DOCSLIB.ORG

Herpes Simplex Virus VI

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Herpes Simplex Virus VI JOURNAL OP VIROLOGY, OCt. 1972, p. 875-879 Vol. 10, No. 4 Copyright © 1972 American Society for Microbiology Printed In U.S.A. Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus VI. Polyadenylic Acid Sequences in Viral Messenger Ribonucleic Acid STEVEN L. BACHENHEIMER AND BERNARD ROIZMAN Department of Microbiology, The University of Chicago, Chicago, Illinois 60637 Received for publication 28 June 1972 Evidence is presented by use of radiolabeling and pancreatic and Ti ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus con- tains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA. The occurrence of polyriboadenylic acid than 99% of the 3H-adenosine was contained in a (poly A) sequences in messenger ribonucleic acid molecule labile in alkali, i.e., in RNA. (mRNA) and heterogeneous nuclear ribonucleic The size of poly A isolated from polyribosomes acid (HnRNA) of a variety of eukaryotic cell of infected cells was determined by co-electro- systems has been well established (3, 4, 5, 11, 12, phoresis on a polyacrylamide gel with uninfected 16). Poly A has also been detected in the niRNA cell 4S RNA (Fig. 1). Both poly A and 4S of adenovirus (14) and vaccinia virus (9, 16), in RNA marker were denatured in 1.1 M formal- the genomic ribonucleic acid (RNA) of tumor dehyde prior to electrophoresis so that their viruses (6, 7, 10), as well as in the RNA of polio- migration and subsequent molecular-weight de- (1) and sindbis (8) viruses. Significant amounts termination would be independent of molecular of poly A were not detected in the genomic conformation (2). The molecular weight of the RNA species of Newcastle disease, influenza, or largest poly A sequence found, calculated from vesicular stomatitis viruses (6). In the instances the relationship of relative distance migrated to where poly A sequences were detected, they were log molecular weight, was 54,500, correspond- characterized as single-stranded ribonucleotide ing to about 160 nucleotides. The heterogeneity sequences resistant to pancreatic and Ti ribo- with respect to the size distribution of poly A nuclease. The isolated poly A sequences char- sequences has been noted by others (1, 10, 13) acteristically migrated in polyacrylamide gels as if in a variety of cell systems, both uninfected and they were 150 to 200 nucleotides in length. In this infected with virus. The heterogeneity may be a report we wish to extend these observations to reflection ofrandomness in the process of poly herpes simplex virus. A attachment to mRNA, or the consequence The evidence that RNA specified by herpes of removal of adenylic acid residues after poly simplex virus contains poly A sequences is based A is in the cytoplasm, or a combination. on two series of experiments. The first series of The objective of the second series of experi- experiments was based on previous work (15, 17, ments was to determine in a more rigorous man- 18) indicating that, by 4 hr after infection, the ner whether poly A sequences were contained in polyribosomes of infected cells were exclusively viral RNA. In these experiments 8P-labeled synthesizing viral proteins, and that most, if not RNA was hybridized to viral DNA immobilized all, cytoplasmic RNA specified by the virus was on filters. The hybridization was carried out in associated with polyribosomes. In the present five times concentrated standard saline citrate and experiments, RNA labeled with 3H-adenosine and 50% formamide at 43 C to insure a minimum of 