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| 2. |
- Landberg, Eva, et al.
(författare)
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Glycosylation of Bile-Salt-Stimulated Lipase from Human Milk : Comparison of Native and Recombinant Forms
- 1997
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 344:1, s. 94-102
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Tidskriftsartikel (refereegranskat)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19–26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types ofN-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short typeO-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.
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| 3. |
- Landberg, Eva, et al.
(författare)
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Temperature effects in high-performance anion-exchange chromatography of oligosaccharides
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 814:1-2, s. 97-104
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Tidskriftsartikel (refereegranskat)abstract
- High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection has been widely used for analysis of mono-, oligo- and polysaccharides. Many factors that affect separation of carbohydrates by HPAEC have been evaluated, however effect of temperature has not been carefully studied. In the present study, neutral and sialylated oligosaccharides from human milk and different types of N-linked oligosaccharides were analysed by HPAEC at temperatures ranging from 13 to 30°C. N-Acetyl neuraminic acid, galacturonic acid and stachyose were also analysed since they have been used as internal standards when analysing various oligosaccharides by HPAEC. All oligosaccharides showed decreased retention times with increased temperature. Even small differences in temperature (i.e. ±5°) resulted in considerable changes in retention times. In addition, individual oligosaccharides showed different relative changes in retention time with increased temperature. By changing the temperature, a switch in elution order of individual oligosaccharides were sometimes found. These results show that retention times relative to an internal standard cannot be used for oligosaccharide identification unless temperature is carefully controlled. Regulation of temperature is also a valuable tool in achieving optimal separation of oligosaccharides by HPAEC.
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| 4. |
- Liljeblad, Mathias, et al.
(författare)
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Detection of low-molecular-weight heparin oligosaccharides (Fragmin™) using surface plasmon resonance
- 1998
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Ingår i: Journal of Molecular Recognition. - : John Wiley and Sons. - 0952-3499 .- 1099-1352. ; 11:1-6, s. 191-193
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Tidskriftsartikel (refereegranskat)abstract
- During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide–protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin™) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore™), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.
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| 6. |
- Lindberg, Gunnar, et al.
(författare)
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Serum sialic acid and its correlates in community samples from Akita, Japan and Minneapolis, USA
- 1997
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Ingår i: International Journal of Epidemiology. - 1464-3685. ; 26:1, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The concentration of serum total sialic acid (S-TSA) is one recently investigated risk marker for cardiovascular mortality and atherosclerosis. Since the mortality from coronary heart disease is higher in the United States than in Japan, one could expect the S-TSA to be higher among Caucasian US citizens than among Japanese citizens, a hypothesis that is tested in this study. DESIGN: Cross-sectional study of population-based samples of Japanese and US Caucasian men and women. SETTING: The rural community Akita, Japan, and the suburbs of Minneapolis, Minnesota. SUBJECTS: These were 75 consecutive men and women from Akita and Minneapolis respectively aged 47-69 years in 1990. People who had smoked cigarettes during the past 5 years; who had a history of diabetes mellitus, liver disease, coronary heart disease, or stroke; or who were taking anticoagulants were excluded. OUTCOME MEASURES: Serum total sialic acid levels in male and female Japanese and US Caucasian subjects with adjustment for age, systolic blood pressure, fibrinogen, triglycerides and in women also for menopausal status. Race and sex-specific correlations with serum total sialic acid for selected cardiovascular risk markers. RESULTS: The entire sialic acid distributions were shifted to the right in Caucasian men and women compared to Japanese men and women. The mean +/- standard deviation concentrations of S-TSA were 54.1 +/- 5.3 mg/dl in Japanese men and 58.7 +/- 5.6 mg/dl in Caucasian men (P < 0.001). In women, the concentrations were 54.8 +/- 5.1 and 63.1 +/- 6.0 mg/dl respectively (P < 0.001). S-TSA level correlated significantly and positively with fibrinogen levels in Caucasian and Japanese men and women and with triglyceride levels in Caucasian and Japanese men and in Caucasian women but not in Japanese women. After adjustment for age, systolic blood pressure, fibrinogen, triglycerides and menopausal status, the sialic acid levels were 2.2 (P = 0.009) and 6.2 (P < 0.001) mg/dl higher in Caucasian compared to Japanese men and women respectively. CONCLUSIONS: Higher S-TSA levels in Caucasians living in Minneapolis compared to Japanese living in Akita, Japan is in concordance with the higher cardiovascular mortality in the US. Differences in S-TSA levels may reflect international differences in the prevalence of atherosclerosis.
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| 8. |
- Lindberg, Gunnar, et al.
(författare)
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The association between serum sialic acid and asymptomatic carotid atherosclerosis is not related to antibodies to herpes type viruses or Chlamydia pneumoniae. The Atherosclerosis Risk in Communities (ARIC) Study Investigators
- 1997
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Ingår i: International Journal of Epidemiology. - 1464-3685. ; 26:6, s. 1386-1391
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Total serum sialic acid is a recently investigated marker for cardiovascular mortality and carotid atherosclerosis. This study tested the hypothesis that past infection by Herpes simplex type 1 or type 2 viruses or Cytomegalovirus or Chlamydia pneumoniae accounts for the association between serum total sialic acid and atherosclerosis. METHODS: Population-based samples of men and women living in four US communities were used in a cross-sectional study. Cases and matched controls were defined by B-mode ultrasound measurements of carotid and popliteal arterial wall thickness. In all, there were 267 case control pairs with information about antibody titres to viruses and 256 pairs with information about antibody titres to Chlamydia pneumoniae. RESULTS: Serum total sialic acid (S-TSA) level was significantly higher in cases with carotid atherosclerosis compared to their controls. The odds ratio for carotid atherosclerosis associated with sialic acid level above 75th percentile was 1.73 (95% confidence interval [CI]: 1.02-2.95) in the sample with information about antibodies to viruses and 1.70 (95% CI: 1.00-2.93) in the sample with information about antibodies to C. pneumoniae. Adjustment for titres of antibodies to viruses and C. pneumoniae had no impact on the relation between sialic acid and carotid atherosclerosis. CONCLUSIONS: From these results, it seems unlikely that previous infection by any of these micro-organisms accounts for the relation between S-TSA level and carotid atherosclerosis.
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| 9. |
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| 10. |
- Påhlsson, Peter, et al.
(författare)
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Role of N-linked glycosylation in expression of E-selectin on human endothelial cells.
- 1995
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 25:9, s. 2452-2459
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Tidskriftsartikel (refereegranskat)abstract
- E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.
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