This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: GAG Isolation The frozen tissue was ground in liquid nitrogen and extracted with 10 ml acetone (twice for 24 h) and once with diethyl ether (5 ml, 30 min) and dried under vacuum. The tissue was resuspended in 2 mL pronase digestion buffer (0.1 M Tris-HCl, pH 8.0, 2 mM CaCl2, 1% Triton X-100), 1.6 mg pronase was added, and the tissue was digested with shaking at 55 ?C (1). After 24 h, a second 1.6 mg aliquot of pronase was added and digestion continued for 24 h. The enzyme was inactivated by heating to 100 ?C for 15 min. The buffer was adjusted to 2 mM MgCl2, benzonase (100 mU) was added, and the sample was incubated for 2 h at 37 ?C. After inactivation of the enzyme (15 min 100 ?C) the undigested tissue was precipitated by centrifugation for 15 min at 12000 g. The supernatant was applied to a DEAE-Sephacel column (2 mL), washed with 20 mL equilibration buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl), and eluted with 6 mL elution buffer (20 mM Tris-HCl pH 7.5, 2 M NaCl). The sample was treated with 1 mL 10 % (w/v) NaBH4 in 2N NaOH, and incubated overnight at 4 ?C (2). The reaction was stopped by adding glacial acetic acid until no bubbles were formed and the pH was neutral. The sample was freeze-dried, desalted using a PD10 column (GE Healthcare), again freeze-dried, and dissolved in 50 ?L water. GAG lyase digestion A 10-?L aliquot of the sample solution was dissolved 50 mM ammonium acetate, pH 7, and treated with 10 ?L of the appropriate GAG lyase solution (1 U/mL). The enzyme solutions used were a) chondroitinases ABC and AC, b) chondroitinase ABC, and c) heparinases I, II, and III. The samples were incubated at 37 ?C overnight and heated to 100 ?C for 2 min. SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6?250 mm Waters Spherisorb analytical column with 5 ?m particle size at 25 ?C using the following gradients: Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. Detection was performed by post-column derivatization as described (3). Briefly, to the eluent from the column was added, from a binary HPLC pump, a 1:1 mixture of 0.25 M NaOH and 1 % 2-cyanoacetamide at 0.5 mL/min. The eluent was then heated to 120 ?C in a 10-m reaction coil, followed by cooling in a 30-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR005351-22
Application #
8361838
Study Section
Special Emphasis Panel (ZRG1-IMST-A (40))
Project Start
2011-02-01
Project End
2012-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
22
Fiscal Year
2011
Total Cost
$1,772
Indirect Cost
Name
University of Georgia
Department
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Hannides, Angelos K; Aller, Robert C (2016) Priming effect of benthic gastropod mucus on sedimentary organic matter remineralization. Limnol Oceanogr 61:1640-1650
Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul et al. (2016) Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 26:360-76
Zhao, Wujun; Zhu, Taotao; Cheng, Rui et al. (2016) Label-Free and Continuous-Flow Ferrohydrodynamic Separation of HeLa Cells and Blood Cells in Biocompatible Ferrofluids. Adv Funct Mater 26:3990-3998
Wu, Liang; Viola, Cristina M; Brzozowski, Andrzej M et al. (2015) Structural characterization of human heparanase reveals insights into substrate recognition. Nat Struct Mol Biol 22:1016-22
Qiu, Hong; Xiao, Wenyuan; Yue, Jingwen et al. (2015) Heparan sulfate modulates Slit3-induced endothelial cell migration. Methods Mol Biol 1229:549-55
Li, Zixuan; Moniz, Heather; Wang, Shuo et al. (2015) High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface. J Biol Chem 290:10729-40
Liu, Lin; Zha, Jingying; DiGiandomenico, Antonio et al. (2015) Synthetic Enterobacterial Common Antigen (ECA) for the Development of a Universal Immunotherapy for Drug-Resistant Enterobacteriaceae. Angew Chem Int Ed Engl 54:10953-7
Czuchry, Diana; Desormeaux, Paul; Stuart, Melissa et al. (2015) Identification and Biochemical Characterization of the Novel ?2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104. J Bacteriol 197:3760-8
Li, Xiuru; Fang, Tao; Boons, Geert-Jan (2014) Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl 53:7179-82
Zhang, Fuming; Moniz, Heather A; Walcott, Benjamin et al. (2014) Probing the impact of GFP tagging on Robo1-heparin interaction. Glycoconj J 31:299-307

Showing the most recent 10 out of 244 publications