scholarly journals Human mononuclear cells contain an endoglycosidase specific for heparan sulphate glycosaminoglycan demonstrable with the use of a specific solid-phase metabolically radiolabelled substrate

1989 ◽  
Vol 264 (3) ◽  
pp. 777-783 ◽  
Author(s):  
R F Sewell ◽  
P E C Brenchley ◽  
N P Mallick
Keyword(s):  
Affinity Chromatography ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Human Spleen ◽  
Human Mononuclear Cells ◽  
Normal Human ◽  
Sepharose 4B ◽  
Heparin Affinity

Xyloside-initiated 35SO4(2-)-labelled glycosaminoglycans were isolated from the medium of cultured bovine glomeruli and covalently coupled to Sepharose 4B to construct a solid-phase substrate suitable for the detection of endoglycosidases. The substrate is rendered specific for heparitinase by prior digestion with chondroitin sulphate ABC lyase and is insensitive to proteinase, neuraminidase and hyaluronidase. Normal human mononuclear cells are shown to contain a heparitinase. This enzyme appears to be cell-associated and can be partially purified from human spleen by heparin affinity chromatography.

scholarly journals Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans

1988 ◽  
Vol 256 (1) ◽  
pp. 29-33 ◽  
Author(s):  
K Karlsson ◽  
U Lindahl ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Inhibitory Effect ◽  
Extracellular Superoxide Dismutase ◽  
Net Charge ◽  
Sulphated Glycosaminoglycans ◽  
Sepharose 4B ◽  
Heparin Affinity ◽  
Secretory Enzyme

The secretory enzyme extracellular superoxide dismutase (EC-SOD) occurs in at least three forms, which differ with regard to heparin affinity: A lacks affinity, B has intermediate affinity, and C has relatively strong affinity. The affinity of EC-SOD C for various sulphated glycosaminoglycans (GAGs) was assessed (a) by determining the concentration of NaCl required to release the enzyme from GAG-substituted Sepharose 4B and (b) by determining the relative potencies of the GAGs to release EC-SOD C from heparan sulphate-Sepharose 4B. Both methods indicated the same order of affinity. Heparin bound EC-SOD C about 10 times as avidly as the studied heparan sulphate preparation, which in turn was 10 and 150 times as efficient as dermatan sulphate and chondroitin sulphate respectively. Chondroitin sulphate showed weak interaction with EC-SOD C at physiological ionic strength. Heparin subfractions with high or low affinity for antithrombin III were equally efficient. The binding of EC-SOD C to heparin-Sepharose was essentially independent of pH in the range 6.5-9; below pH 6.5 the affinity increased, and beyond pH 9.5 there was a precipitous fall in affinity. The inhibitory effect of NaCl on the binding of EC-SOD C to GAGs indicates that the interaction is of electrostatic nature. EC-SOD C carries a negative net charge at neutral pH, and it is suggested that the binding occurs between the negative charges of the GAG sulphate groups and a structure in the C-terminal end of the enzyme that has a cluster of positive charges. These results are compatible with the notion that heparan sulphate proteoglycans on cell surfaces or in the intercellular matrix may serve to bind EC-SOD C in tissues.

scholarly journals Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns

1986 ◽  
Vol 235 (2) ◽  
pp. 469-479 ◽  
Author(s):  
B C Wightman ◽  
E A Weltman ◽  
L A Culp
Keyword(s):  
Extracellular Matrix ◽  
Affinity Chromatography ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Heparan Sulphate Proteoglycan ◽  
3T3 Cells ◽  
Platelet Factor ◽  
Sulphate Proteoglycan ◽  
Adhesion Sites ◽  
Chondroitin Sulphate Proteoglycan

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.

scholarly journals Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.

1979 ◽  
Vol 27 (3) ◽  
pp. 794-796 ◽  
Author(s):  
M J McElrath ◽  
R M Galbraith ◽  
R C Allen
Keyword(s):  
Human Serum ◽  
Solid Phase ◽  
Islet Cells ◽  
Pancreatic Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Normal Human ◽  
Liver Biopsies ◽  
Alpha 1 Antitrypsin ◽  
Sepharose 4B

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.

