Localisation and characterization of acid mucopolysaccharides in the early chick blastoderm

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 169-178
Author(s):  
Ch. Vanroelen ◽  
L. Vakaet ◽  
L. Andries
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Middle Layer ◽  
Ventral Side ◽  
Chick Blastoderm ◽  
Cellulose Acetate Electrophoresis ◽  
Acid Mucopolysaccharides ◽  
Extracellular Compartment ◽  
Early Specialization ◽  
Testicular Hyaluronidase

Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms (16 h of incubation) were labelled with tritiated glucosamine and/or ]35S]sulphate. The incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the ventral side of the Upper Layer. A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e. hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.

scholarly journals Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils

1991 ◽  
Vol 275 (1) ◽  
pp. 67-73 ◽  
Author(s):  
S R Nelson ◽  
M Lyon ◽  
J T Gallagher ◽  
E A Johnson ◽  
M B Pepys
Keyword(s):  
Light Chain ◽  
Amyloid Fibrils ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Cellulose Acetate Electrophoresis ◽  
Amyloid A ◽  
Isolation And Characterization ◽  
Cscl Density Gradient ◽  
A Minor

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

scholarly journals Evidence for the existence of a multichain proteoglycan of heparan sulphate

1970 ◽  
Vol 117 (4) ◽  
pp. 699-702 ◽  
Author(s):  
L. Jansson ◽  
U. Lindahl
Keyword(s):  
Potassium Chloride ◽  
Aortic Wall ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Heparan Sulphate ◽  
Gel Chromatography ◽  
Dermatan Sulphate ◽  
Fraction I ◽  
Two Peaks ◽  
Testicular Hyaluronidase

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.

scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

Dermatan sulphate promotes neuronal differentiation in mouse and human stem cells

2020 ◽  
Author(s):  
Chika Ogura ◽  
Kazumi Hirano ◽  
Shuji Mizumoto ◽  
Shuhei Yamada ◽  
Shoko Nishihara
Keyword(s):  
Stem Cells ◽  
Neurite Outgrowth ◽  
Neuronal Differentiation ◽  
Neural Progenitor Cells ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Embryonic Stem ◽  
Hydroxyl Group ◽  
Human Stem Cells ◽  
Dermatan Sulphate

Abstract Dermatan sulphate (DS), a glycosaminoglycan, is present in the extracellular matrix and on the cell surface. Previously, we showed that heparan sulphate plays a key role in the maintenance of the undifferentiated state in mouse embryonic stem cells (mESCs) and in the regulation of their differentiation. Chondroitin sulphate has also been to be important for pluripotency and differentiation of mESCs. Keratan sulphate is a marker of human pluripotent stem cells. To date, however, the function of DS in mESCs has not been clarified. Dermatan 4 sulfotransferase 1, which transfers sulphate to the C-4 hydroxyl group of N-acetylgalactosamine of DS, contributes to neuronal differentiation of mouse neural progenitor cells. Therefore, we anticipated that neuronal differentiation would be induced in mESCs in culture by the addition of DS. To test this expectation, we investigated neuronal differentiation in mESCs and human neural stem cells (hNSCs) cultures containing DS. In mESCs, DS promoted neuronal differentiation by activation of extracellular signal-regulated kinase 1/2 and also accelerated neurite outgrowth. In hNSCs, DS promoted neuronal differentiation and neuronal migration, but not neurite outgrowth. Thus, DS promotes neuronal differentiation in both mouse and human stem cells, suggesting that it offers a novel method for efficiently inducing neuronal differentiation.

scholarly journals Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry

2015 ◽  
Vol 13 (4) ◽  
pp. 510-517 ◽  
Author(s):  
Ana Paula Cleto Marolla ◽  
Jaques Waisberg ◽  
Gabriela Tognini Saba ◽  
Daniel Reis Waisberg ◽  
Fernando Beani Margeotto ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
Electrospray Ionization Mass Spectrometry ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Ionization Mass Spectrometry ◽  
Ionization Mass ◽  
Neoplastic Tissue ◽  
Colorectal Tissue ◽  
Neoplastic Tissues

ABSTRACT Objective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’st test. Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic 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.

Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540

Zygote ◽  
2007 ◽  
Vol 15 (3) ◽  
pp. 225-232 ◽  
Author(s):  
A.-S. Bergqvist ◽  
J. Ballester ◽  
A. Johannisson ◽  
N. Lundeheim ◽  
H. Rodríguez-Martínez
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Negative Control ◽  
Dermatan Sulphate ◽  
Flow Cytometric ◽  
Merocyanine 540 ◽  
Incubation Duration ◽  
Flow Cytometric Study ◽  
Oviductal Fluid

SummaryGlycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0–30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.

scholarly journals The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1968 ◽  
Vol 109 (4) ◽  
pp. 517-526 ◽  
Author(s):  
M B Mathews ◽  
L. Decker
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
High Molecular Weight ◽  
Chondroitin Sulphate ◽  
Fibril Formation ◽  
Acid Mucopolysaccharide ◽  
Acid Mucopolysaccharides ◽  
Electrostatic Binding ◽  
Fibril Aggregates ◽  
Chondroitin Sulphate A

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide–proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7·6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate–proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2·5μg./ml. were unusual in decreasing aggregation, but heparin at 0·25μg./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ‘normal’ collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide–proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.

scholarly journals Structure and binding properties of collagen type XIV isolated from human placenta.

1993 ◽  
Vol 120 (2) ◽  
pp. 557-567 ◽  
Author(s):  
J C Brown ◽  
K Mann ◽  
H Wiedemann ◽  
R Timpl
Keyword(s):  
Human Placenta ◽  
Matrix Protein ◽  
Cell Attachment ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Extracellular Matrix Protein ◽  
Neutral Salt ◽  
Heparin Binding ◽  
Collagen Vi ◽  
Sulphate Proteoglycan

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.

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