Thomas N. Huckerby

Galantamine inhibits β-amyloid aggregation and cytotoxicity

The ability of galantamine (Reminyl) to inhibit the aggregation and toxicity of the beta-amyloid peptide (Abeta) was investigated. Galantamine showed concentration-dependent inhibition of aggregation of both Abeta 1-40 and Abeta 1-42, as determined by an ELISA method. Electron microscope studies of Abeta 1-40 incubated in the presence of galantamine revealed fibrils that were disordered and clumped in appearance. MTT and lactate dehydrogenase assays, employing SH-SY5Y human neuroblastoma cells, showed that galantamine reduced the cytotoxicity induced by Abeta 1-40. Galantamine also dramatically reduced Abeta 1-40-induced cellular apoptosis in these cells. There is some evidence that galantamine may not be acting purely as a symptomatic treatment. Disease-modifying effects of the drug could be due to an additional effect on Abeta aggregation and/or toxicity.

An appraisal of NMR methods for study of end-groups derived from initiators in radical polymerizations

Abstract Various studies by NMR spectroscopy of end-groups in polymers and copolymers are reviewed, particular attention being paid to groups formed during radical polymerization from initiators enriched with carbon-13. The sensitivity of the end-group analysis is not as high as that achievable when the initiator is labelled with a radioactive isotope but the specificity is incomparably better. It is possible to identify the incorporated initiator fragments and also to make deductions about the nature and stereochemistry of the monomeric units close to the end-groups. For quantitative work requiring comparison of the numbers of initiator fragments and monomeric units, the conditions for acquisition of the NMR spectrum of the polymer must be selected with care.

Periodate oxidation and alkaline degradation of heparin-related glycans

Abstract Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d -Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L -Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino- d -glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.

Human aggrecan keratan sulfate undergoes structural changes during adolescent development

Alkaline borohydride-reduced keratan sulfate chains were isolated from human articular cartilage aggrecan from individuals of various ages (0–85 years old). The chains were structurally characterized using 1H NMR spectroscopy, gel permeation chromatography, and oligosaccharide profiling (after digestion with the enzymes keratanase and keratanase II). The results show that from birth to early adolescence (0–9 years) the levels of α(1–3)-fucosylation, α(2–3)-sialylation, and galactose sulfation increase. Also, the weight-average molecular weight of the chains increases. During maturation (9–18 years) the levels of fucosylation and galactose sulfation continue to increase and α(2–6)-sialylation of the chains occurs. In adult life (18–85 years) there is little change in the weight-average molecular weight of the chains, and the levels of fucosylation, sialylation, and sulfation remain fairly constant.

Fast-atom-bombardment mass-spectrometric strategies for sequencing sulphated oligosaccharides

Abstract A strategy is presented for the structural analysis of sulphated oligosaccharides. The oligosaccharides are permethylated to leave sulphate groups intact, and the products examined by negative f.a.b.-m.s. The fragmentation observed from such compounds in the negative mode is described for the first time. The sulphates are then chemically replaced by acetyl groups, so producing a derivative that is examined in the positive mode. This procedure yields sequence data and defines the residues on which the sulphates were originally located. The strategy is illustrated using glycosaminoglycan fragments.

Human Corneal Keratan Sulfates

The keratan sulfate-containing proteoglycans were isolated from fourteen pooled human corneas (thirteen from 61- to 86-year-olds, plus one from a 12-year-old). These proteoglycans were subjected to digestion with the enzyme keratanase II, and the released oligosaccharides, which included nonreducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and identified on two separate ion-exchange columns. Both of the latter had been calibrated with samples, most of which had been derived from bovine corneal keratan sulfate (Tai, G.-H., Huckerby, T. N., and Nieduszynski, I. A. (1996) J. Biol. Chem. 271, 23535–23546) and all of which had been fully characterized by NMR spectroscopic analysis. The capping structures identified in human corneal keratan sulfates occurred in the relative proportions: NeuAcα(2-6)- >NeuAcα(2-3)- >GalNAc(S)β(1-3)-. The other groups of capping structures which had been identified in bovine corneal keratan sulfate, i.e. NeuGcα(2-3)-, NeuGcα(2-6)-, GlcNAc(S)β(1-3)- were absent, although the possibility of the presence of some Galα(1-3)- structures could not be excluded. In addition, the human sample showed significantly higher levels of α(1-3)-fucosylated repeat region structures than did the bovine sample, and it is not clear whether this reflects a species or age dependence as the bovine corneas were from young animals, whereas the human corneas were predominantly from an older group. The charge densities and keratan sulfate chain sizes of the human and bovine keratan sulfate-containing proteoglycans were seen to be similar.

