Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.     

Gene survival in the Asian wild horse (Equus przewalskii): I. Dependence of gene survival in the calgary breeding group pedigree

The joint probability distribution of the number of distinct (not identical by descent) genes from each founder of the Equus przewalskii population that survive in the five horses of the Calgary Zoological Gardens breeding group has been calculated. The dependence structure of this distribution is investigated, and informative marginal distributions are given, among them the distributions of the genetic contributions of each founder to the Calgary horses and the distribution of wild-type genes in these horses. The dependence pattern is found to be complex; there is no substitute for exact calculation of the full joint probability distribution of numbers of surviving genes. Probabilities of gene survival give a more complete summary of the genetic structure of a set of individuals than is provided by more routine measures such as heterozygosity or founder contributions. The feasibility of computing these probabilities for small groups of current individuals descended from few founders via long and complex pedigrees, provides a new approach to assessing such groups, and could be used also in selecting animals to form the founder stock of propagules for future reintroduction programs.     

31P-NMR study of high-energy phosphorylated compounds metabolism and intracellular pH in the perfused rat heart

Using 31P-NMR and haemodynamical measurements, this work assesses different aspects of myocardial preservation improvement during a global ischaemia, based on a simultaneous and correlated study of high-energy phosphorylated compounds, intracellular pH and left ventricular function. Isolated perfused working rat hearts were subjected to 2 or 3 h of hypothermic ischaemia followed by 30 or 45 min of reperfusion. A study of the influence of pH and buffer used in cardioplegic solutions has demonstrated a better preservation of high-energy phosphates and an improved functional recovery when using a pH 7.0, glutamate - containing solution. Protection provided by cardioplegia can be enhanced by the appropriate use of a fluorocarbon-oxygenated cardioplegic reperfusate. The use of nifedipine, a calcium antagonist, in the cardioplegic solutions, does not provide any additional protection under hypothermic conditions.     

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures.

Abstract: Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-³H]alkyl- sn -glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1–20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyksn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl-, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl- compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.     

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.