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E Buddecke

Publications and source records attributed to E Buddecke.

At least 55 records · Page 3Linked to original sources
PubMed1986

Isolation and characterization of proteoglycans and glycosaminoglycans from human chondrosarcoma.

The proteoglycans and glycosaminoglycans of four human chondrosarcomas with different degrees of malignancy (I-III) have been studied. The hydrodynamic size of proteoglycan subunits and the tissue concentration of total glycosaminoglycans decreased with increasing grade of malignancy. The glycosaminoglycan distribution pattern of all chondrosarcomas showed a similar ratio of chondroitin-4-sulfate:chondroitin-6-sulfate but an increasing portion of keratan sulfate from grade I (6.5%) to grade III (19.2%). Determinations of the molecular weight (Mr values) of glycosaminoglycans were made after 3H labeling by alkaline reduction of proteoglycans in the presence of NaB3H4. The Mr of [3H]chondroitin sulfate isomers decreased markedly from grade I (35,500) to grade III (15,100) while the chain length of [3H]keratan sulfate showed minor variations (Mr 5600-6200). The previously reported decrease in the molecular weight of keratan sulfate with increasing degree of malignancy (S. Pal, W. Strider, R. Margolis, G. Gallo, S. Lee-Huang, and L. Rosenberg, 1978, J. Biol. Chem. 253, 1279-1289) was not observed.

Chondrosarcoma↗
PubMed1986

Increased activity of chondroitin sulfate-synthesizing enzymes during proliferation of arterial smooth muscle cells.

Cultured arterial smooth muscle cells incorporate [35S]sulfate into the extracellular chondroitin sulfate/dermatan sulfate containing proteoglycans at a higher rate in the phase of logarithmic growth than do non-dividing cells. The cell growth-dependent decrease in 35S incorporation with increasing cell density is accompanied by a decrease in the activity of chondroitin sulfate-synthesizing enzymes. The specific activity of xylosyl transferase, N-acetylgalactosaminyl transferase I and chondroitin sulfotransferase declines as the cells proceed from low to high densities. The corresponding correlation coefficients are 0.86, 0.91 and 0.89. The ratio of C-6OH/C-4OH sulfation of chondroitin shows a cell proliferation-dependent decrease indicating an inverse correlation of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase activity. The observed changes in the expression of enzyme activities are thought to have some implications in the pathogenesis of arteriosclerosis, the initial stages of which are characterized by proliferation of arterial smooth muscle cells.

Animals↗
PubMed1986

Compartmentation and characterization of different proteoglycans in bovine arterial wall.

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

Animals↗
PubMed1986

Purification and characterization of a 3'-phosphoadenylylsulfate:chondroitin 6-sulfotransferase from arterial tissue.

A 3'-phosphoadenylylsulfate:chondroitin sulfotransferase (EC 2.8.2.5) was purified to homogeneity (about 760-fold) from the cytosolic fraction of calf arterial tissue by Con A-Sepharose, ion exchange and affinity chromatography. The enzyme has a molecular mass of 38000 Da, optimal activity at pH 6.0 (100%) and 7.25 (75%), requires divalent cations for maximal activity (Mn2+ greater than Mg2+, Ca2+) and exhibits specificity towards desulfated chondroitin sulfate and oligosaccharides derived therefrom. The enzyme transfers sulfate groups from [35S]phosphoadenylylsulfate exclusively to C-6 OH groups of N-acetylgalactosamine units of the acceptor substrates. Maximal sulfate transfer occurs at 2mM chondroitin disaccharide units (100%), the transfer rates decreasing with decreasing chain length in the order deca (55%), octa (17%) and hexasaccharides (4%). Lineweaver-Burk plots revealed equal maximal velocities for chondroitin, deca-, octa- and hexasaccharide, but decreasing Km values. Chondroitin 4-sulfate has 21% of the acceptor potency exhibited by chondroitin, whereas dermatan sulfate, heparan sulfate and hyaluronate and the chondroitin tetrasaccharide showed no acceptor properties. Analysis of the reaction products formed by prolonged enzymatic sulfation of a reduced chondroitin hexasaccharide [GlcA-GalNAc]2-GlcA-GalNAc-ol revealed that the preterminal N-acetylgalactosamine from the non-reducing end and the internal N-acetylgalactosamine but not the N-acetylgalactosaminitol were sulfated and that no hexasaccharide disulfate was formed by the action of chondroitin 6-sulfotransferase. Chondroitin 6-sulfotransferase is considered to possess a binding region capable of accommodating a nonsulfated oligosaccharide sequence of at least six sugars and is believed to act in the course of chondroitin sulfate synthesis in cooperation with, but shortly after, the enzymes involved in the chain elongation reaction.

Animals↗
PubMed1985

High-uptake and low-uptake forms of proteoglycans secreted by arterial smooth muscle cells.