Glycoproteins and glycosaminoglycans of cultured normal human epidermal keratinocytes

1983 ◽  
Vol 61 (1) ◽  
pp. 325-338
Author(s):  
K.W. Brown ◽  
E.K. Parkinson
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Sodium Dodecyl ◽  
Immunological Methods ◽  
Epidermal Keratinocytes ◽  
Immunoperoxidase Staining ◽  
Human Epidermal Keratinocytes ◽  
Molecular Weights ◽  
Keratinocyte Cell ◽  
Normal Human

[3H]glucosamine has been used to label metabolically keratinocyte cell-surface glycoconjugates. The major labelled bands identified on sodium dodecyl sulphate/polyacrylamide gels had apparent molecular weights of greater than 250 000, and 150 000-80 000. Most of these components were trypsin-sensitive, indicating that the label was protein-bound. Some of the labelled components were shown to be proteoglycans and the labelled glycosaminoglycans released from them by trypsin were identified as hyaluronic acid (54%), heparan sulphate (33%) and chondroitin sulphate (13%). Specific immunological methods (immunoperoxidase staining and immunoprecipitation) showed that keratinocytes produced fibronectin. Immunoperoxidase staining showed keratinocytes produce only small ‘stitches’ of fibronectin at cell edges; no large fibrils were seen nor any staining over or between cells.

scholarly journals Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1971 ◽  
Vol 124 (4) ◽  
pp. 677-683 ◽  
Author(s):  
P.-H. Iverius
Keyword(s):  
Cyanogen Bromide ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Direct Analysis ◽  
Dermatan Sulphate ◽  
Peptide Residue ◽  
Agarose Beads ◽  
Freeze Dried ◽  
Coupling Procedure ◽  
Sepharose 4B

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.

scholarly journals The nature of the non-ultrafilterable glycosaminoglycans of normal human urine

1971 ◽  
Vol 122 (3) ◽  
pp. 373-384 ◽  
Author(s):  
E. Wessler
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Molar Ratios ◽  
Sodium Barbital ◽  
Normal Human ◽  
Normal Men ◽  
Barbital Buffer ◽  
Acidic Glycosaminoglycans

1. The non-ultrafilterable acidic glycosaminoglycans from pooled urine of normal men, aged about 20, were isolated and characterized. The isolation procedure included digestion with sialidase and pronase, and fractionation by stepwise elution from an ECTEOLA-cellulose column. The glycosaminoglycans in each fraction were separated from each other by preparative electrophoresis in sodium barbital buffer and in barium acetate. 2. Approximate relative amounts of the different glycosaminoglycans were: chondroitin sulphate 60%, chondroitin 2%, hyaluronic acid 4%, dermatan sulphate 1%, heparan sulphate 15% and keratan sulphate 18%. Chondroitin sulphate–dermatan sulphate hybrids seemed to occur in trace amounts. 3. Chondroitin sulphate, heparan sulphate and keratan sulphate were heterogeneous with respect to degree of sulphation. Two distinct groups of chondroitin sulphate fractions were found, with sulphate/hexosamine molar ratios of about 0.5 and 1 respectively. The sulphate/hexosamine molar ratios in the heparan sulphate fractions varied from 0.5 to 0.9; the N-sulphate/hexosamine ratio was about 0.5 in all fractions. The sulphate/hexosamine molar ratios in the keratan sulphate fractions varied from 0.2 to 0.7.

scholarly journals Tuftsin induces tissue factor-like activity in human mononuclear cells and in monocytic cell lines

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 814-819 ◽  
Author(s):  
A Kornberg ◽  
R Catane ◽  
S Peller ◽  
S Kaufman ◽  
M Fridkin
Keyword(s):  
Tissue Factor ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Monocytic Cell ◽  
Human Mononuclear Cells ◽  
Monocytes And Macrophages ◽  
Normal Human ◽  
Natural Stimulator ◽  
Coagulation Pathway ◽  
Lymphoid Cell Lines

Abstract Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF- like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

γ-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias

1995 ◽  
Vol 70 (5) ◽  
pp. 237-242 ◽  
Author(s):  
M. Täger ◽  
A. Ittenson ◽  
A. Franke ◽  
A. Frey ◽  
H. G. Gassen ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Mononuclear Cells ◽  
Cellular Expression ◽  
Normal Human ◽  
Glutamyl Transpeptidase
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