Biomedical subjects

K G Rice

Publications and source records attributed to K G Rice.

65 records · Page 4Linked to original sources

PubMed1988

Mapping and quantification of the major oligosaccharide components of heparin.

A new method of determining the oligosaccharide composition of commercial glycosaminoglycan heparin is described in which heparin was first depolymerized using heparin lyase (EC 4.2.2.7), and then analysed by a single h.p.l.c. step. All 20 of the porcine and bovine heparins examined were found to contain a small number of major oligosaccharide components, which on average comprised 86% of their mass. The five most abundant oligosaccharides have defined chemical structures. Although the relative abundance of oligosaccharides varied, the heparins examined were surprisingly similar. Porcine, bovine, low-Mr, and high and low antithrombin III (ATIII)-affinity heparins, however, each had distinctly different proportions of these major oligosaccharide components. The concentrations of one of these five oligosaccharides, containing a portion of the ATIII binding site, correlated with the anticoagulant activity of the ATIII-affinity-fractionated porcine-mucosal heparins from which it was derived. An additional oligosaccharide of undetermined structure was found in significant quantities in both bovine heparin and high ATIII-affinity porcine-mucosal heparin. The correlation between oligosaccharide concentration and anticoagulant activity suggests that the oligosaccharide is derived from a structural variant of the ATIII-binding site. Finally, for the heparins examined chondroitin/dermatan sulphate formed 0.6-7.4% of their mass.

Animals↗

PubMed1988

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro.

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.

Animals↗

PubMed1987

Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

Animals↗

PubMed1986

Structure and activity of a unique heparin-derived hexasaccharide.

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.

Animals↗

PubMed1985

Evidence of random structural features in the heparin polymer.

The first use of computer-simulation studies to examine heparin's structure has been reported. The product distributions obtained when porcine mucosal heparins were depolymerized with heparinase have been compared to computer-simulated distributions. The modeled distribution was relatively unaffected by the polydispersity and molecular weight of heparin. However, the percent of heparinase-cleavable glycosidic linkages and their distribution throughout the polymer resulted in a marked change in the simulated product distribution. The similarity between experimentally observed and computer-simulated product distributions is consistent with the random distribution of heparinase-cleavable sites in porcine mucosal heparin. Finally, a random distribution of N-acetyl residues with respect to heparinase-cleavable sites was experimentally observed.

Animals↗

PubMed1985

High-performance liquid chromatographic separation of heparin-derived oligosaccharides.

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.

Chromatography, Gel↗

PubMed1985

Structure of heparin-derived tetrasaccharides.

The structure of heparin was examined by characterizing a disaccharide and five of the more than a dozen tetrasaccharide components obtained by its depolymerization with flavobacterial heparinase. Enzymic depolymerization of porcine mucosal heparin results in a mixture of di-, tetra-, hexa- and higher oligo-saccharides. The di- and tetra-saccharide components represent 75mol/100mol of these heparin fragments. Ion-exchange chromatography indicates the presence of only one disaccharide, deltaIdu2S(1----4)-alpha-D-GlcNS6S (where Idu is iduronic acid, deltaIdu is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, GlcA is glucuronic acid and S is sulphate), but results in the isolation of five major and at least seven minor tetrasaccharide components. The structures of the disaccharide and five major tetrasaccharides were determined by chemical, enzymic, electrophoretic and spectroscopic methods, including 13C, 1H n.m.r. and fast atom bombardment-m.s. The structure of these five tetrasaccharides are: delta Idu2S(1----4)-alpha-D-GlcNS6S(1---4)-alpha-L-Idu2S(1-- --4)-alpha -D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS6S(1----4)-beta-D-GlcA(1--- -4)- alpha-D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS(1----4)-beta-D-GlcA delta Idu2S(1----4)-alpha-D-GlcNAc(1----4)-beta-D-GlcA(1----4)- alpha-D-GlcNS6S; and delta Idu2S(1----4)-alpha- D-GlcNAc(1----4)-alpha-L-Idu(1----4)-alpha-D-GlcNS6S. Biological activity for the disaccharide and the five major tetrasaccharides was examined, and none of them were found to possess significant anticoagulant activity.

