Biomedical subjects

A A Bergwerff

Publications and source records attributed to A A Bergwerff.

At least 19 recordsLinked to original sources

PubMed2006

Clinical efficacy of local administration of ceftiofur in a Staphylococcus aureus infection in tissue cages in ponies.

Ceftiofur concentrations in an infected and uninfected environment were compared and the efficacy of locally administered ceftiofur was evaluated in an experimental infection with Staphylococcus aureus in tissue cages. Eight ponies had tissue cages (TCs) implanted s.c. on each side of the neck. Into one of the cages 150 mg of ceftiofur was administered and fluid samples were taken to determine ceftiofur concentrations. After 1 week the other TC was infected with S. aureus and subsequently treated with 150 mg ceftiofur administered locally into the TC once daily for 21 days. Samples of fluid were taken to determine ceftiofur concentrations and for bacterial counts. Ceftiofur concentrations did not differ significantly in the infected and uninfected environments after single dose of 150 mg of ceftiofur. Concentrations were considerably in excess of the minimum inhibitory concentration (MIC) of the S. aureus strain used. A marked decrease of viable bacteria in tissue cage fluid (TCF) occurred. In five of seven ponies; however, the infection was not eliminated and abscess formation occurred. Therefore, local application of ceftiofur alone is not advisable for infections with S. aureus in secluded sites in horses, but should be used only with adjunctive therapy.

Animals↗

PubMed2006

Ceftiofur derivatives in serum, uterine tissues, cotyledons, and lochia after fetal membrane retention.

The objective of the study was to determine concentrations of ceftiofur derivatives after subcutaneous application of ceftiofur hydrochloride in cows with retained fetal membranes. Concentrations of ceftiofur derivatives detected as desfuroylceftiofuracetamide were determined in blood serum, endometrium, caruncles, cotyledons, and lochia during 72 h. After induction of parturition, 2 primiparous and 4 multiparous cows having retained fetal membranes for at least 12 h were studied. All cows received 3 consecutive injections (C1 to C3; 24 h apart) of 1-mg ceftiofur equivalents per kilogram of body weight as ceftiofur hydrochloride sterile suspension. Samples of blood, endometrium, caruncles, cotyledons, and lochia were collected immediately before each injection (0 h) and again at 4, 12, and 24 h after C1, C2, and C3. Blood samples were collected from coccygeal vessels. Caruncles were removed from the uterine lumen by manual extirpation and separated from cotyledons. Endometrial tissue (0.5 g) was collected by using Kenny's biopsy apparatus. For all samples, concentrations of potentially active ceftiofur derivatives were quantified using an HPLC assay. Within 2 h (serum), 4 h (endometrium), and 12 h (caruncles, cotyledons, lochia) after C1 and during the entire study period, mean concentration of ceftiofur derivatives exceeded the reported minimum drug concentrations required to inhibit the growth of 90% of isolates for relevant bacteria such as Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium pyogenes. Only in single samples did concentrations decrease temporarily below the reported minimum drug concentrations required to inhibit the growth of 90% of isolates.

Animals↗

PubMed2004

Proficiency studies on the determination of paralytic shellfish poisoning toxins in shellfish.

Paralytic shellfish poisoning toxins are produced by dinoflagellates. Shellfish filtering these unicellular algae will accumulate the toxins and pose a health risk when consumed by man. In the European Union, paralytic shellfish poisoning toxins in bivalve molluscs are regulated at a maximum content of 80 microg/100 g (91/492/EEC). The current reference method in the European Union is the mouse bioassay, but alternative methods including the liquid chromatography methodology are preferred for ethical reasons. Analyses of suspected shellfish batches revealed, however, unacceptable differences in results reported by a small group of Dutch laboratories all using liquid chromatography methods with precolumn derivatization, followed by fluorescence detection. Therefore, a series of proficiency studies were undertaken among these laboratories. In the first three studies, participants were more or less allowed their own choice of method execution details. This approach yielded unsatisfactory results. A fourth study was then initiated in which a standardized method was mandatory. Two types of test material were used in the fourth study: lyophilized Cardium tuberculatum material containing saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX), and lyophilized mussel material containing dc-STX. The latter material was investigated in an interlaboratory study involving 15 participants and was considered as the reference material. Among the four laboratories, coefficients of variation (ANOVA) for C. tuberculatum material were 10% (n = 11) and 9% (n = 12) for STX and dc-STX, respectively, and for the reference material was 8% (n = 12) for dc-STX. The joint efforts showed that variability in analysis results between laboratories that all apply more or less the same method can be drastically improved if the methodology is rigorously standardized.

