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A Linker

Publications and source records attributed to A Linker.

At least 37 records · Page 2Linked to original sources
PubMed1987

Changes in glomerular heparan sulfate in puromycin aminonucleoside nephrosis.

Changes in glomerular anionic sites in puromycin aminonucleoside nephrosis (PAN) in the rat are controversial. The authors examined glomerular anionic sites in PAN by in vivo staining with polyethyleneimine (PEI). They also quantitated and characterized glomerular heparan sulfate (HS), which is known to be a major glomerular polyanion in PAN, using in vivo incorporation of 35S-sulfate. PAN rats had a mean protein excretion of 96 +/- 23 mg per 24 hours. Staining of anionic sites with PEI showed 15.3 +/- 2.8 sites per 1000-nm length of glomerular basement membrane in controls, 13.7 +/- 1.9 sites in PAN rats (P greater than 0.05), and 50% of rats with early PAN had absent staining. Total 35S-sulfate incorporation was similar in both the controls and established PAN rats (2900 +/- 150 dpm/mg dry wt of glomeruli versus 3005 +/- 260, P greater than 0.05) but decreased in early PAN rats (2025 +/- 148). The percentage of 35S-sulfate incorporated into chondroitin sulfate was similar in all three groups of animals. HS uronic acid was also similar (1.8 +/- 0.2 g/mg dry wt of glomeruli versus 1.7 +/- 0.3, P greater than 0.05) but decreased in early PAN (1.1 +/- 0.2). The distribution of 35S-sulfate activity within the HS subfractions was examined by ion-exchange chromatography and showed a shift in percent present from 1.0 M to 1.25 M fraction in established and early PAN animals (control 1.0 M 37% +/- 3.2% versus PAN 19% +/- 3.4%, P less than 0.01, and 1.25 M 36% +/- 2.9% versus 53% +/- 2.9%, P less than 0.01). These results demonstrate that glomerular heparan sulfate is unchanged in established PAN but decreased in early PAN. SO4 incorporation is unchanged in established PAN and diminished in early PAN. Thus, early in PAN HS synthesis is impaired, but in established PAN the HS is normal, and changes in glomerular HS cannot explain the increased permeability.

Animals↗
PubMed1987

Presence and role of glycosaminoglycans in amyloidosis.

Though the presence of glycosaminoglycans in amyloid deposits has been recognized for a long time their role in the pathogenesis of the disorder has remained elusive. As shown here, liver and spleen of human patients with secondary amyloidosis contain 5 to 10 times the amount of glycosaminoglycans as normal organs. Of the three major glycosaminoglycans measured, the heparan sulfate fraction showed the largest increase. In mice where amyloidosis was induced by the injection of casein and enhancing factor (accelerated model) 35SO4-labeled, or Alcian blue stained glycosaminoglycans appeared as early as and at the same location as proteins detected by Congo red staining which was about 2 days after initiation of the procedure. When glycosaminoglycan synthesis was followed in liver and spleen slices of control and experimental animals a significant increase in rate was found in the spleen of the experimental mice. Though there was an increase in heparan sulfate synthesis the major contribution to the overall increase was made by the chondroitin sulfates in the accelerated as well as in the standard induction model. In addition, unlike in the human disorder the chondroitin sulfates were the major glycosaminoglycans which had accumulated in the spleens of animals which had amyloidosis induced by the long term standard procedure (6 weeks) as measured by isolation and uronic acid analysis. The data presented here show that glycosaminoglycans appear to play an important and perhaps direct role in the process of amyloid deposition in the human disease as well as in the experimentally induced disorder in animals.

Amyloidosis↗
PubMed1987

Glycosaminoglycans of BALB/c 3T3 and B16.F10 melanoma cells. Metabolic differences.

When the glycosaminoglycans (GAGs) of BALB/c 3T3 and B16.F10 melanoma cells were compared during parallel studies, substantial differences were found in the cellular content, rates of turnover, and extent of intrinsic sulfated GAG degradation between the two cell types. The 3T3 cells had more than 10-fold more metabolically-labeled [35S]-GAGs per cell under steady state conditions than did the melanoma cells, and in pulse-chase studies they turned this material over at a much slower rate (apparent respective half-times of 30 hours and 8 hours). Moreover, the labeled material released into the medium during the chase periods was approximately 43% low molecular weight polymeric product, eluting in the included volume of Sephadex G50, for the 3T3 cells, as compared with 79% for the B16.F10 line. For both cell lines approximately 86% of total cell layer-associated polymeric GAG was accessible to GAG-degrading enzymes under conditions which did not lyse the cells, indicating that the majority of cellular GAGs is present external to the plasma membrane. The extensive digestion of sulfated GAGs in evidence for the melanoma cells suggests the involvement of constitutively high cellular levels of GAG-degrading enzymes.

Animals↗
PubMed1986

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers.

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.

Animals↗
PubMed1986

Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulphates.

