial patterns for up to Editor: Dimitrios Karamichos, Oklahoma State 9 weeks in culture. H&E histological sections showed that the systems seeded with the University Center for Health Sciences, UNITED hCSSCs displayed ECM production throughout the entire thickness of the constructs.
    [Show full text]
  • Application of Collagen I and IV in Bioengineering Transparent Ocular Tissues
    REVIEW published: 26 August 2021 doi: 10.3389/fsurg.2021.639500 Application of Collagen I and IV in Bioengineering Transparent Ocular Tissues Yihui Song 1†, Morgan Overmass 1†, Jiawen Fan 2, Chris Hodge 1,3,4, Gerard Sutton 1,3,4, Frank J. Lovicu 1,5 and Jingjing You 1,6* 1 Save Sight Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia, 2 Key Laboratory of Myopia of State Health Ministry, Department of Ophthalmology and Vision Sciences, Eye and Ear, Nose, and Throat (ENT) Hospital, Shanghai Medical College, Fudan University, Shanghai, China, 3 New South Wales (NSW) Tissue Bank, Sydney, NSW, Australia, 4 Vision Eye Institute, Chatswood, NSW, Australia, 5 Discipline of Anatomy and Histology, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia, 6 School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, Australia Collagens represent a major group of structural proteins expressed in different tissues and display distinct and variable properties. Whilst collagens are non-transparent in the skin, they confer transparency in the cornea and crystalline lens of the eye. There are 28 types of collagen that all share a common triple helix structure yet differ in the composition Edited by: of their α-chains leading to their different properties. The different organization of collagen Zhilian Yue, fibers also contributes to the variable tissue morphology. The important ability of collagen University of Wollongong, Australia to form different tissues has led to the exploration and application of collagen as a Reviewed by: biomaterial. Collagen type I (Col-I) and collagen type IV (Col-IV) are the two primary Tiago H.
    [Show full text]
  • Safety and Efficacy of Combination of Suberoylamilide Hydroxyamic Acid
    www.nature.com/scientificreports OPEN Safety and efcacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro‑fbrotic changes in human corneal epithelial cells Rohit Shetty1,7, Nimisha Rajiv Kumar2,7, Murali Subramani3, Lekshmi Krishna3, Ponnalagu Murugeswari3, Himanshu Matalia1, Pooja Khamar1, Zelda V. Dadachanji1, Rajiv R. Mohan4,5,6*, Arkasubhra Ghosh2* & Debashish Das3* Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse efects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT‑qPCR, immunofuorescence and western blot for markers of apoptosis and fbrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFβ induced fbrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse efects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fbrotic markers without causing any adverse efect on cellular properties.
    [Show full text]
  • Keratocan Is Expressed by Osteoblasts and Can Modulate Osteogenic Differentiation John C
    University of Connecticut OpenCommons@UConn UCHC Articles - Research University of Connecticut Health Center Research 10-2011 Keratocan Is Expressed By Osteoblasts And Can Modulate Osteogenic Differentiation John C. Igwe University of Connecticut School of Medicine and Dentistry Qi Gao University of Connecticut School of Medicine and Dentistry Tomislav Kizivat University of Connecticut School of Medicine and Dentistry Ivo Kalajzic University of Connecticut School of Medicine and Dentistry Follow this and additional works at: https://opencommons.uconn.edu/uchcres_articles Part of the Medicine and Health Sciences Commons Recommended Citation Igwe, John C.; Gao, Qi; Kizivat, Tomislav; and Kalajzic, Ivo, "Keratocan Is Expressed By Osteoblasts And Can Modulate Osteogenic Differentiation" (2011). UCHC Articles - Research. 140. https://opencommons.uconn.edu/uchcres_articles/140 NIH Public Access Author Manuscript Connect Tissue Res. Author manuscript; available in PMC 2013 February 17. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Connect Tissue Res. 2011 October ; 52(5): 401–407. doi:10.3109/03008207.2010.546536. Keratocan is expressed by osteoblasts and can modulate osteogenic differentiation John C. Igwea, Qi Gaob, Tomislav Kizivata, Winston W. Kaoc, and Ivo Kalajzica aDepartment of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA bDepartment of Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA cDepartment of Ophthalmology, University of Cincinnati, Cincinnati, Ohio, USA Abstract Keratocan is an extracellular matrix protein that belongs to the small leucine-rich proteoglycan family which also includes the lumican, biglycan, decorin, mimecan and fibromodulin. Members of this family are known to play a role in regulating cellular processes such as proliferation and modulation of osteoprogenitor lineage differentiation.