Free-radical aqueous slurry polymerizations of acrylonitrile: 1. End-groups and other minor structures in polyacrylonitriles initiated by ammonium persulfate/sodium metabisulfite

Abstract Polyacrylonitriles prepared by aqueous free-radical slurry polymerization at 40°C using ammonium persulfate and sodium metabisulfite as redox initiator have been examined by FT i.r. and by high-field 13 C and 1 H n.m.r. They have been shown to contain small quantities of acrylamide and acrylic acid units and to possess predominantly sulfonate and non-sulfur containing end-groups derived principally from transfer to bisulfite ion during the polymerization. Other end-groups present in low concentration are sulfate and hydroxyl; the former probably arises from initiation by sulfate radical anions and the latter either from transfer to water or by adventitious hydrolysis of some of the sulfate end-groups. Because they contain few groups capable of initiating intramolecular cyclization, the polyacrylonitriles are white with no sign of discoloration.

Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anion-exchange chromatography.

We report the isolation, characterization and quantification of five octasaccharides, four hexasaccharides and two tetrasaccharides, derived from the chondroitin sulphate (CS) linkage region of 6-8-year-old bovine articular cartilage aggrecan, following digestion with chondroitin ABC endolyase. Using a novel high-pH anion-exchange chromatography (HPAEC) method, in conjunction with one- and two-dimensional (1)H-NMR spectroscopy, we have identified the following basic structure for the CS linkage region of aggrecan: DeltaUA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)GalNAc[0S/4S/6S](beta1-4)GlcA(beta1-3)Gal[0S/6S](beta1-3)Gal(beta1-4)Xyl, where DeltaUA represents 4,5-unsaturated hexuronic acid, and 4S and 6S represent an O-ester sulphate group on C-4 and C-6 respectively. The octa-, hexa- and tetra-saccharide linkage region fragments were used to develop a HPAEC fingerprinting method, with detection at A(232 nm), and a linear response to approx. 0.1 nmol of substance. The sulphation patterns of CS linkage regions, of up to octasaccharide in size, from articular and tracheal cartilage aggrecan were examined. The results show that in articular cartilage, for the majority (53%) of octasaccharides the 2-deoxy-2-N-acetyl amino-D-galactose (GalNAc) residues closest to the linkage region are both 6-sulphated; however, in a significant portion (34%), one or more of these GalNAc residues are unsulphated, and in 8% both are unsulphated. Approximately 10-18% of the chains have a 4-sulphated GalNAc in the first disaccharide, and 12% have a sulphated linkage region Gal residue. No evidence was found for uronic acid sulphation. These data show that there is a significant increase in the incidence of unsulphated and 4-sulphated GalNAc residues adjacent to the linkage region compared with the rest of the chain. Bovine tracheal cartilage linkage regions displayed very similar sulphation profiles to those from articular cartilage, despite the presence of a higher level of GalNAc 4-sulphation within the repeat region of the main CS chain.

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides.

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high‐field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4‐O‐sulfated and have the structure: ΔUA(β1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S‐ol, whereas one from bovine tracheal cartilage CS comprised only 6‐O‐sulfated residues and had the structure: ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol. No oligosaccharide showed any uronic acid 2‐sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(β1–4)GlcA‐ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: ΔUA(β1–3)GalNAc4S(β1–4)GlcA‐ol and ΔUA(β1–3)GalNAc6S(β1–4)GlcA‐ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol and GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.

The influence of polymerization conditions on the tacticity of poly(N-vinyl-2-pyrrolidone)

Abstract The tacticities of polyvinylpyrrolidones prepared under various free radical polymerization conditions have been examined by 13 C n.m.r. at both 25 and 75 MHz. The spectra reveal, contrary to previous reports, that the polymerization solvent influences polymer structure to a small extent. Polymers prepared in water are slightly more syndiotactic than those prepared in organic solvents or in bulk. This difference is attributed to the influence of hydrogen bonding.