We analysed the distribution, structural characteristics, antithrombin-III-binding properties and anticoagulant activities of heparins and heparan sulphates isolated from the tissues of a wide range of vertebrates. Heparin has a curiously limited distribution, since it was absent from lower aquatic vertebrate species, present in only certain organs such as intestine in many higher vertebrates, and completely absent from the rabbit among mammals examined. The heparins were structurally diverse, and they exhibited a broad range of anticoagulant activities, from approx. 50% to 150% of average commercial heparins. Although there was a rough correlation between the anticoagulant potency of the starting isolate and the proportional content of material exhibiting high-affinity binding to the proteinase inhibitor antithrombin III, activities of high-affinity fractions from heparins low in activity overlapped those of low-affinity fractions from highly active heparins. Heparan sulphates, which in contrast were isolated from nearly all vertebrate organs, contained high-affinity subfractions constituting up to 5% of the starting material and possessing anticoagulant potencies of 2-30 units/mg. In consideration of the heparin data, we infer that its biological function is either species-specific or may be served by other molecular elements, and that there exists considerable diversity in the antithrombin-III-binding sequence of heparin. The more-generally distributed glycosaminoglycan heparan sulphate possesses within its variable structure a small high-affinity subfraction with low anticoagulant potency, whether isolated from aorta or other tissues. Although heparan sulphate appears to have an essential function at the cellular level, we suggest that this is probably not that of providing heparin-like antithrombotic effects on vascular surfaces.

Animals↗
PubMed1985

Growth-related variations in the glycosaminoglycan synthesis of ultraviolet light-induced murine cutaneous fibrosarcoma cells.

Glycosaminoglycan synthesis was studied in cell populations of ultraviolet light-induced murine cutaneous fibrosarcoma cells under conditions of varying growth rates in vitro. After labeling with the precursors, 3H-glucosamine and 35SO4, sulfated glycosaminoglycans recoverable by direct proteolysis of the culture monolayers increased approximately 5-fold on a per cell basis from sparsely populated, exponential cell cultures (greater than 85% of cells in S, G2, or M phases) to stationary cultures inhibited by high cell density (greater than 50% of cells in G1). Within this cell surface-associated material, the relative ratio of heparan sulfate to the chondroitin sulfates was approximately 60/40% under conditions of exponential growth; in the growth-arrested cultures, the reverse ratio was found. The substratum attached material, obtained from the flask surface after ethyl glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-mediated detachment of the monolayers, contained relatively more hyaluronic acid, heparan sulfate, and chondroitin sulfates in the most actively proliferating cultures compared with the growth-inhibited cell populations. Furthermore, heparan sulfate and the chondroitin sulfates, which were enriched in the substratum material and in the cell pellet of exponential cultures, showed a relative shift to the cell surface-associated compartment (releasable by mild trypsinization after EGTA-mediated cell detachment) and to the compartment loosely associated with the pericellular matrix (i.e., released into the supernatant during detachment of the monolayers in the presence of EGTA). These results demonstrate that a variety of differences in the quantities, relative compositional ratios, and cell compartment distributions of hyaluronic acid and sulfated glycosaminoglycans occur in fibrosarcoma cell populations which vary in their rate of cell growth consequent to cell density in culture.

Animals↗
PubMed1984

Structural studies on heparin. Tetrasaccharides obtained by heparinase degradation.

Three tetrasaccharides representing major structural sequences of heparin were isolated in good yield and characterized after degradation of heparin by purified flavobacterial heparinase. N-Desulfation was necessary to achieve good separation of these closely related compounds from each other. One of the tetrasaccharides was shown to be derived from the fully sulfated repeating segments; to contain L-iduronic acid and six sulfate groups, and have the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4)-(6-SO4). The second contained a D-glucuronic acid unit that was nonsulfated instead of the L-iduronic acid, and the third, obtained in a fairly low yield, contained five sulfate groups, three of which being located on the disaccharide at the nonreducing end, and having the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4). All tetrasaccharides had a sulfated, unsaturated uronic acid unit at the nonreducing end, confirming that the heparinase requires sulfated L-iduronic acid units for activity.

Carbohydrate Sequence↗
PubMed1984

Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans.

The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.

Cells, Cultured↗
PubMed1984

Isolation and characterization of an alginase from mucoid strains of Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa which produce an alginate-like slime polysaccharide were shown to also synthesize an intracellular enzyme which can degrade these polysaccharides and the seaweed alginic acids. The enzyme acts as an eliminase introducing delta 4,5 unsaturation into the uronic acid moiety. It appears to be a polymannuronide lyase which degrades the polysaccharides, depending on their uronic acid composition, to a series of oligosaccharides, the smallest of which is a disaccharide. L-Guluronic acid linkages are not split. The Pseudomonas alginase resembles other bacterial alginases and enzymes from molluscs but differs in some important properties, such as extent of degradation and linkage preference. Nonmucoid forms of the organism produce detectable but much lower amounts of enzyme.

Glycoside Hydrolases↗
PubMed1983

Decreased de novo synthesis of glomerular proteoglycans in diabetes: biochemical and autoradiographic evidence.