    [Show full text]
  • A Novel Keratocan Mutation Causing Autosomal Recessive Cornea Plana
    A Novel Keratocan Mutation Causing Autosomal Recessive Cornea Plana Ordan J. Lehmann,1,2 Mohamed F. El-ashry,1,2 Neil D. Ebenezer,1 Louise Ocaka,1 Peter J. Francis,1 Susan E. Wilkie,1 Reshma J. Patel,1 Linda Ficker,3 Tim Jordan,1 Peng T. Khaw,3 and Shomi S. Bhattacharya1 PURPOSE. Mutations in keratocan (KERA), a small leucine-rich exhibits the more severe phenotype and includes the presence proteoglycan, have recently been shown to be responsible for of a degree of corneal opacity and sclerocornea (OMIM cases of autosomal recessive cornea plana (CNA2). A consan- 181700). Two mutations in keratocan (KERA; OMIM 603288), guineous pedigree in which cornea plana cosegregated with a small leucine-rich proteoglycan (SLRP), have recently been microphthalmia was investigated by linkage analysis and direct shown to be responsible for autosomal recessive cornea plana sequencing. in a series of patients of Finnish and American extraction. The METHODS. Linkage was sought to polymorphic microsatellite cause of the autosomal dominant form of the disease remains markers distributed around the CNA2 and microphthalmia loci to be determined. (arCMIC, adCMIC, NNO1, and CHX10) using PCR and nonde- The combined phenotype of cornea plana and microphthal- naturing polyacrylamide gel electrophoresis before KERA was mia has not been previously described. Microphthalmia is a directly sequenced for mutations. heterogeneous group of developmental anomalies in which the eye fails to attain its normal dimensions with the axial RESULTS. Positive lod scores were obtained with markers en- 4 compassing the CNA2 locus, the maximum two-point lod diameter reduced to less than the age-adjusted 5th centile.
    [Show full text]
  • Proteoglycan Form and Function: a Comprehensive Nomenclature of Proteoglycans
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Matrix Biol Manuscript Author . Author manuscript; Manuscript Author available in PMC 2016 May 06. Published in final edited form as: Matrix Biol. 2015 March ; 42: 11–55. doi:10.1016/j.matbio.2015.02.003. Proteoglycan form and function: A comprehensive nomenclature of proteoglycans Renato V. Iozzo1 and Liliana Schaefer2 1Department of Pathology, Anatomy and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA 19107, USA 2Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Frankfurt am Main, Germany Abstract We provide a comprehensive classification of the proteoglycan gene families and respective protein cores. This updated nomenclature is based on three criteria: Cellular and subcellular location, overall gene/protein homology, and the utilization of specific protein modules within their respective protein cores. These three signatures were utilized to design four major classes of proteoglycans with distinct forms and functions: the intracellular, cell-surface, pericellular and extracellular proteoglycans. The proposed nomenclature encompasses forty-three distinct proteoglycan-encoding genes and many alternatively-spliced variants. The biological functions of these four proteoglycan families are critically assessed in development, cancer and angiogenesis, and in various acquired and genetic diseases where their expression is aberrant. Keywords Proteoglycan; Glycosaminoglycan; Cancer growth; Angiogenesis; Growth factor modulation Introduction It has been nearly 20 years since the original publication of a comprehensive classification of proteoglycan gene families [1]. For the most part, these classes have been widely accepted. However, a broad and current taxonomy of the various proteoglycan gene families and their products is not available.
    [Show full text]
  • Fragmentation of Decorin, Biglycan, Lumican and Keratocan Is Elevated
    Available online http://arthritis-research.com/content/10/4/R79 ResearchVol 10 No 4 article Open Access Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues James Melrose1, Emily S Fuller1, Peter J Roughley2, Margaret M Smith1, Briedgeen Kerr3, Clare E Hughes3, Bruce Caterson3 and Christopher B Little1 1Raymond Purves Research Laboratory, Institute of Bone & Joint Research, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Reserve Road, St. Leonards, NSW 2065, Australia 2Genetics Unit, 1529 Cedar, Rm 338, Shriners Hospital for Children, McGill University, Montreal, Quebec H3G 1A6, Canada 3School of Molecular and Medical Biosciences, PO Box 911, University of Cardiff, Cardiff CF1 3US, UK Corresponding author: James Melrose, [email protected] Received: 23 Apr 2008 Revisions requested: 10 Jun 2008 Revisions received: 18 Jun 2008 Accepted: 14 Jul 2008 Published: 14 Jul 2008 Arthritis Research & Therapy 2008, 10:R79 (doi:10.1186/ar2453) This article is online at: http://arthritis-research.com/content/10/4/R79 © 2008 Melrose et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction The small leucine-rich proteoglycans (SLRPs) catabolites in osteoarthritic hip than in knee articular cartilage. modulate tissue organization, cellular proliferation, matrix Fragmentation of all SLRPs in normal age-matched, adhesion, growth factor and cytokine responses, and sterically nonfibrillated knee articular cartilage was less than in fibrillated protect the surface of collagen type I and II fibrils from articular cartilage from the same knee joint or total knee proteolysis.