Chromatography, Liquid↗

PubMed2002

Pharmacokinetics of ceftiofur in plasma and uterine secretions and tissues after subcutaneous postpartum administration in lactating dairy cows.

A study was conducted to measure concentrations of potentially active ceftiofur derivatives, in plasma, in uterine tissues (endometrium and caruncles) and in uterine secretions at different time points after a single subcutaneous administration of ceftiofur hydrochloride (Excenel RTU Sterile Suspension) at the dose of 1 mg/kg body weight in Holstein-Friesian dairy cows. The animals (n=4) were injected within 24 h of calving, after expulsion of the foetal membranes. Plasma, lochial fluid, caruncles and endometrium were collected before ceftiofur hydrochloride administration and at 1, 2, 4, 8, 12 and 24 h after treatment. For each cow the concentrations of ceftiofur in the biological matrices were quantified using an high-performance liquid chromatography (HPLC) assay. The limit of quantification of the method was 0.1 microg/mL for plasma and 0.1 microg/g for lochial fluid, caruncles and endometrium. The concentrations of potentially active ceftiofur derivatives detected in plasma reached a maximum of 2.85 +/- 1.11 microg/mL at 2 h and decreased to 0.64 +/- 0.14 microg/mL at 24 h after administration. In lochial fluid, these concentrations reached a maximum of 0.97 +/- 0.25 microg/g at 4 h and decreased to 0.22 +/- 0.21 microg/g at 24 h after administration. In endometrium, these concentrations reached a maximum of 2.23 +/- 0.82 microg/g at 4 h and decreased to 0.56 +/- 0.14 microg/g at 24 h following the injection, whereas these levels in caruncles were 0.96 +/- 0.45 and 0.60 +/- 0.39 microg/g obtained at 8 and 24 h, respectively. At the dose of 1 mg/kg body weight in healthy dairy cows, subcutaneous administration of ceftiofur (as ceftiofur hydrochloride) after parturition results in concentrations of ceftiofur derivatives in uterine tissues and in lochial fluid that exceed the reported minimal inhibitory concentrations (MICs) for the common pathogens (Escherichia coli, Fusobacterium necrophorum, Bacteroides spp., and Arcanobacterium pyogenes) associated with acute puerperal metritis.

Animals↗

PubMed1998

Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide.

The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.

Amino Acid Sequence↗

PubMed1998

HPLC determination of residues of spectinomycin in various tissue types from husbandry animals.

An HPLC method was developed for the determination of bacteriostatic aminocyclitol spectinomycin (SP) in animal tissue products. These products included chicken eggs and edible fat, kidney, liver, muscle tissues from calf, poultry, pig and sheep. Residues of SP were extracted from homogenized tissue and egg-derived material with 25 mM citrate of pH 4.0, trichloroacetic acid and dichloromethane. The extract was purified and concentrated over a carboxylic acid-bonded solid-phase extraction (SPE) column. The SPE-eluate was analysed by cation-exchange HPLC involving a two-column switching system, post-column derivatization and fluorescence detection. Spectinomycin could be successfully determined at levels of 0.05 mg kg-1 and higher. Recoveries from spiked tissue material and from spiked egg material were in excess of 74% and did not show a concentration or tissue-type dependence. Precision of the elution position and signal response was better than 2%. Matrix effects and interference from lincomycin were less than 7 and 2%, respectively, on the signal response. Spectinomycin was shown to be stable at -20 degrees C in combined egg yolk and white over a test period of 12 weeks and in calf and sheep muscle tissue over a test period of 10 days. SP was, however, not stable at this temperature over a period of 12 months in chicken muscle tissue. Incurred SP residues were successfully determined in kidney and muscle tissue at the injection site of pigs administered with two doses of 15 mg kg-1 body weight SP with an intermittent withdrawal period of 15 days. Kidney showed higher concentrations and more persistent residues of SP than muscle tissue at the injection site.

Animals↗

PubMed1995

Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Sialylation patterns and branch location of dimeric N-acetyllactosamine units.