14C-uridine was isolated from polyribosomes of breakage of RNA and to test for the covalent infected cells and digested with pancreatic and Ti attachment of poly A to virus-specified RNA. ribonuclease. As shown in Table 1, the residual Preliminary experiments designed to test the ribonuclease-resistant RNA contains 20% of the effects of various hybridization procedures on adenosine but only 1% of the uridine of poly- the integrity of the poly A-mRNA linkage have ribosomal RNA. To establish that the residual shown that, in general, the lower the temperature 3H-adenosine was in RNA, polyribosomal RNA at which the hybridization is carried out, the was digested with ribonuclease and then hydro- greater the stability ofthe RNA. At the conditions lyzed with 0.6 N KOH. As shown in Table 1, more selected for the hybridization tests, 77% of the 875 876 NOTES J. VIROL. TABLE 1. Presence of polyadenylic acid sequenices 14 - u in polyribosomal RNA of herpes virus- 3H-Aid inifected cellsa 1000 r250 3H-Adenosine 14C-Uridine Polyribosomal RNA Counts/ Percent Counts/ Per- 800 200 min min cent Expt 1 600 150 Input 10,200 100 3,100 100 After ribonuclease 2,037 20 33 1 Expt 2 400 100 Input 51,000 100 15, 500 100 I\ Wbe,^~~ >k10 After ribonuclease 107 0.2 34 0.2 \z + KOH 200 50 a 2 X 108 A monolayer of HEp-2 cells, infected Oa* oao,o'd o\ \ with 10 plaque-forming units of herpes simplex virus subtype 1 (HSV-1) per cell was labeled nI nm from 3 to 4.5 hr postinfection with 10 ,Ci of 10 20 30 40 + 3H-adenosine per ml in 25 ml of Eagle minimal fraction no. essential medium supplemented with 1% dialyzed calf serum. Cells were washed in phosphate- FIG. 1. Acrylamide gel electropherogram ofpoly A buffered saline and disrupted by resuspension in isolated from polyribosomes of infected cells. Inifected- RSB [0.01 M NaCl, 0.01 M tris(hydroxymethyl)- cell polyribosomes, labeled with 3H-adenzosine, were aminomethane (Tris), pH 7.5, 0.0015 M MgCl2J prepared, and poly A was isolated as described in the containing 0.5% Nonidet P-40 at 4 C for 15 min. legend to Table 1. After digestion with pancreatic Nuclei were removed by centrifugation at low and Ti ribonuclease, sodium dodecyl sulfate was speed. The cytoplasmic extract was layered over a added to a final concentration of 0.5%, and 100 ,g 40% (w/w) sucrose-RSB cushion, and poly- of yeast RNA was added as carrier. The solubilized ribosomes were collected as a pellet by centrifuga- digest was phenol-extracted, anid a Tris blffer, pH 9.0, tion at 102,000 X g for 2.5 hr at 5 C in a T65 rotor. wash of the interface and pheniol phase was combined The polyribosome pellet was resuspended in with the original aqueous phase an1d chloroform-ex- TSM (0.15 M NaCl, 0.01 M Tris, pH 7.5, 0.001 M tracted. Uninfected cell RNA, labeled with 14C-uridine MgCl2) containing 0.5% sodium dodecyl sulfate, and soluble in 2 M LiCl was added to the combinied and was extracted with phenol. The interphase and aqueous phases, precipitated with 7.5%7 trichloroacetic phenol phase were washed with Tris buffer (0.1 M acid, anid washed with 80% ethanol. The precipitate Tris, pH 9.0, 0.01 M KCI, 0.002 M MgCl2), to insure was resuspended in 50 jAliters of buffer containing that RNA containing poly A sequences was not 0.09 M Na2HPO4, 0.01 M NaH2PO4, pH 7.6, 1.1 m specifically lost during the extraction. The aqueous formaldehyde, heated to 65 C for 15 min, quick- phases were combined and extracted with chloro- cooled, and applied to a 10-cm, 10% acrylamide gel form-2% isoamyl alcohol, and RNA was pre- made up in 0.018 M Na2HPO4, 0.002 M NaH2PO4, cipitated with two volumes