Animals↗

PubMed1985

Small heparin fragments regulate the amplification pathway of complement.

Heparin is a highly sulfated, polydisperse and heterogeneous glycosaminoglycan which has been well characterized for its ability to regulate multiple sites in the complement cascade. Although previous studies demonstrated the relationship between degree of sulfation, particularly O-sulfation, and complement inhibiting capacity, they left unclear the relationship between the size of the heparin molecule and its ability to inhibit complement. Therefore, although the structure-activity relationship for heparin is well understood for anticoagulant activity, it is ill defined for the complement system. The present studies were designed to examine depolymerized heparin to determine which fragments were capable of inhibiting amplification pathway activation. We found that as the size of the molecule increases the ability to regulate complement increases; below 1000 Da the fragments were essentially inactive and above 3500 Da they had the same activity as does commercial heparin. Furthermore, we examined the five major tetrasaccharides of heparin and found that the degree of sulfation did correlate with the ability to inhibit complement. These studies have for the first time begun to examine the minimal structural requirements for heparin to regulate complement.

Complement Activation↗

PubMedDate not supplied

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers.

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.

Amino Acid Sequence↗

PubMedDate not supplied

Targeted gene delivery with a low molecular weight glycopeptide carrier.

A low molecular weight glycopeptide carrier was prepared by coupling a tyrosinamide-triantennary oligosaccharide to dp19 poly-L-lysine resulting in a 1:1 conjugate. The glycopeptide carrier complexed with plasmid DNA as evidenced by displacement of intercalated dye, light scattering by condensed DNA, and immobility of complexed DNA upon agarose gel electrophoresis. DNA-carrier complexes were endocytosed into HepG2 cells via the asialoglycoprotein receptor due to recognition of terminal galactose residues on the oligosaccharide. The resulting luciferase reporter gene expression was dramatically influenced by the solubility of complexes, the extent of complexation, and the presence of the lysosomotropic agent chloroquine. The results suggest that low molecular weight glycopeptides may be suitable for further development as well-defined DNA carriers for receptor-mediated gene delivery in vivo.

Carbohydrate Sequence↗

PubMedDate not supplied

Peptide-mediated gene delivery: influence of peptide structure on gene expression.

Cationic peptides possessing a single cysteine, tryptophan, and lysine repeat were synthesized to define the minimal peptide length needed to mediate transient gene expression in mammalian cells. The N-terminal cysteine in each peptide was either alkylated or oxidatively dimerized to produce peptides possessing lysine chains of 3, 6, 8, 13, 16, 18, 26, and 36 residues. Each synthetic peptide was studied for its ability to condense plasmid DNA and compared to polylysine19 and cationic lipids to establish relative in vitro gene transfer efficiency in HepG2 and COS7 cells. Peptides with lysine repeats of 13 or more bound DNA tightly and produced condensates that decreased in mean diameter from 231 to 53 nm as lysine chain length increased. In contrast, peptides possessing 8 or fewer lysine residues were similar to polylysine19, which bound DNA weakly and produced large (0.7-3 microns) DNA condensates. The luciferase expression was elevated 1000-fold after HepG2 cells were transfected with DNA condensates prepared with alkylated Cys-Trp-Lys18 (AlkCWK18) versus polylysine19. The gene transfer efficiencies of AlkCWK18 and cationic lipids were equivalent in HepG2 cells but different by 10-fold in COS 7 cells. A 40-fold reduction in particle size and a 1000-fold amplification in transfection efficiency for AlkCWK18 DNA condensates relative to polylysine19 DNA condensates suggest a contribution from tryptophan that leads to enhanced gene transfer properties for AlkCWK18. Tryptophan-containing cationic peptides result in the formation of small DNA condensates that mediate efficient nonspecific gene transfer in mammalian cells. Due to their low toxicity, these peptides may find utility as carriers for nonspecific gene delivery or may be developed further as low molecular weight DNA condensing agents used in targeted gene delivery systems.

DNA↗