The N-linked carbohydrate chains of recombinant human erythropoietin expressed in CHO cells were quantitatively released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, separated from the remaining O-glycoprotein by gel-permeation chromatography, and subsequently fractionated via FPLC on Mono Q, HPLC on Lichrosorb-NH2 and high-pH anion-exchange chromatography on CarboPac PA1. The purified sialylated oligosaccharides were analyzed by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. When necessary, oligosaccharides were treated with endo-beta-galactosidase (and N-acetyl-beta-glucosaminidase) followed by 1H-NMR analysis of the incubation products, to obtain additional structural information. Di-, tri-, tri'- and tetraantennary N-acetyllactosamine-type oligosaccharides occur which can be completely (major) or partially (minor) sialylated. Three different types of alpha 2-3-linked sialic acids are present, namely, N-acetylneuraminic acid (95%), N-glycolylneuraminic acid (2%) and N-acetyl-9-O-acetylneuraminic acid (3%). In the case of partial sialylation, a non-random distribution of the sialic acids over the branches is observed. One or two extra N-acetyllactosamine units, being exclusively located in the branches attached to the alpha 1-6-linked Man residue, can be present in completely or partially sialylated di-, tri'-, and tetraantennary oligosaccharides. Tetraantennary oligosaccharides with N-acetyllactosamine repeats could be digested quantitatively with endo-beta-galactosidase from Bacteroides fragilis, whereas under the same conditions tri' antennary oligosaccharides hardly reacted (< 15%). Using endo-beta-galactosidase from Escherichia freundii, these tri'antennary oligosaccharides could be digested more extensively (> 75%). The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment, and purified via FPLC on Mono Q and HPLC on Lichrosorb-NH2. Two O-glycans were found, namely, Neu5Ac alpha 2-3Gal beta 1-3GalNAc-ol and Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol.

Amino Sugars↗

PubMed1995

The major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements.

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.

Acetylglucosamine↗

PubMed1995

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.

Animals↗

PubMed1994

The immunologically reactive part of immunopurified circulating anodic antigen from Schistosoma mansoni is a threonine-linked polysaccharide consisting of --> 6)-(beta-D-GlcpA-(1 --> 3))-beta-D-GalpNAc-(1 --> repeating units.

The gut-associated excretory antigen CAA (circulating anodic antigen) from adult Schistosoma mansoni worms was isolated by immunoaffinity chromatography. Amino acid analysis following alkaline borohydride treatment indicated that CAA is a glycoprotein, O-glycosylated at Thr. The primary structure of the released O-glycan moiety was investigated by one- and two-dimensional, homo- and heteronuclear 1H and 13C NMR spectroscopy. It was found that the major carbohydrate chains have a novel polysaccharide structure, consisting of a branched disaccharide repeating unit containing 2-acetamido-2-deoxy-beta-D- galactopyranose (beta-D-Galp-NAc) and beta-D-glucopyranuronic acid (beta-D-GlcpA). [formula: see text] The major antigenic character of CAA arises from this novel polysaccharide, which was shown to be an absolutely specific diagnostic marker in schistosomiasis. The cross-reactivity of CAA with anti-CCA (circulating cathodic antigen) monoclonal antibodies is caused by the presence of a small amount of O-linked CCA-poly-Lewis x carbohydrate chains on the CAA protein chain.

Animals↗

PubMed1994

The immunologically reactive O-linked polysaccharide chains derived from circulating cathodic antigen isolated from the human blood fluke Schistosoma mansoni have Lewis x as repeating unit.

The gut-associated excretory antigen circulating cathodic antigen (CCA) was isolated by immunoaffinity chromatography from adult Schistosoma mansoni worms, which were collected from infected golden hamsters. This antigen is probably involved in protection of the schistosome gut and is increasingly used in highly sensitive and specific immunodiagnostic assays. Amino acid analysis before and after alkaline borohydride treatment of CCA and monosaccharide analysis indicated that CCA is O-glycosylated mostly via GalNAc-Thr. After reductive alkaline treatment, the O-linked carbohydrate chains were fractionated by gel-permeation chromatography, followed by normal-phase HPLC on LiChrosorb-NH2. Carbohydrate-positive fractions were investigated by one-dimensional and two-dimensional 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and collision-induced-dissociation tandem mass spectrometry. The analyses showed that the low-molecular-mass O-linked oligosaccharide alditols (the minor fraction) consist of disaccharides to hexasaccharides having the Gal beta (1-3)GalNAc-OL core in common. The major carbohydrate fraction comprises a population of polysaccharides, containing Lewis x repeating units (-3)Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-). CCA-specific monoclonal antibodies and IgM antibodies in patient sera recognized the fucosylated O-linked carbohydrate antigenic structures. Since CCA evokes a strong IgM antibody response and carbohydrate structures containing repeating Lewis x units are found on circulating neutrophils, it is proposed that the antigenic poly-Lewis x polysaccharide of CCA is involved in the induction of auto-immunity against granulocytes, resulting in the mild to moderate neutropenia observed during schistosome infection.