of absolute ethanol 1.1 sm formaldehyde. After electrophoresis for 3.25 hr and stored at -20 C. The precipitate was col- at 4 ma, the gel was fractioniated into 2-mm slices. lected by centrifugation and resuspended in The slices were swelled in 0.6 ml of soluenie (Packard TSM buffer. In experiment 1, a sample of the Instrument Co., Inc.) at 60 C for 2 hr and theni as- RNA was digested with deoxyribonuclease I sayed for radioactivity in 10 ml of a toluene-base (50 jg/ml) for 30 min at 37 C. Ethylenediamine- scintillant. tetraacetic acid (EDTA) was then added to a final concentration of 0.01 M, and enough 10 X SSC (1.5 M NaCl, 0.15 M sodium citrate) was added RNA retained its ability to bind to membrane to give a final concentration of 0.3 M NaCl. Pan- filters (Millipore Corp.). After 20 hr of hybridiza- creatic ribonuclease (2,ug/ml) and Ti ribonuclease tion, the RNA was eluted from the filter and (5 units/ml) were added and allowed to digest analyzed for its ability to bind to Millipore filters the RNA for 60 min at 37 C. The digest was pre- in the presence of 0.5 M KCI (11). As shown in cipitated with an equal volume of 15c%o trichloro- Table 2, of 50,000 counts/min of cytoplasmic acetic acid, collected on a membrane filter, dried, viral RNA tested, 41% bound to the filter, and counted. In experiment 2, a sample of RNA whereas 59%O passed through the filter. These was digested as described above, and then hy- drolyzed for 18 hr at 37 C in 0.6 N KOH. The data indicate that poly A is covalently linked to hydrolysate was then precipitated, potassium viral RNA. perchlorate was spun out at low speed, and any Consistent with previous reports (13, 14), the residual material in the supernatant was collected experiment described above and other experi- on a filter for counting. ments suggest that, in herpesvirus-infected cells VOL. 10, 1972 NOTES 877 TABLE 2. Binding of viral RNA to cellulose 3H-adenosine, the appearance of label in poly nitrate filtersa A-rich RNA binding to filters lags behind the appearance of the label in total nuclear RNA Total RNA RNA bound Viral RNA tested (counts/ to filters tof (Fig.
Load more

Recommended publications
  • To Study Mutant P53 Gain of Function, Various Tumor-Derived P53 Mutants
    Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By Shama K Khokhar M.Sc., Bilaspur University, 2004 B.Sc., Bhopal University, 2002 2007 1 COPYRIGHT SHAMA K KHOKHAR 2007 2 WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES Date of Defense: 12-03-07 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY SHAMA KHAN KHOKHAR ENTITLED Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science Madhavi P. Kadakia, Ph.D. Thesis Director Daniel Organisciak , Ph.D. Department Chair Committee on Final Examination Madhavi P. Kadakia, Ph.D. Steven J. Berberich, Ph.D. Michael Leffak, Ph.D. Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies 3 Abstract Khokhar, Shama K. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2007 Differential effect of TAp63γ mutants on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. p63, a member of the p53 gene family, known to play a role in development, has more recently also been implicated in cancer progression. Mice lacking p63 exhibit severe developmental defects such as limb truncations, abnormal skin, and absence of hair follicles, teeth, and mammary glands. Germline missense mutations of p63 have been shown to be responsible for several human developmental syndromes including SHFM, EEC and ADULT syndromes and are associated with anomalies in the development of organs of epithelial origin.
