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Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.

High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.

Previous studies have shown that human leukocyte interferon (IFN), IFN-gamma, and recombinant (r) IFN-alpha-A protect cultured human cells against the invasiveness of salmonellae, shigellae, and enteroinvasive Escherichia coli. To differentiate between effect on endocytosis and adhesiveness, HEp-2 cells were pretreated with cytochalasin B to block endocytosis, and thereafter treated with IFN. Although a significant reduction of adhesion of bacteria to HEp-2 cells was observed, this reduction was far too small to explain the total reduction of bacterial invasiveness. Thus, the anti-invasive effect of IFN on HEp-2 cells is due mostly to inhibition of endocytosis. The IFN effect was not expressed at 4 degrees C, and the ribosomal 60S subunit inactivators, cycloheximide, abrin, and shigella toxin, completely blocked the expression of the IFN-induced anti-invasive state of the cell. Thus, the IFN effect is dependent on continuous ribosomal activity. Our results indicate that the major effect of IFN on bacterial invasiveness is on endocytosis, and that the action is mediated through the effect of IFN-induced protein synthesis.

Previous studies have shown that interferons specifically interact with HEp-2 cells and reduce invasiveness by Salmonella and Shigella. In this study an effect of interferons on the adhesiveness of a strictly extracellular bacterium was demonstrated. HEp-2 cells were treated with human leukocyte interferon, recombinant interferon alpha-A or recombinant interferon gamma and the adhesiveness of Pseudomonas aeruginosa to the cells was examined using a light microscopy method. A recent isolate and a variant of the same strain subcultivated for 12 months were used. Treatment of cells with human leukocyte interferon or recombinant interferon alpha-A reduced the adhesiveness of both variants. When recombinant interferons were used a dose dependent effect was observed, whereas the effect after treatment with human leukocyte interferon was not dose dependent. Adhesiveness of the bacteria to HEp-2 cells was reduced after subcultivation of the bacteria but was reconstituted by adding an extracellular filtrate from the recent isolate. This extracellular filtrate inhibited the effect of human leukocyte interferon on the adhesiveness of bacteria in the cell cultures. The human leukocyte interferon effect on bacterial adhesiveness was neutralized by anti-interferon globulin. Treatment of HEp-2 cells with recombinant human gamma interferon caused an enhanced adhesiveness of the recent isolate, but reduced the adhesiveness of the subcultivated variant strain.

Infection of adult CBA mice with Rauscher or Moloney leukaemia virus concomitantly with caseination significantly accelerated spleen amyloid development in irradiated, bone-marrow protected mice, but had no effect on untreated, adult thymectomized or thymectomized irradiated mice. Spleen tissue of mice infected with Moloney virus had the highest titre in the mice with accelerated amyloid development.

A prospective, randomized study was carried out, creating a pneumoperitoneum with 100% CO2 or with 5% CO2 in air and evaluating the effect(s) of these gas phases on fertilization, cleavage and pregnancy outcome in this in-vitro fertilization and embryo transfer programme. There was no significant difference in the fertilization rate when either 100% CO2 or 5% CO2 in air was used for insufflation during laparoscopy. Further, embryonic development and pregnancy rates also indicated no significant differences between the two groups. Similarly, the oocytes which were retrieved and replaced with spermatozoa in the gamete intra-Fallopian transfer programme using either 100% or 5% CO2, gave similar pregnancy rates. Furthermore, the fertilization and cleavage rates of the supernumerary oocytes were not significantly different in both groups.

