The patterns of the complex- and oligomannose-type glycans of uromodulin (Tamm-Horsfall glycoprotein) in the course of pregnancy.

Uromodulin was isolated from urine of three pregnant women. Urine of each donor was collected at subsequent stages of their pregnancy and at one month after gestation. Each batch of uromodulin was enzymatically N-deglycosylated and the released N-glycans were isolated, quantified and profiled by high-pH anion-exchange chromatography. In the course of pregnancy no significant changes were detected in the negative charge distribution stemming from sialic acid and sulfate residues on the complex-type carbohydrate chains of uromodulin. Furthermore, no significant changes in the molar ratio between Man6GlcNAc2 and Man7GlcNAc2 were found in the course of pregnancy, only uromodulin from non-pregnant periods showed small differences.

Effects of acute (-)-hydroxycitrate supplementation on substrate metabolism at rest and during exercise in humans.

BACKGROUND: (-)-Hydroxycitrate (HCA), a competitive inhibitor of ATP-citrate lyase, should reduce the extramitochondrial acetyl-CoA pool. It has been hypothesized that HCA ingestion can reduce malonyl-CoA concentrations and consequently increase fatty acid oxidation in vivo. OBJECTIVE: This study investigated the acute effects of HCA supplementation on substrate utilization at rest and during exercise in endurance-trained humans. DESIGN: Ten cyclists [x+/- SD) age: 24 +/- 2 y, weight: 73 +/- 2 kg, maximal oxygen uptake: 4.95 +/- 0.11 L/min, maximal work output (W:max): 408 +/- 8 W] were studied at rest and during 2 h of exercise at 50% W:max on 2 occasions. Both 45 and 15 min before exercise and 30 and 60 min after the start of exercise, 3.1 mL/kg body wt of an HCA solution (19 g/L) or placebo was ingested. Total fat and carbohydrate oxidation rates were assessed. Blood samples were collected at 15-min intervals at rest and every 30 min during exercise. RESULTS: Plasma HCA concentrations increased after HCA ingestion up to 0.39 +/- 0.02 mmol/L (82.0 +/- 4.8 mg/L). However, no significant differences in total fat and carbohydrate oxidation rates were observed between trials. Accordingly, plasma glucose, glycerol, and fatty acid concentrations did not differ between trials. Plasma lactate concentrations were significantly lower in the HCA than in the placebo trial after 30 min of exercise but at the end of the exercise period they did not differ between trials. CONCLUSION: HCA, even when provided in large quantities, does not increase total fat oxidation in vivo in endurance-trained humans.

Conformational analysis of the Sd(a) determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations.

The conformational behaviour of the spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-[alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)- beta-D-GlcpNAc-(1-->O)(CH2)5NH2 (1) and the two mimics beta-D-Galp-(1-->4)-[alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-bet a-D-GlcpNAc-(1-->O)(CH2)5NH2 (2) and beta-D-GlcpNAc-(1-->4)-[alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)- beta-D-GlcpNAc-(1-->O)(CH2)5NH2 (3) were investigated by 1H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D 1H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1-3 are rather rigid, especially the beta-D-Hex(NAc)-(1-->4)-[alpha-Neu5Ac-(2-->3)]-beta-D-Galp fragments. The alpha-Neu5-Ac-(2-->3)-beta-D-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1-3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.

Glycosylation sites and site-specific glycosylation in human Tamm-Horsfall glycoprotein.

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex-type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.

Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein.

The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HPLC on LiChrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA-1. Characterizations were performed using 500-MHz and 600-MHz 1H-NMR spectroscopy, in combination with sialidase treatments. The type of characterized N-glycans ranged from monosulfated to trisulfated N-glycans, whereby the sulfate groups were present as 3-O-sulfated Gal (Gal3S) and 4-O-sulfated GalNAc (GalNAc4S). A compilation of the established structures is shown below. [structure in text]

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature.

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N -acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N-acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N -acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N-acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.

Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy.

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N-glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.

Observations of energetic ions from comet giacobini-zinner.

During the encounter with comet Giacobini-Zinner, the energetic particle anisotropy spectrometer on the International Cometary Explorer spacecraft observed large fluxes of energetic ions, believed to result principally from ionization of the cometary atmosphere followed by pickup and acceleration by the ambient flow of the solar wind. These heavy cometary ions were observed from approximately 1 day before closest approach to about 2(1/2) days afterward. Three regimes of differing ion characteristics have been identified. An outer region with a scale of approximately 10(6) kilometers contains variable fluxes of antisolar-streaming pick-up ions in the undisturbed solar wind. In the middle region, of approximately 10(5) kilometers, fluxes have less large-scale variability and broader angular and energy distributions. This region is separated from the outer zone by a sharp transition. The inner region has a scale of approximately 10(4) kilometers and is characterized by reduced fluxes and complex angular distributions.