    [Show full text]
  • HOXB6 Homeo Box B6 HOXB5 Homeo Box B5 WNT5A Wingless-Type
    5 6 6 5 . 4 2 1 1 1 2 4 6 4 3 2 9 9 7 0 5 7 5 8 6 4 0 8 2 3 1 8 3 7 1 0 0 4 0 2 5 0 8 7 5 4 1 1 0 3 6 0 4 8 3 7 4 7 6 9 6 7 1 5 0 8 1 4 1 1 7 1 0 0 4 2 0 8 1 1 1 2 5 3 5 0 7 2 6 9 1 2 1 8 3 5 2 9 8 0 6 0 9 5 1 9 9 2 1 1 6 0 2 3 0 3 6 9 1 6 5 5 7 1 1 2 1 1 7 5 4 6 6 4 1 1 2 8 4 7 1 6 2 7 7 5 4 3 2 4 3 6 9 4 1 7 1 3 4 1 2 1 3 1 1 4 7 3 1 1 1 1 5 3 2 6 1 5 1 3 5 4 5 2 3 1 1 6 1 7 3 2 5 4 3 1 6 1 5 3 1 7 6 5 1 1 1 4 6 1 6 2 7 2 1 2 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S HOXB6 homeo box B6 HOXB5 homeo box B5 WNT5A wingless-type MMTV integration site family, member 5A WNT5A wingless-type MMTV integration site family, member 5A FKBP11 FK506 binding protein 11, 19 kDa EPOR erythropoietin receptor SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 RAD52 RAD52 homolog S.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Supplementary Table 2
    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • An NKX2-1/ERK/WNT Feedback Loop Modulates Gastric Identity And
    RESEARCH ARTICLE An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma Rediet Zewdu1,2, Elnaz Mirzaei Mehrabad1,3, Kelley Ingram1,4, Pengshu Fang1,4, Katherine L Gillis1,4, Soledad A Camolotto1,2, Grace Orstad1,4, Alex Jones1,2, Michelle C Mendoza1,4, Benjamin T Spike1,4, Eric L Snyder1,2,4* 1Huntsman Cancer Institute, Salt Lake City, United States; 2Department of Pathology, University of Utah, Salt Lake City, United States; 3School of Computing, University of Utah, Salt Lake City, United States; 4Department of Oncological Sciences, University of Utah, Salt Lake City, United States Abstract Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2- 1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.
    [Show full text]
  • To Nucleotide Sequence Determinations (Pancreatic Dnase I/U1 Ribonuclease/U2 Ribonuclease/Pancreatic Ribonuclease A/Ribosubstituted DNA) GARY V
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5017-5021, December 1974 Deoxysubstitution in RNA by RNA Polymerase In Vitro: A New Approach to Nucleotide Sequence Determinations (pancreatic DNase I/U1 ribonuclease/U2 ribonuclease/pancreatic ribonuclease A/ribosubstituted DNA) GARY V. PADDOCK, HOWARD C. HEINDELL AND WINSTON SALSER Biology Department and Molecular Biology Institute, University of California at Los Angeles, 405 Hilgard Ave., Los Angeles, Calif. 90024 Communicated by Charles Yanofsky, August 12, 1974 ABSTRACT Deoxynucleotides have been incorporated substituted RNA. We have observed that Mn++ ion will not into RNA synthesized in vitro by RNA polymerase with only cause DNA polymerase to synthesize ribosubstituted either double-stranded or single-stranded DNA as a to synthesize template. By use of this technique to block or promote DNA but will also cause RNA polymerase cleavage at a particular phosphodiester bond, a variety of deoxysubstituted RNA. Subsequently we have discovered a specific cleavages may be obtained with the available ribo- number of other papers dealing with the synthesis of deoxy- nucleases and deoxyribonuclease I. These methods should substituted RNA (12-14). These papers did not, however, greatly increase the ease and rapidity of nucleotide se- point out the applicability of the approach to nucleotide quence determinations. sequencing and did not include the controls we have found The introduction of radiographic approaches by Sanger and essential for demonstrating that complete deoxysubstitution his colleagues (1-4) greatly increased the power of nucleotide has occurred. sequencing techniques, but there remain certain obstacles In this preliminary paper we discuss the theory by which whose solution could result in impressive further increases in substitution of deoxynucleotides becomes an aid in nucleotide the rapidity with which large sequences may be determined.