    [Show full text]
  • Labned.Com Bovine
    Antibodies Predicted to Bind Homologous Bovine Targets This is a catalog of all antibodies predicted to react with bovine proteins in addition to their validated reactivities. This catalog was assembled by identifying proteins targeted by LabNed antibodies that share 99% sequence homology withtheir bovine counterparts. Table of Contents High Affinity Antibodies .................................................................................... 1 High Sensitivity ELISA Kits .................................................................................... 31 High Affinity Antibodies LabNed supplies only the highest quality antibodies. Our high-affinity polyclonal and monoclonal antibodies are thoroughly validated by Western Blotting, Immunohistochemistry and ELISA. This is our comprehensive catalog of our antibody products predicted to react with bovine proteins, sorted in alphabetical order by target gene name. Applications Gene Name Product Name Reactivity WB ABAT Anti-ABA Human, Mouse, Rat WB ABCA1 Anti-ABCA1 Human, Mouse, Rat WB ABCA1 Anti-ABCA1 Human, Mouse, Rat IHC-P, WB ABCB11 Anti-ABCB11 Human, Mouse, Rat IHC-P, WB ABCC1 Anti-MRP1 Human, Mouse, Rat WB ABCC4 Anti-MRP4 Human, Mouse, Rat IHC-P, WB ABCD3 Anti-PMP70 Human, Mouse, Rat WB ABCE1 Anti-ABCE1 Human, Mouse, Rat WB ABCG1 Anti-ABCG1 Human, Mouse, Rat WB ABCG2 Anti-ABCG2 Human, Mouse, Rat WB ABCG4 Anti-ABCG4 Human, Mouse, Rat WB ABCG5 Anti-ABCG5 Human IHC-P, ICC, WB ABI1 Anti-ABI1 Human, Mouse, Rat WB ABI2 Anti-ABI2 Human, Mouse, Rat WB ACO2 Anti-Aconitase 2 Human, Mouse, Rat
    [Show full text]
  • Role of Lumican in Cancer Cells and Adjacent Stromal Tissues in Human Pancreatic Cancer
    537-543 25/7/07 14:59 Page 537 ONCOLOGY REPORTS 18: 537-543, 2007 537 Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer TOSHIYUKI ISHIWATA1, KAZUMITSU CHO1,2, KIYOKO KAWAHARA1, TETSUSHI YAMAMOTO1, YURI FUJIWARA1, EIJI UCHIDA2, TAKASHI TAJIRI2 and ZENYA NAITO1 1Department of Pathology, Integrative Oncological Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602; 2Surgery for Organ and Biological Regulation (Department of Surgery I), Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan Received February 20, 2007; Accepted April 30, 2007 Abstract. Lumican is a member of a small leucine-rich lumican-negative cancer cells (p=0.286), but patients with proteoglycan family and its overexpression has been reported lumican-positive stromal tissues had shorter survival than in carcinoid tumor, breast, colorectal, neuroendocrine cell, those with lumican-negative stromal tissues (p=0.062). These uterine cervical and pancreatic cancers. The expression of results suggest that lumican in stromal tissues plays an lumican in stromal tissues in breast cancer is associated with a important role in the growth and invasion of pancreatic cancer. high tumor grade, a low estrogen receptor expression level and young age. Lumican expression in the cytoplasm in Introduction advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic Members of the small leucine-rich proteoglycan (SLRP) cancer, but the role of lumican in pancreatic cancer is still not family have small core proteins of approximately 40 kD and well understood. In this study, we aimed to clarify the role of possess 6-10 leucine-rich repeating units flanked by disulfide- lumican in pancreatic cancer.
    [Show full text]