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: Chondroitinase digestion A solution (100 ?L total volume) containing 0.1 mg/mL GAG, 50 mM NH4OAc buffer, pH 6 (kratanase and hyaluronidase) or pH 7 (chondroitinase and heparinase), and 0.1 mU/mL or 20 mU/mL (hyaluronidase only) GAG lyase was incubated at 37 ?C for 24 h. In the case of hyaluronidase digestion, the samples were incubated for an additional 2 h at 55 ?C. The enzyme was inactivated by heating to 100 ?C for 2 min and the samples were centrifuged prior to HPLC analysis. The following enzymes were used: Chondroitinase ABC (F. heparinum, Sigma) Chondroitinase AC (A. aurescens, Sigma) Heparinases I, II, and III (F. heparinum, Grampian) Hyaluronidase (S. hyalurolyticus, Northstar) Keratanase II (Bacillus sp., Northstar) 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. Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. The flow rate was 1.0 mL/min. Injection volume was 10 ?L. Detection was performed by post-column derivatization. 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 50-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm. Commercial standard disaccharides (Dextra Laboratories) were used for calibration for heparinase and chondroitinase digestion mixtures. For the other mixtures enzyme digested authentic keratan sulfate (AMS Biotechnology) and hyaluronic acid (Sigma) samples were used as standards.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR005351-22
Application #
8361857
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
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
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
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
Zarnowski, Robert; Westler, William M; Lacmbouh, Ghislain Ade et al. (2014) Novel entries in a fungal biofilm matrix encyclopedia. MBio 5:e01333-14

Showing the most recent 10 out of 245 publications