    [Show full text]
  • Cell-Based Osteoprotegerin Therapy for Debris-Induced Aseptic Prosthetic Loosening on a Murine Model
    Gene Therapy (2010) 17, 1262–1269 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt ORIGINAL ARTICLE Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model L Zhang1,2, T-H Jia2, ACM Chong1, L Bai1,HYu1, W Gong2, PH Wooley1 and S-Y Yang1 1Orthopaedic Research Institute, Via Christi Regional Medical Center, Wichita, KS, USA and 2Department of Orthopaedic Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan, China Exogenous osteoprotegerin (OPG) gene modification membranes existed ubiquitously at bone–implant interface appears a therapeutic strategy for osteolytic aseptic in control groups, whereas only observed sporadically in OPG loosening. The feasibility and efficacy of a cell-based OPG gene-modified groups. Tartrate-resistant acid phosphatase+ gene delivery approach were investigated using a murine osteoclasts and tumor necrosis factor a,interleukin-1b, model of knee prosthesis failure. A titanium pin was CD68+ expressing cells were significantly reduced in implanted into mouse proximal tibia to mimic a weight- periprosthetic tissues of OPG gene-modified mice. No bearing knee arthroplasty, followed by titanium particles transgene dissemination or tumorigenesis was detected in challenge to induce periprosthetic osteolysis. Mouse fibro- remote organs and tissues. Data suggest that cell-based blast-like synoviocytes were transduced in vitro with either ex vivo OPG gene therapy was comparable in efficacy with AAV-OPG or AAV-LacZ before transfused into the osteolytic in vivo local gene transfer technique to deliver functional prosthetic joint 3 weeks post surgery. Successful transgene therapeutic OPG activities, effectively halted the debris- expression at the local site was confirmed 4 weeks later after induced osteolysis and regained the implant stability in this killing.
    [Show full text]
  • Figure S1. GO Analysis of Genes in Glioblastoma Cases That Showed Positive and Negative Correlations with TCIRG1 in the GSE16011 Cohort
    Figure S1. GO analysis of genes in glioblastoma cases that showed positive and negative correlations with TCIRG1 in the GSE16011 cohort. (A‑C) GO‑BP, GO‑CC and GO‑MF terms of genes that showed positive correlations with TCIRG1, respec‑ tively. (D‑F) GO‑BP, GO‑CC and GO‑MF terms of genes that showed negative correlations with TCIRG1. Red nodes represent gene counts, and black bars represent negative 1og10 P‑values. TCIRG1, T cell immune regulator 1; GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function. Table SI. Genes correlated with T cell immune regulator 1. Gene Name Pearson's r ARPC1B Actin‑related protein 2/3 complex subunit 1B 0.756 IL4R Interleukin 4 receptor 0.695 PLAUR Plasminogen activator, urokinase receptor 0.693 IFI30 IFI30, lysosomal thiol reductase 0.675 TNFAIP3 TNF α‑induced protein 3 0.675 RBM47 RNA binding motif protein 47 0.666 TYMP Thymidine phosphorylase 0.665 CEBPB CCAAT/enhancer binding protein β 0.663 MVP Major vault protein 0.660 BCL3 B‑cell CLL/lymphoma 3 0.657 LILRB3 Leukocyte immunoglobulin‑like receptor B3 0.656 ELF4 E74 like ETS transcription factor 4 0.652 ITGA5 Integrin subunit α 5 0.651 SLAMF8 SLAM family member 8 0.647 PTPN6 Protein tyrosine phosphatase, non‑receptor type 6 0.641 RAB27A RAB27A, member RAS oncogene family 0.64 S100A11 S100 calcium binding protein A11 0.639 CAST Calpastatin 0.638 EHBP1L1 EH domain‑binding protein 1‑like 1 0.638 LILRB2 Leukocyte immunoglobulin‑like receptor B2 0.629 ALDH3B1 Aldehyde dehydrogenase 3 family member B1 0.626 GNA15 G protein
    [Show full text]
  • Deoxyribonuclease I