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans, designated as proteoglycan A and B, into the culture medium. They are isolated as immunologically distinct monomers with relative molecular masses of 280 X 10(3) and 180 X 10(3) and are characterized as chondroitin-sulfate-rich (A) and dermatan-sulfate-rich (B) proteoglycans. Both proteoglycan A and B were labelled with [35S]sulfate and used for studies of endocytosis. Uptake of proteoglycan B by arterial smooth muscle cells shows saturable kinetics. At saturation (500 microM) one cell may endocytose up to 1.5 X 10(6) proteoglycan B molecules/h. Proteoglycan A is internalized at a 10-fold lower rate. No saturation kinetics were observed at high proteoglycan A concentrations (500 microM). Endocytosis of proteoglycan B in the presence of an excess of proteoglycan A and vice versa suggest that proteoglycan A and B do not compete for the same receptor site. Free hyaluronate or chondroitin sulfate do not inhibit the uptake of proteoglycan B or A. The results suggest that proteoglycan B is internalized by arterial smooth muscle cells via a high-affinity receptor-mediated process, whereas proteoglycan A is taken up by fluid endocytosis and/or by low-affinity endocytotic processes.

Animals↗
PubMed1985

Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate.

Two N-acetylgalactosaminyltransferases, designated I and II, have been purified from the microsomal fraction of calf arterial tissue and separated on Bio-Gel A. N-Acetylgalactosaminyltransferase I was purified 450-fold. It requires Mn2+ for maximal activity and transfers N-acetylgalactosamine residues from UDP-[1-3H]GalNAc in beta-glycosidic configuration to the non-reducing terminus of the acceptor substrates GlcA(beta 1-3)Gal(beta 1-3)Gal, GlcA(beta 1-3)Gal(beta 1-4)Glc and GlcA(beta 1-3)Gal. Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase II, which transfers N-acetylgalactosamine from UDP-[1-3H]GalNAc to the non-reducing glucuronic acid residues of oligosaccharide acceptor substrates. Maximum transfer rates were obtained with a decasaccharide derived from chondroitin. Longer or shorter-chain chondroitin oligosaccharides are less effective acceptor substrates. All reaction products formed by N-acetylgalactosaminyltransferases I and II are substrates of beta-N-acetylhexosaminidase, which splits off the transferred [1-3H]GalNAc completely. In the microsomal fraction N-acetylgalactosaminyltransferase II had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme II loses much of its activity during the purification procedure and undergoes rapid thermodenaturation. GlcA-Gal-Gal is a characteristic sequence of the carbohydrate-protein linkage region of proteochondrioitin sulfate. The acceptor capacity of this trisaccharide suggests that N-acetylgalactosaminyltransferase I is involved in the synthesis of the carbohydrate-protein linkage region. Since N-acetylgalactosaminyltransferase II is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.

Animals↗
PubMed1985

Proliferation, morphology, and low-density lipoprotein metabolism of arterial endothelial cells cultured from normal and diabetic minipigs.

Aortic endothelial cells from control and streptozotocin diabetic minipigs were cultured. Both groups of cells exhibited the typical cobblestone-like appearance and gap junction formation. Endothelial cells derived from diabetic minipigs differed, however, from those from control animals by a higher rate of proliferation and a higher percentage of large and often multinucleated cells. In these cells the specific binding of low-density lipoproteins (LDL) to coated pits on the cell surface, the LDL uptake, and the intracellular transport of LDL to lysosomes were visualized by gold-labeled LDL complexes. The binding, internalization, and degradation of LDL by subconfluent, non-contact-inhibited endothelial cells was quantified using 125I-labeled LDL. The LDL metabolism of endothelial cells derived from diabetic animals was increased by about 40% compared to endothelial cells derived from nondiabetic animals.

Animals↗
PubMed1985

Alteration of glycosyltransferase activities during proliferation of cultivated arterial endothelial cells and smooth muscle cells.

The activities of glycosyltransferases involved in the biosynthesis of N-linked glycans of glycoproteins in cultivated endothelial and smooth muscle cells have been analysed. Both cell types contain galactosyl-, N-acetylglucosaminyl-, sialyl- and fucosyltransferases in decreasing order of activity, but the specific enzyme activity of glycosyltransferases is higher in endothelial cells. These differences are due neither to elevated glycosidase activities nor to enhanced hydrolysis of nucleotide sugars. Cell growth and differentiation have substantial influence on glycosyltransferase expression. Within 5 days after plating of endothelial cells all glycosyltransferase activities increase 3-10-fold. The highest activities are found in confluent cultures. In smooth muscle cells, however, the activities of all transferases except N-acetylglucosaminyltransferase, whose activity does not change during the logarithmic growth phase, increase by only 20-50%. Glycosidases are subject to variations but the specific activities are not strictly correlated to cell proliferation. Four days after the beginning of cultivation the activities of alpha-fucosidase and beta-N-acetylhexosaminidase are at a minimum in endothelial cells, whereas the activities of these enzymes attain their maxima in smooth muscle cells. The results suggest that the biosynthesis of glycoproteins in endothelial and smooth muscle cells is subject to growth-dependent regulation. Thus, substantial alterations of the cellular glycoprotein pattern are expected to occur during cell proliferation.

