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Biomedical subjects

S M Haslam

Publications and source records attributed to S M Haslam.

At least 19 recordsLinked to original sources

Preliminary study to examine the utility of using foot burn or hock burn to assess aspects of housing conditions for broiler chicken.

1. Eleven broiler chicken farms, representing 4 production system types, were visited during the last 5 d of the flock cycle: bird and flock details were recorded. Litter friability was assessed at 9 sites within the house, atmospheric ammonia was measured at three sites and bird cleanliness was assessed on a numerical rating scale. 2. For these flocks, hock burn, foot burn and breast burn were measured at the processing plant by standardised assessors. 3. Significant correlations were identified between the percentage of birds with foot burn and average litter score, average house ammonia concentrations and feather score. 4. No correlation was found between the percentage of birds with hock burn or breast burn and average litter scores, average ammonia concentrations or feather score. 5. No correlation was found between stocking density and foot burn, hock burn or breast burn.6. If confirmed, these findings may have implications for the draft EU Broiler Directive, for which it is proposed that permitted stocking density on farm may be determined by the incidence and severity of contact dermatitis measured on plant.

Animal Husbandry↗

Isolation and structural determination of novel sulfated hexasaccharides from squid cartilage chondroitin sulfate E that exhibits neuroregulatory activities.

Squid cartilage chondroitin sulfate E (CS-E) exhibits various biological activities, including anticoagulant activities, lymphoid regulatory activities, and neuroregulatory activities [Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413]. These activities are expressed through molecular interactions with specific proteins, including heparin cofactor II, selectins, CD44, chemokines, and the heparin-binding growth factor midkine. Hence, the sugar sequence information is essential for a better understanding of the CS-E functions. Previously, several novel tetrasaccharides containing the unreported 3-O-sulfated glucuronic acid (GlcA) were isolated after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, hexasaccharides were isolated to obtain more detailed sequence information, especially around the GlcA(3-O-sulfate) residue, and were characterized by fast atom bombardment mass spectrometry and 500 or 600 MHz (1)H NMR spectroscopy. The findings demonstrate one tetrasulfated and five pentasulfated hexasaccharide sequences, five of them being novel. They were composed of three disaccharide building units of either A [GlcA(beta1-3)GalNAc(4-O-sulfate)], E [GlcA(beta1-3)GalNAc(4,6-O-disulfate)], K [GlcA(3-O-sulfate)(beta1-3)GalNAc(4-O-sulfate)], L [GlcA(3-O-sulfate)(beta1-3)GalNAc(6-O-sulfate)], or M [GlcA(3-O-sulfate)(beta1-3)GalNAc(4,6-O-disulfate)], forming E-A-A, M-A-A, K-L-A, E-E-A, K-K-A, and A-M-A hexasaccharide sequences. The K-L tetrasaccharide sequence is to date unreported. The isolated sequences appear to indicate the occurrence of an unreported GlcA 3-O-sulfotransferase specific for chondroitin sulfate. The obtained sequence information will be useful for investigating the structure-function relationship and biosynthesis of CS-E.

Animals↗

Mass spectrometric strategies: providing structural clues for helminth glycoproteins.

Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as 'self' or 'foreign' by the immune system of the host.

Glycoproteins↗

Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode.

This paper reports the first rigorous evidence for the existence of N-linked oligosaccharides in Dictyocaulus viviparus, an economically important nematode that parasitises cattle. Structural strategies based upon fast atom bombardment mass spectrometry were employed to examine detergent extracts of homogenised adult D.viviparus for their N-glycan content. These revealed that detergent-soluble material is rich in high mannose, truncated and complex-type families of N-linked oligosaccharides. Importantly, the most abundant antennae in the complex-type structures were shown to carry the Lewis(x)epitope (Galbeta1-4(Fucalpha1-3)GlcNAc). Although the Lewis(x)moiety occurs in other helminths such as schistosomes, nematodes have previously been thought to lack this epitope. The Lewis(x)epitopes in D.viviparus are carried on bi-, tri-, and tetraantennary glycans and are therefore candidates for recognition events requiring multivalent ligands. There is compelling evidence from schistosome research that glycoconjugates containing Lewis(x)structures are immunomodulators. We propose that the Lewis(x)-rich glycans identified in this study might similarly be involved in D.viviparus host interactions.