    Catalog Number: 100574, 100575, 190062 Deoxyribonuclease I CAS #: 9003-98-9 Description: Deoxyribonuclease from beef pancreas, DNase I, was first crystallized by Kunitz.10,11 It is an endonuclease which splits phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The average chain of limit digest is a tetranucleotide.19 DNase I acts upon single chain DNA29, and upon double-stranded DNA and chromatin. In the latter case, although histones restrict susceptibility to nuclease action, over a period of time nearly all chromatin DNA is acted upon. According to Mirsky and Silverman20, this could result from the looseness of histone attachment to DNA. They found that lysine-rich histones more effectively block DNase access to DNA than arginine-rich histones. Billing and Bonner2 suggest that DNase attacks the histone-free strand of chromatin DNA. Schmidt, et. al.28 indicate that hydrolysis of the histone-free region of DNA strands accounts for the initial rapid action of the enzyme on chromatin. Bollum3 reports degradation of synthetic homopolymer complexes by DNase I. The intracellular functions of the enzyme are probably controlled by a DNase inhibitor17, which according to Lazarides and Lindberg13 is actin. Molecular weight: 31,000.18 Composition: There are four deoxyribonucleases of beef pancreas: A, B, C, and D.14, 25, 27 Five have been reported by Junowicz and Spencer.7 They are glycoproteins differing from each other either in carbohydrate side-chain or polypeptide component.14, 25 DNase A is the predominant form26; its amino acid sequence has been reported.16 Optimum pH: 7.8.
    [Show full text]
  • Skeletal Stem and Progenitor Cells Maintain Cranial Suture Patency and Prevent Craniosynostosis
    ARTICLE https://doi.org/10.1038/s41467-021-24801-6 OPEN Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis Siddharth Menon1,2,3,4, Ankit Salhotra 1,2,3, Siny Shailendra1,2,3, Ruth Tevlin1,2,3, Ryan C. Ransom1,2,3, Michael Januszyk1,2,3, Charles K. F. Chan 1,2,3,4, Björn Behr5, Derrick C. Wan1,2,3, ✉ ✉ Michael T. Longaker 1,2,3,4 & Natalina Quarto 1,2,3,6 Cranial sutures are major growth centers for the calvarial vault, and their premature fusion 1234567890():,; leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/− mouse model of coronal cra- niosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co- transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/− mice. Our study reveals that decrease and/or imbalance of skeletal stem/pro- genitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis. 1 Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 2 Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, USA.
    [Show full text]
  • Deoxyribonuclease I, Bovine (D2821)
    Deoxyribonuclease I, bovine recombinant, expressed in Pichia pastoris lyophilized powder Catalog Number D2821 Storage Temperature 2–8 °C CAS RN 9003-98-9 Optimal pH: EC 3.1.21.1 The optimal pH of DNase I activity is dependent on the Synonyms: DNase I, divalent ion present. In the presence of both Mg2+ and Deoxyribonucleate 5¢-Oligonucleotidohydrolase Ca2+, the optimal pH is between 7–8, while in the absence of Ca2+, the optimal pH is between 5.5–6.0.6 Product Description 1% Deoxyribonuclease I (DNase I) is found in most cells Extinction Coefficient: E280 = 11.1 and tissues. In mammals, the pancreas is one of the best sources for the enzyme. Pancreatic DNase I was This recombinant bovine DNase I is a glycoprotein, the first isolated DNase. produced without the addition of any animal-derived materials. It is supplied as a lyophilized powder DNase I is an endonuclease that acts on containing a glycine stabilizer. phosphodiester bonds adjacent to pyrimidines to produce polynucleotides with terminal 5¢-phosphates. Molecular mass: ~39 kDa A tetranucleotide is the smallest average digestion product. DNase I hydrolyzes single- and double- Specific activity: ³4,000 units/mg protein stranded DNA. In the presence of Mg2+ ions, DNase I attacks each strand of DNA independently and the Unit definition: One unit will produce a change in A260 of cleavage sites are random. If Mn2+ ions are present, 0.001 per minute per ml at pH 5.0 at 25 °C using DNA, both DNA strands are cleaved at approximately the Type I or III, as the substrate.
    [Show full text]