Animals↗

PubMed1993

Conversion of GalNAc beta(1-4)GlcNAc beta-OMe into GalNAc beta (1-4)-[Fuc alpha(1-3)]GlcNAc beta-OMe using human milk alpha 3/4-fucosyltransferase. Synthesis of a novel terminal element in glycoprotein glycans.

Incubation of GalNAc beta(1-4)GlcNAc beta-OMe with GDP-Fuc in the presence of human milk alpha 3/4-fucosyltransferase resulted in the formation of GalNAc beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OMe. Under conditions that led to complete alpha 3-fucosylation of Gal beta(1-4)GlcNAc beta-OEt, GalNAc beta(1-4)GlcNAc beta-OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one- and two-dimensional 1H-NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Gal beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OEt and GalNAc beta(1-4)[Fuc alpha(1-3)]GlcNAc beta-OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C-2', i.e. a hydroxyl function versus a N-acetyl group.

Carbohydrate Sequence↗

PubMed1993

Primary structure of N-linked carbohydrate chains of a human chimeric plasminogen activator K2tu-PA expressed in Chinese hamster ovary cells.

A recombinant human plasminogen activator hybrid variant K2tu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH2, and analysed by 500-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri- and tri'-antennary (1%), trisialylated tri- and tri'-antennary (28%), and tetrasialylated tetra-antennary (18%) structures, all having fucose in alpha(1-6)-linkage at the Asn-bound N-acetylglucosamine. Sialic acid occurred exclusively in alpha(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94%), N-glycolylneuraminic acid (3%), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K2tu-PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K2tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences. Profiling studies on different K2tu-PA production batches demonstrated that the structures of N-linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.

Amino Acid Sequence↗

PubMed1992

Human urokinase contains GalNAc beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-2) as a novel terminal element in N-linked carbohydrate chains.

Structural analysis of enzymically released N-linked carbohydrate chains of human urokinase (urinary-type plasminogen activator) by 1H NMR spectroscopy and FAB-MS demonstrated that the N-linked oligosaccharides on the only N-glycosylation site contain diantennary structures with the novel GalNAc beta (1-4) [Fuc alpha (1-3)]GlcNAc beta (1-2) element in the upper or the lower branch.

Carbohydrate Sequence↗

PubMed1992

The carbohydrate chains of the beta subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates.

The N-linked carbohydrate chains of the beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text]

Acetylglucosamine↗

PubMed1992

1H-NMR structural determination of the N-linked carbohydrate chains on glycopeptides obtained from the bean lectin phytohemagglutinin.

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, is a N-linked glycoprotein with one high-mannose-type and one xylose-containing oligosaccharide side chain per polypeptide. The high-mannose-type glycan is attached to Asn12 and the complex-type glycan to Asn60 [Sturm, A. & Chrispeels, M. J. (1986) Plant Physiol. 81, 320-322]. The structures of the oligosaccharides were elucidated from two glycopeptides obtained from the lectin by Pronase digestion, affinity chromatography on concanavalin-A--Sepharose and gel-filtration chromatography on a column of BioGel P-4. The N-linked glycan structures were investigated by 500-MHz 1H-NMR spectroscopy and were established to be: [formula; see text]

Carbohydrate Conformation↗

PubMed1992

Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-L-methionine: N-acylneuraminate 8-O-methyltransferase and CMP-N-acetylneuraminate monooxygenase activities.

Mass spectrometric and NMR spectroscopic analyses of bound sialic acids from the starfish Asterias rubens revealed the presence of N-acetylneuraminic acid (4%), N-acetyl-8-O-methylneuraminic acid (12%), N-acetyl-9-O-acetyl-8-O-methylneuraminic acid (less than 1%), N-glycoloylneuraminic acid (19%), N-glycoloyl-8-O-methylneuraminic acid (47%), and N-glycoloyl-9-O-acetyl-8-O-methylneuraminic acid (18%). Analysis of sialo-oligomeric material, obtained after mild acid hydrolysis, demonstrated that N-glycoloyl-8-O-methylneuraminic acid can occur as di- and tri-oligomers, linked through the anomeric center and the N-glycoloyl moiety, Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me and Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me-alpha (2----O5)-Neu5Gc8Me. Studies on the biosynthesis of N-acyl-8-O-methylneuraminic acid in A rubens, using the tracer S-adenosyl-L-[methyl-14C]methionine, showed that N-acylneuraminate 8-O-methyltransferase activity was present predominantly in the membrane fraction. CMP-N-acetylneuraminic acid monooxygenase activity was found in the soluble protein fraction, in agreement with investigations on the corresponding vertebrate enzyme.

Animals↗

PubMed1992

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.

Animals↗