Animals↗

Characterization of the N-linked oligosaccharides of megalin (gp330) from rat kidney.

Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.

Amidohydrolases↗

Phosphorylcholine-containing N-glycans of Trichinella spiralis: identification of multiantennary lacdiNAc structures.

Although the presence of phosphorylcholine (PC) in Trichinella spiralis is well established, the precise structure of the PC-bearing molecules is not known. In this paper, we report structural studies of N-glycans released from T.spiralis affinity-purified antigens by peptide N-glycosidase F. Three classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and eight N-acetylhexosamine residues. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine.

Acetylation↗

Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein.

ES-62 is an abundant phosphorylcholine-containing secreted glycoprotein of the filarial nematode Acanthocheilonema viteae. Using an antiserum directed against the parasite molecule, 3 cDNAs of size, approximately 1.5-1.6 kbp were isolated from an A. viteae expression library. Sequence analysis in combination with N-terminal amino acid sequencing of purified ES-62 revealed that each clone contained a full-length cDNA for ES-62 corresponding to 474 amino acid residues but differed in their 5' and 3' untranslated regions. Characterisation of the 5' end of ES-62 mRNA using 5' rapid amplification of cDNA ends showed that it coded for a signal sequence. Several tryptic peptides were independently sequenced using quadruple-time-of-flight mass spectrometry and used to confirm the cDNA sequence. The mature protein was found to contain three potential N-linked glycosylation sites. Comparison of the derived amino acid sequence of ES-62 with the SwissProt database identified a sequence (between amino acid residues approximately 250 and 350 of mature ES-62) with significant similarity to several bacterial/fungal aminopeptidases. Incubation of ES-62 with leucine-7-amino-4-methylcoumarin as substrate confirmed that ES-62 possessed aminopeptidase activity.

Amino Acid Sequence↗

Immunogenic glycoconjugates implicated in parasitic nematode diseases.

Parasitic nematodes infect billions of people world-wide, often causing chronic infections associated with high morbidity. The greatest interface between the parasite and its host is the cuticle surface, the outer layer of which in many species is covered by a carbohydrate-rich glycocalyx or cuticle surface coat. In addition many nematodes excrete or secrete antigenic glycoconjugates (ES antigens) which can either help to form the glycocalyx or dissipate more extensively into the nematode's environment. The glycocalyx and ES antigens represent the main immunogenic challenge to the host and could therefore be crucial in determining if successful parasitism is established. This review focuses on a few selected model systems where detailed structural data on glycoconjugates have been obtained over the last few years and where this structural information is starting to provide insight into possible molecular functions.

Animals↗

Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers.

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

Animals↗

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]

Carbohydrate Conformation↗

Structural analysis of sequences O-linked to mannose reveals a novel Lewis X structure in cranin (dystroglycan) purified from sheep brain.

The Lewis X epitope, Galbeta1-4(Fucalpha1-3)GlcNAc-R, has been implicated in cell-cell recognition events in a number of systems including the central nervous system and is expressed on diverse glycoconjugates including cell adhesion molecules, glycolipids, and the proteoglycan phosphacan. Although Lewis X sequences 3-linked to mannose have been described within proteoglycan fractions of mammalian brain, these have not been reported in other contexts and have been widely believed to be peculiar constituents of brain proteoglycans. In the present paper, we confirm the existence of Lewis X structures O-linked to mannose within the mammalian brain, demonstrate that these structures are present on a well defined mucin-like glycoprotein, cranin (dystroglycan), and report studies suggesting that the linkages involved may be predominantly 2-linked to mannose. Mannose-linked Lewis X is the latest in an increasing list of oligosaccharide recognition "tags" that have been shown to be expressed on cranin (dystroglycan) purified from brain.

Animals↗

Novel tetrasaccharides isolated from squid cartilage chondroitin sulfate E contain unusual sulfated disaccharide units GlcA(3-O-sulfate)beta1-3GalNAc(6-O-sulfate) or GlcA(3-O-sulfate)beta1-3GalNAc.