The specificity of fetal calf liver and ovine and porcine submaxillary gland N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase was investigated with acceptors of low and high molecular weight containing O-glycosidically linked carbohydrate chains. It appeared that fetal calf liver microsomes were able to transfer sialic acid to C-6 of GalNAc in NeuAc(alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc-R, in which the aglycon could be protein as well as p-nitrophenol (Nph). The substrates Gal(beta 1 leads to 3)GalNAc-R and GalNAc-R were inactive as acceptor with this enzyme source. Ovine and porcine submaxillary gland microsomes were both active with GalNAc-protein, Gal(beta 1 leads to 3)GalNAc-protein, NeuAc[alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc-protein and NeuAc(alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc alpha-Nph, but not with GalNAc alpha-Nph and Gal(beta 1 leads to 3)GalNAc alpha-Nph. The N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase which had been purified to homogeneity from porcine submaxillary gland [Sadler, J. E., Rearick, J. I. and Hill, R. L. (1979) J. Biol. Chem. 254, 5934-5941], was able to sialylate all three protein acceptors, but was virtually inactive with each of the three p-nitrophenyl glycosides. Our studies indicate that two N-acetylgalactosaminide alpha 2 leads to 6 sialtransferases exist acting on O-glycosidically linked carbohydrate chains. The first enzyme, present in fetal calf liver, has a narrow specificity with regard to the oligosaccharide structure, but shows no specificity for the aglycon. Based on its specificity this enzyme can be designated as an [alpha-N-acetylneuraminosyl 2 leads to 3-beta-galactosyl 1 leads to 3]-alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. The second enzyme, present in porcine submaxillary gland, has an absolute requirement for protein as the aglycon. Once this condition is fulfilled, the enzyme is able to transfer sialic acid to each of the three oligosaccharide chains and thus the enzyme is an alpha-N-acetylgalactosaminylprotein alpha 2 leads to 6 sialyltransferase. The data also seem to suggest that ovine and porcine submaxillary gland microsomes contain, in addition to the latter enzyme activity, the alpha 2 leads to 6 sialyltransferase with the strict oligosaccharide specificity.

With Percoll density gradients, blasts from peripheral blood and bone marrow could be separated with a significant enrichment, and very often with a high degree of purity. This allowed a study of selected cases, where the separated sample exhibited chromosome abnormalities and/or an abnormal DNA content distribution (as measured by DNA-flow cytometry). The anomalies were shown to be associated with the separated blast fraction.

Porcine liver microsomes are capable of transferring sialic acid from CMP-NeuAc to [14C]galactosylated ovine submaxillary asialo-mucin, porcine submaxillary asialo/afuco-mucin and ganglioside GM1. The specificity of the porcine liver sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) towards the first acceptor, [14C]Gal-GalNAc-protein, was investigated by means of methylation studies on the oligosaccharides changes cleft-off from the sialylated product glycoprotein by beta-elimination under reductive conditions. It appeared that sialic acid was transferred solely to position C-3 of galactose residues on Gal beta(1 leads to 3)GalNAc disaccharide units. Transfer to GalNAc residues was completely absent. Competition experiments and heat activation studies suggested that the same enzyme also converts ganglioside GM1 to ganglioside GD1a. Therefore, this porcine liver sialyltransferase can be designated as a Gal beta(1 leads to 3)GalNAc-R alpha(2 leads to 3) sialyltransferase.

With the use of a monoclonal anti-glycophorin A antibody and flow cytometric cell sorting, an erythroleukemic bone marrow sample was separated in highly purified erythroblast and myeloblast fractions. Similar karyotypic anomalies were found in both cell populations as in the unseparated bone marrow. This study confirms that acute nonlymphocytic leukemia can originate at the level of a multipotential hemopoietic stem cell.

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.

Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors we studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely, since all glycans are susceptible to endo-beta-N-acetylglucosaminidase H. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. In both cases secretion of the protein is not affected. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37 degrees C. At 26 degrees C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.

Sixty-five neutral oligosaccharides were analyzed by high-pressure liquid chromatography on an amine-modified silica column (Lichrosorb-NH2). By systematic comparison of the retention times, it was possible to attribute chromatographic behavior to specific structural features. It appeared that retention times increase with the number of sugar residues. The presence of a fucose or an N-acetylglucosamine residue results in a decreased retention time, in particular when the latter sugar is at the reducing end. A dramatic increase in retention time is shown by oligosaccharides having a 1----6 linkage, regardless of whether this linkage is involved in a branch. Less important features are the nature of the component sugars other than N-acetylglucosamine and fucose, the anomeric configuration of the sugars, and the presence of a reduced terminal sugar.