31 research outputs found
'Immunosequencing' of heparan sulfate from human cell lines and rat kidney: the (GlcNS6S-IdoA2S)(3) motif, recognized by antibody NS4F5, is located towards the non-reducing end
No full textItem does not contain fulltextHS (heparan sulfate) is a long linear polysaccharide, variably modified by epimerization and sulfation reactions, and is organized into different domains defined by the extent of modification. To further elucidate HS structural organization, the relative position of different HS structures, identified by a set of phage-display-derived anti-HS antibodies, was established. Two strategies were employed: inhibition of HS biosynthesis using 4-deoxy-GlcNAc, followed by resynthesis, and limited degradation of HS using heparinases. Using both approaches, information about the position of antibody-defined HS structures was identified. The HS structure recognized by the antibody NS4F5, rigorously identified as (GlcN6S-IdoA2S)(3), was found towards the non-reducing end of the HS chain
Construction and evaluation of an antibody phage display library targeting heparan sulfate
No full textContains fulltext :
220758.pdf (Publisher’s version ) (Open Access
Use of flow cytometry for characterization and fractionation of cell populations based on their expression of heparan sulfate epitopes
No full textItem does not contain fulltextThe ability to characterize alterations in heparan sulfate (HS) structure during development or as a result of loss or mutation of one or more components of the HS biosynthetic pathway is essential for broad understanding of the effects these changes may have on cell/tissue function. The use of anti-HS antibodies provides an opportunity to study HS chain composition in situ, with a multitude of different antibodies having been generated that recognize subtle differences in HS patterning, with the number and positioning of sulfate groups influencing antibody binding affinity. Flow cytometry is a valuable technique to enable the rapid characterization of the changes in HS-specific antibody binding in situ, allowing multiple cell types to be directly compared. Additionally fluorescent-activated cell sorting (FACS) allows fractionation of cells based on their HS-epitope expression
Heterogeneity of heparan sulfates in human lung.
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58299.pdf (Publisher’s version ) (Open Access)Heparan sulfates (HS), a class of glycosaminoglycans, are long linear complex polysaccharides covalently attached to a protein core. The HS molecules are made up of repeating disaccharides onto which modification patterns are superimposed. This results in a large structural heterogeneity and forms the basis of specific interactions of HS toward a vast array of proteins, including growth factors and proteases. To study HS heterogeneity in the lung, we used phage display technology to select seven antibodies against human lung HS. Antibodies reacted with HS/heparin, but not with other glycosaminoglycans or polyanions. Sulfate groups were essential for antibody binding. The amino acid sequence of the antibodies was established, the complementarity determining region 3 of the heavy chain containing basic amino acids. The antibodies defined HS epitopes with a characteristic tissue distribution. Antibody EV3A1 primarily stained macrophages. Other antibodies primarily stained basement membranes, but with different preference toward type of basement membrane. Antibody EV3C3 was the only antibody which clearly reacted with bronchiolar epithelial cells. In human lung parenchyma, basic fibroblast growth factor and vascular endothelial growth factor were largely bound by HS. Some antibodies blocked a basic fibroblast growth factor-binding site of HS, and one antibody blocked a vascular endothelial growth factor-binding site of heparin. Taken together, these data suggest a specific role for HS epitopes in human lung. The antibodies obtained may be valuable tools to study HS in pulmonary diseases
A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity
No full textItem does not contain fulltextGlycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials
Sequencing of glycosaminoglycans with potential to interrogate sequence-specific interactions
No full textTechnologies to sequence nucleic acids/proteins are widely available, but straightforward methodologies to sequence complex polysaccharides are lacking. We here put forward a strategy to sequence glycosaminoglycans, long linear polysaccharides involved in many biochemical processes. The method is based on the covalent immobilization and (immuno)chemical characterization of only those size-separated saccharides that harbor the original reducing end of the full-length chain. Using this methodology, the saccharide sequence of the chondroitin sulfate chain of the proteoglycan bikunin was determined. The method can be performed in any standard biochemical lab and opens studies to the interaction of complex saccharide sequences with other biomolecules
Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells
No full textIncreasing the effectiveness of the capacity usage at rolling stock service locations
No full textTrains consist of one or more railway vehicles called rolling stock, which need interior and exterior cleaning and small technical checks on a daily basis. These services are executed at service locations (SLs). Scheduling rolling stock servicing tasks during an operational day is important to guarantee the fulfilment of servicing deadlines. Public transport companies face large scheduling problems, especially those with 24-hour-a-day operation. The expected increase in transport frequencies enhances the need for improving scheduling servicing tasks during an operational day. Therefore, the Rolling Stock Servicing Scheduling Problem (RS-SSP) is modelled comprising a MILP model. Complying with the planned timetable, the RS-SSP maximises the RS units being serviced during daytime. The RS-SSP allows RS exchanges between RS units having completed servicing and operating RS units requiring servicing. Due to this RS Exchange Concept, the number of RS units visiting the SL during daytime can be increased. The proposed RS-SSP model has been tested on a real-life case from the Dutch railways. For multiple scenarios, the model was able to exchange all running RS. Consequently, the capacity usage at SLs can be increased by the RS-SSP by shifting some of the excessive workload to daytime, and thus solving the capacity shortages.Transport and PlanningAlgorithmic
Human single chain antibodies against heparin: selection, characterization, and effect on coagulation.
No full textHeparin, located in mast cells and basophilic granulocytes, is widely used as an anticoagulant. It belongs to a class of linear polysaccharides called glycosaminoglycans (GAGs). Using phage display technology, we have selected 19 unique human antiheparin antibodies. Some antibodies react almost exclusively with heparin, others also react with the structurally related heparan sulfate, and some with chondroitin sulfate. In all cases, sulfate groups are essential for binding. For activity of some antibodies, O-sulfation is more important than N-sulfation. Antibodies are reactive with heparin in mast cells. Each antibody showed a defined staining pattern on cryosections of rat kidney, pancreas, and testis. Enzymatic digestion with glycosidases on tissue sections further indicated that the antibodies are specific for GAGs. All antibodies recognize a unique epitope. The effect of the antibodies on heparin as an anticoagulant was also studied. There were 3 antibodies that were very effective inhibitors of heparin action in the activated partial thromboplastin time (APTT) clotting assay, and their effect was related to the amount of heparin bound. Some antibodies reacted strongly with the pentasaccharide, which interacts with antithrombin III. The human antibodies selected represent unique tools to study the structure, location, and function of heparin and related GAGs, and some may be used as blocking agents
Dynamic Expression of Genes Involved in Proteoglycan/Glycosaminoglycan Metabolism during Skin Development
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195443.pdf (Publisher’s version ) (Open Access