We previously isolated novel tetrasaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K and demonstrated that the disaccharide units containing 3-O-sulfated glucuronic acid were decomposed by chondroitinase ABC digestion (Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 26745-26754). The findings indicated the necessity to re-evaluate the disaccharide compositions of chondroitin sulfate preparations purified from other biological sources and analyzed using the above enzyme. In this study, to evaluate squid cartilage chondroitin sulfate E a series of even-numbered oligosaccharides were isolated after exhaustive digestion with sheep testicular hyaluronidase and subsequent fractionation by gel chromatography. The tetrasaccharide fraction was subfractionated by high performance liquid chromatography on an amine-bound silica column. Systematic structural analysis of five major fractions, h, l, m, n, and q, by fast atom bombardment mass spectrometry, enzymatic digestions in conjunction with capillary electrophoresis, and 500-MHz 1H NMR spectroscopy revealed one disulfated, three trisulfated, and one tetrasulfated tetrasaccharide structure: fraction h, GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S); fraction l, GlcA(3S)beta1-3GalNAc(6S)beta1-4GlcAbeta1-3GalNAc(4S); fraction m, GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S); fraction n, GlcAbeta1-3GalNAc(4S,6S)beta1-4GlcAbeta1-3GalNAc(4S); and fraction q, GlcA(3S)beta1-3GalNAc(4S,6S)beta1-4GlcAbeta1-3GalNAc(4S), where 3S, 4S, and 6S represent 3-O-, 4-O- and 6-O-sulfate, respectively. The structures found in fractions h and m as well as the unsaturated counterpart of that found in fraction n have been reported, whereas those in fractions l and q are novel in that they contained unusual disulfated and trisulfated disaccharide units where GlcA(3S) is directly linked to GalNAc(6S) and GalNAc(4S,6S), respectively. These novel tetrasaccharide sequences are distinct from those found in other chondroitin sulfate isoforms and may play key roles in the biological functions and activities of chondroitin sulfate E not only from squid cartilage but also from mammalian cells and tissues.

Animals↗

A novel pentasaccharide sequence GlcA(3-sulfate)(beta1-3)GalNAc(4-sulfate)(beta1-4)(Fuc alpha1-3)GlcA(beta1-3)GalNAc(4-sulfate) in the oligosaccharides isolated from king crab cartilage chondroitin sulfate K and its differential susceptibility to chondroitinases and hyaluronidase.

Novel sulfated tetrasaccharide structures containing 3-O-sulfated GlcA were isolated recently from king crab cartilage chondroitin sulfate K [Sugahara, K., Tanaka, Y., Yamada, S., Seno, N., Kitagawa, H., Haslam, S. M., Morris, H. R., & Dell, A. (1996) J. Biol. Chem. 271, 26745-26754]. In this study, we prepared a series of oligosaccharides from the same source after exhaustive digestion with testicular hyaluronidase and determined the structures of a pentasaccharide, two hexasaccharides, and two heptasaccharides by means of fast atom bombardment mass spectrometry and 500-MHz 1H-NMR spectroscopy. All the oligosaccharides had the following hitherto unreported structures including a novel glucuronate 3-O-sulfate: GlcA(3S)(beta1-3)GalNAc(4S)(beta1-4)GlcA(3S)(beta1-3)GalNAc( 4S)(beta1-4)GlcA(beta1-3)GalNAc(4S), GlcA(3S)(beta1-3)GalNAc(4S)(beta1-4)GlcA(3S)(beta1-3)GalNAc( 4S)(beta1-4)GlcA(3S)(beta1-3)-GalNAc(4S), GlcA(3S)(beta1-3)GalNAc(4S)(beta1-4)(Fuc alpha1-3)GlcA(beta1-3)GalNAc(4S), GlcA(3S)(beta1-3)-GalNAc(4S)(beta1-4)(Fuc alpha1-3)GlcA(beta1-3)GalNAc(4S)(beta1-4)GlcA(beta1-3)GalNAc (4S), and GlcA(3S)(beta1-3)GalNAc(4S)(beta1-4)GlcA(3S)(beta1-3)GalNAc( 4S)(beta1-4)(Fuc alpha1-3)GlcA(beta1-3)GalNAc(4S), where 3S or 4S represent 3-O- or 4-O-sulfate, respectively. Furthermore, the three latter structures contained a novel combination of both 3-O-sulfated and 3-O-fucosylated GlcA residues. The pentasaccharide with 3-O-fucosylated GlcA at the internal position remained totally resistant to chondroitinase AC-II, whereas it was degraded by chondroitinase ABC into a disaccharide unit containing GlcA(3S) derived from the nonreducing side and a trisaccharide unit containing fucose from the reducing side.

Animals↗