62,796 research outputs found
William J. Gerhardt M.D.
No full textThis oral history may be streamed from the Winkler Center websiteAn interview of William J. Gerhardt M.D. August 9, 2012. Introduction by John McDonough M.D. Interview by John McDonough M.D. and Joan Linhardt M.D. This video was a part of the Henry R. Winkler Center Oral History Series
Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations
No full textA simple, accurate and robust quantitative CE method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced, i.e., GlcN from Hep and GalN from OSCS, were derivatized with anthranilic acid (AA) and separated by means of CE in approx. 10 min with high sensitivity detection at 214 nm (limit of detection (LOD) of approx. 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity allowing for direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination, since chondroitin ABC lyase is unable to act on this semi-synthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS while a formulated contaminated Hep was calculated to have 39.7% OSCS
Separation of Glycosaminoglycan Derived Oligosaccharides by Capillary Electrophoresis Using Reverse Polarity
No full textAnalytical Biochemistry, 221, 182-188,Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.A comparative study on compositional analysis of two sets of eight unsaturated disaccharide standards derived from heparin/heparan sulfate and chondroitin/dermatan sulfate was carried out using capillary electrophoresis performed in both normal and reverse polarity modes. While these heparin/heparan sulfate disaccharides (S. A. Ampofo, H. M. Wang, and R. J. Linhardt (1991) Anal. Biochem. 199, 249-255) and chondroitin/dermatan sulfate disaccharides (A. Al-Hakim and R. J. Linhardt (1991) Anal. Biochem. 195, 68-73) have previously been fractionated using normal polarity capillary electrophoresis, multiple buffer systems and conditions were required to separate certain disaccharide isomers and these separations often resulted in poor peak symmetry and significant tailing. This paper demonstrates that reverse polarity capillary electrophoresis completely resolves disaccharide mixtures into all components using a single buffer, 20 mM phosphoric acid-sodium phosphate at pH 3.48. This improved resolution is due primarily to an increase in the sharpness of peaks and improved peak symmetry. Separation of heparin-derived oligosaccharides, ranging from disaccharide to hexasaccharide, had also previously been reported using normal polarity capillary electrophoresis (U.R. Desai, H.M. Wang, S.A. Ampofo, and R.J. Linhardt (1993) Anal. Biochem. 213, 120-127). This paper now demonstrates the separation of 13 heparin-derived oligosaccharides of sizes ranging from disaccharide to tetradecasaccharide using both reverse and normal polarities. An enzymatic digestion of bovine lung heparin containing many of these larger oligosaccharides was also compared in both normal and reverse polarity modes. Mixtures containing oligosaccharides primarily differing in size (number of saccharide units) were better resolved using normal polarity.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1006/abio.1994.139
Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2-mers to 16-mers. Comparison with hyaluronic acid oligomers
No full textThis study reports for the first time the complete LC-ESI-MS and MS/MS spectra performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only for the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly-charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]-2 product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There is no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Nonsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern of deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study we were unable to distinguish between unsulfated chondroitin and HA
Capillary electrophoresis of complex natural polysaccharides
No full textComplex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs) (chondroitin/dermatan sulfate, heparin/heparan sulfate and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. Capillary electrophoresis (CE), due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and tandem MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed
Purification and Characterization of Heparin Lyases from Flavobacterium heparinum
No full textJournal of Biological Chemistry, 267, 24347-24355,Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate
Regulation Activity of Heparin in Complement System
No full textin the Chemistry and Biology of Heparin and Heparan Sulfate, Elsevier Ltd., Oxford, H. G. Garg, R. J. Linhardt, and C. A. Hales (Eds.), Chapter 11, pp.313-343Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.Heparin (HP) can bind to a variety of proteins, including growth factors, pro-inflammatory chemokines and cytokines, extracellular matrix proteins, and complement proteins. HP has a variety of biological activities, many of which are of interest because of their potential therapeutic utility. By regulating the activity of HP-binding proteins, HP and the related glycosaminoglycan (GAG), heparan sulfate (HS), can influence various biological processes giving HP therapeutic applications as an antithrombotic, antiatherosclerotic, anticomplement, antiinfective, anticancer, and anti-inflammatory agent. Monosaccharide and disaccharides with structural similarities to dextran did not cause a detectable decrease in C3b-factor H binding, while sugar polymers caused large decreases in the affinity between C3b and factor H as a result of the polysaccharide occupying the binding site in C3b or in factor H, preventing their interaction. HP and the structurally similar HS regulate multiple steps in the complement system including ones in both the classical and alternative pathways. Quantitative data in the form of association rates, dissociation rates, and affinity constants for complex formation are provided for many of these interactions.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/B978-008044859-6/50012-
High performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides
No full textGlycosaminoglycans (GAGs) have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity. Since the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential in developing a structure-activity relationship. Electrospray ionization mass spectrometry (MS) is particularly promising for the analysis of GAG-derived oligosaccharides due to its relative soft ionization capacity. Furthermore, on-line high performance liquid chromatography (HPLC)-MS greatly enhances the analysis of complex mixtures of GAG-derived oligosaccharides providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerisation, is presented together with their HPLC separation using volatile reversed phase ion-pairing reagents and on-line electrospray ionisation MS structural identification. This analysis provides an oligosaccharide map together with sequence from a reading frame beginning at the non-reducing end of the GAG chains. The preparation of oligosaccharides can be done in 10 h with subsequent HPLC analysis in 1-2 h and HPLC-MS analysis taking 2 h
Influence of Heparin Chemical Modifications on its Antiproliferative Properties
No full textin the Chemistry and Biology of Heparin and Heparan Sulfate, Elsevier Ltd., Oxford, H. G. Garg, R. J. Linhardt, and C. A. Hales (Eds.), Chapter 18, pp 513-532Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.This chapter focuses on the mechanisms contributing to heparin inhibition of smooth muscle cell growth. Chronic pulmonary hypertension is characterized by structural changes in the pulmonary vasculature, which along with variable degrees of vasoconstriction, are responsible for the high pulmonary vascular resistance and associated right heart failure. Circulating HP binds to endothelial cells and is taken up by the reticuloendothelial system where it enters a cellular pool to be released at a later stage. Fully sulfated HP and other glycosaminoglycan (GAGS) are prepared by treating tributylammonium salt of these with sulfur trioxide. Sulfo groups in HP appear to play an important role in the growth inhibitory effect on smooth muscle cell proliferation. Removal of N-sulfo groups from HP reportedly negates its growth inhibitory effect on smooth muscle cells (SMCs). No appreciable difference was found between heparin and fully sulfated heparin on the growth of pulmonary artery smooth muscle cells. Chondroitin and dermatan sulfates stimulated the pulmonary artery SMCs. Hyaluronan was not antiproliferative but full sulfation made HA strongly antiproliferative against pulmonary SMCs.https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1016/B978-008044859-6/50019-
Breast cyst fluid heparan sulphate is distinctively N-sulphated depending on apocrine or flattened type
No full textBreast cyst fluid (BCF) contained in gross cists is involved with its many biomolecules in different stages of breast cystic development. Type I apocrine and type II flattened cysts are classified based on biochemical, morphological and hormonal differences, and their different patterns of growth factors and active biocompounds may require different regulation. In a previous paper, hyaluronic acid in a very low content and chondroitin sulphate/dermatan sulphate were identified and characterized in BCF. In this new study, various apocrine and flattened BCFs were analyzed for HS concentration and disaccharide pattern. Apocrine HS was found specifically constituted of N-acetyl groups contrary to flattened HS richer in N-sulphate disaccharides with an overall N-acetylated/N-sulphated ratio significantly increased in apocrine compared with flattened (13.5 vs 3.7). Related to this different structural features, the charge density significantly decreased (~-30%) in apocrine versus flattened BCFs. Finally, no significant differences were observed for HS amount (~0.9-1.3 μg ml(-1) ) between the two BCF types even if a greater content was determined for flattened samples. The specifically N-sulphated sequences in flattened BCF HS can exert biologic capacity by regulating growth factors activity. On the other hand, we cannot exclude a peculiar regulation of the activity of biomolecules in apocrine BCF by HS richer in N-acetylated disaccharides. In fact, the different patterns of growth factors and active biocompounds in the two types of cysts may require different regulation by specific sequences in the HS backbone possessing specific structural characteristics and distinctive chemical groups
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