The experimental model of streptozotocin-induced diabetes in rats was utilized to determine the biosynthetic and biochemical alterations in the proteoglycans of the glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) in diabetic nephropathy. Isolated kidneys from diabetic and control groups of animals were radiolabeled in an organ perfusion apparatus with [35S]sulfate of high specific activity (greater than 1,200 Ci/mmol; 1 Ci = 3.7 x 10(10) Bq) and processed for electron microscopic autoradiography, and the proteoglycans of the glomerular extracellular matrices were characterized. The results indicate that [35S]sulfate incorporation into glomerular extracellular matrices of diabetic animals was 30-40% less than that of the control group; however, no differences in the biochemical properties of the de novo synthesized proteoglycans from either group were observed. The relevance of the decreased de novo synthesis of sulfated proteoglycans of glomerular extracellular matrices is discussed in terms of increased glomerular permeability to plasma proteins and reduction in the glomerular filtration rate.

Animals↗
PubMed1982

An unusual heparan sulfate isolated from lobsters (Homarus americanus).

An unusual heparan sulfate was isolated from lobsters (Homarus americanus). The polysaccharide has a composition and properties intermediate to heparin and the more common heparan sulfates. The sulfate and D-glucuronic acid content is high, while anticoagulant activity is low. The major repeating unit appears to consist of D-glucuronic acid and D-glucosamine N-O-disulfate. Some N-acetyl groups are present, the content of L-iduronic acid O-sulfate is low, and monosulfated or nonsulfated disaccharide-repeating units (very common in heparan sulfates) appear to be very rare. The data obtained again emphasize the heterogeneity of heparan sulfates and the need for adequate characterization when dealing with unusual or unexplored sources.

Animals↗
PubMed1981

Characterization of heparan sulfate isolated from drug glomerular basement membranes.

Glycosaminoglycans were isolated from whole dog kidney and from purified glomerular basement membrane. Heparan sulfate was found to be the major component in the whole organ and the only glycosaminoglycan detected in significant amounts in the glomerular basement membranes. The heparan sulfate from basement membranes was characterized by isolation and analyses and found to be more homogeneous in charge content and molecular weight than the polymer from the whole kidney and to resemble one of the subfractions. It has a lower total sulfate and N-sulfate content than the same polysaccharide isolated from rat basement membranes. The findings suggest that there are several subgroups of heparan sulfate polyanions in the kidney and that the heparan sulfate associated with glomerular basement membranes in line with its specific function may be different from other kidney heparan sulfates.

Animals↗
PubMed1980

Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion.

Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan-sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.

Animals↗
PubMed1979

Structure of heparan sulphate oligosaccharides and their degradation by exo-enzymes.

Oligosaccharides obtained from heparan sulphate by nitrous acid degradation were shown to be degraded sequentially by beta-D-glucuronidase or alpha-L-iduronidase followed by alpha D-N-acetylglucosaminidase. Structural analysis of the tetrasaccharide fraction showed the following. (1) N-Acetylglucosamine is preceded by a non-sulphated uronic acid residue that can be either D-glucuronic of L-iduronic acid, but followed by a glucuronic acid residue. (2) The N-acetylglucosamine in the major fraction is sulphated. (3) Very few if any of the uronic acid residues are sulphated (4). The results indicate that the area of the heparan sulphate chain where disaccharides containing N-acetylglucosamine and N-sulphated glucosamine residues alternate is higher in sulphate content than expected and that the sulphate groups are mainly located on the hexosamine units.

Acetylglucosaminidase↗
PubMed1979

Structure of the antithrombin-binding site in heparin.

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.

Animals↗
PubMed1978

Gelatinous transformation of the bone marrow.

A study of so-called gelatinous transformation of the bone marrow in 14 patients is presented. The condition is characterized by the presence of extracellular "gelatinous" material, fat atrophy, and associated focal marrow hypoplasia and is often mistaken as representing edema, necrosis, or amyloid. Histochemical studies indicate that the extracellular substance is hyaluronic acid. Though the lesion is usually associated with severe weight loss and cachexia, it appears that factors other than malnutrition must play a significant role in the pathogenesis of gelatinous transformation. Anemia, frequently seen in these patients, does not appear to be a consequence of gelatinous transformation.

Adipose Tissue↗
PubMed1977

Specificity of flavobacterial glycuronidases acting on disaccharides derived from glycosaminoglycans.

The specificity of the unusual flavobacterial glycuronidases that act on disaccharides containing delta4,5-unsaturated uronic acids was reinvestigated. The results show that the enzyme that hydrolyses the uronidic bond in disaccharides from hyaluronic acid and the chondroitin sulphates appears to be mainly specific for beta-D-(1 leads to 3)-derived linkages. The enzyme that hydrolyses the uronidic bond in a variety of disaccharides obtained from heparan sulphate and heparin appears to be specific for beta-D-(1 leads to 4)- and alpha-L-(1 leads to 4)- derived linkages. Thus the glycuronidases seem to be specific for linkage position rather than anomeric configuration, as had been thought previously. In addition, the data confirm other evidence that the major glucuronidic linkages in heparan sulphate and heparin have the beta-D-configuration, and the iduronidic linkages the alpha-L-configuration.

Chondroitin Sulfates↗