Fractionation and identification of heparin and other acidic mucopolysaccharides by a new discontinuous electrophoretic method

Bianchini, P; Nader, Helena B.; Takahashi, Hélio K.; Osima, B; Straus, Anita H.; Dietrich, Carl P.

doi:10.1016/s0021-9673(00)84747-0

Cited by 56 publications

(27 citation statements)

References 14 publications

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“…Agarose-gel electrophoresis is a rapid and simple analytical technique to separate GAGs in mixtures, in particular HE, into two species, slow-moving, more sulfated and higher molecular mass, and fast-moving, lower molecular mass and charge density (14,15,36), HS, which has the same electrophoretic mobility of the fast-moving species, DS, and CS (14,15,36). This paper illustrates a simple method for recovering of sulfated GAGs at the microgram level after their agarose-gel separation for further analysis and characterization, such as disaccharide pattern, charge density, and molecular mass evaluation.…”

Section: Discussionmentioning

confidence: 99%

Disaccharide Analysis and Molecular Mass Determination to Microgram Level of Single Sulfated Glycosaminoglycan Species in Mixtures Following Agarose-Gel Electrophoresis

Volpi

1999

Analytical Biochemistry

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Section: Discussionmentioning

confidence: 99%

Disaccharide Analysis and Molecular Mass Determination to Microgram Level of Single Sulfated Glycosaminoglycan Species in Mixtures Following Agarose-Gel Electrophoresis

Volpi

1999

Analytical Biochemistry

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“…Analysis of the Acidic Polysaccharides by Agarose Gel Electrophoresis-Agarose gel electrophoresis of the acidic polysaccharides was performed in 0.6% agarose gels (7.5 ϫ 10 cm, 0.2 cm thick) prepared in four different buffers as follows: 0.05 M 1,3-diaminopropane acetate buffer, pH 9.0; discontinuous buffer 0.04 M barium acetate, pH 4.0; 0.05 M diaminopropane acetate, pH 9.0; 0.05 M KCl-HCl buffer, pH 2.0, or 0.06 M Tris acetate buffer, pH 8.0, as described previously (21,22). Aliquots of the fractions (about 50 g) were applied to the gel and subjected to electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Structural and Hemostatic Activities of a Sulfated Galactofucan from the Brown Alga Spatoglossum schroederi

Rocha

Moraes

Trindade

et al. 2005

Journal of Biological Chemistry

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144

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The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked ␤-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked ␣-fucose and one or two nonsulfated, 4-linked ␤-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.

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“…To distinguish heparin from heparan sulfate and other GAGs, discontinuous electrophoresis was also used. The gel was run for 10 min in 0.04 M barium acetate, pH 5.8, at 5°C (29). The gel was then transferred to another chamber containing diaminopropane acetate buffer and equilibrated for 15 min at 5°C.…”

Section: Identification and Quantification Of Gagsmentioning

confidence: 99%

Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells

Vial

Oliver

Jamur

et al. 2003

The Journal of Immunology

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Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcεRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK−/− embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing β7 integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcεRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.

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Fractionation and identification of heparin and other acidic mucopolysaccharides by a new discontinuous electrophoretic method

Cited by 56 publications

References 14 publications

Disaccharide Analysis and Molecular Mass Determination to Microgram Level of Single Sulfated Glycosaminoglycan Species in Mixtures Following Agarose-Gel Electrophoresis

Disaccharide Analysis and Molecular Mass Determination to Microgram Level of Single Sulfated Glycosaminoglycan Species in Mixtures Following Agarose-Gel Electrophoresis

Structural and Hemostatic Activities of a Sulfated Galactofucan from the Brown Alga Spatoglossum schroederi

Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells

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