Animals↗
PubMed1985

Glucocorticoid-stimulated biosynthesis of low density lipoprotein receptor in cultured fibroblasts.

Preincubation of human skin fibroblasts in the presence of 10(-6)-10(-5) mol/l glucocorticoids (dexamethasone) causes a concentration and time-dependent increase of receptor-mediated internalisation of [125I]LDL. This increase is due to a glucocorticoid-specific stimulation by 40-50% of LDL receptor synthesis as demonstrated by an increased incorporation of [35S]methionine into immune precipitated receptor protein. In contrast the rate of synthesis of total cell protein and of lysosomal cathepsin D is not significantly influenced by dexamethasone. The increased LDL receptor synthesis is accompanied by an enhanced synthesis of cholesterol from [2-3H]mevalonolactone and [1-14C]acetate. The glucocorticoid-induced enhancement of LDL receptor and cholesterol synthesis is abolished by preincubation of the cells with dexamethasone in combination with 25-hydroxycholesterol.

Acetates↗
PubMed1985

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. II. Binding sites of proteoglycans on the cell surface.

Exogenous proteoglycans stained for electron microscopy with colloidal gold and/or cuprolinic blue bind to the surface of cultured arterial smooth muscle cells at two different sites. (I) About 20% of the proteoglycans adsorbed to the cells from the culture medium interact as monomeric and multimeric proteoglycans with smooth or coated membrane areas. (II) The bulk of exogenous proteoglycans exhibits high affinity binding to cell membrane-associated 10 nm fibrils containing or being closely associated with fibronectin and to collagen. It is suggested that the self association of proteoglycans and their binding to the cell membrane and to cell surface-associated fibronectin and collagen are important for maintaining an appropriate micro-environment for the cultured cells.

Arteries↗
PubMed1984

Influence of muzolimine on arterial wall elastin.

Muzolimine, 3-amino-1-(3,4-dichloro-alpha-methylbenzyl)-2- pyrazolin -5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60-300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolimine for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastin under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.

Animals↗
PubMed1984

Purification and characterization of 3'-phosphoadenylylsulfate: N-desulfoheparan sulfate sulfotransferase from arterial tissue.

A 3'-phosphoadenylsulfate: N-desulfoheparan sulfate sulfotransferase (EC 2.8.2.12) was purified 450-fold from the microsomal fraction of calf arterial tissue and separated from 3'-phosphoadenylylsulfate:chondroitin sulfotransferase (EC 2.8.2.5) activity. The enzyme has optimal activity at neutral pH, requires divalent cations (Mn2+, Mg2+, Ca2+) for maximal activity and exhibits specificity towards N-desulfoheparan sulfate, N,O-desulfoheparan sulfate and oligosaccharides derived therefrom. N,O-desulfoheparan sulfate tetrasaccharides serve as acceptor substrates only if the nonreducing terminus is occupied by glucuronic acid (not iduronic acid). The N,O-desulfoheparan sulfate sulfotransferase transfers [35S]sulfate from 3'-phosphoadenylyl[35S]sulfate to the 2-amino groups and to the 6-hydroxy groups of glucosamine units of the acceptor substrates. The ratio of N/O-sulfation ranged between 3:1 and 2:1. O-[35S]Sulfated unsaturated disaccharides were obtained from enzymatically labelled [35S]N-desulfoheparan sulfate by heparitinase degradation and subsequent deamination. Evidence for the O-sulfation at C-6 of the glucosamine units was provided by isolation of anhydromannose [35S]monosulfate, which was formed from uronosylanhydromannose [35S]monosulfate by beta-glucuronidase treatment. An N-desulfo-N-[1-14C]lacetylheparan sulfate deacetylase activity was copurified with the N-desulfoheparan sulfate sulfotransferase.

Animals↗
PubMed1984

Chemical changes of human knee joint menisci in various stages of degeneration.

Human knee joint menisci graded according to degenerative alterations were submitted to chemical analysis. Normal menisci contained 72% water, 0.12% DNA, 22% collagen, and 0.8% total glycosaminoglycans with the following glycosaminoglycan distribution pattern: 40% chondroitin 6-sulphate, 10-20% chondroitin 4-sulphate, 20-30% dermatan sulphate, and 15% keratan sulphate. The water content increased with increasing degeneration, whereas the collagen and glycosaminoglycan contents decreased with relative increase of chondroitin 6-sulphate.

Adolescent↗
PubMed1984

Characterization of proteoglycans synthesized by cultured arterial smooth muscle cells of the rat.

Arterial smooth muscle cells cultured from rat aorta were labelled with sodium [35S]-sulfate in combination with either [3H]glucosamine or [3H]mannose. The newly synthesized hyaluronate and sulfated proteoglycans obtained from the growth medium (M-PG) and extracted from the cell layer (C-PG) with 4M guanidinium chloride in the presence of proteinase inhibitors were purified by sequential fractionation on Sepharose 4B CL, equilibrium density gradient centrifugation and ion exchange chromatography under dissociative conditions. Gel filtration of M-PG resulted in the separation of free hyaluronate and two size classes of [35S]-proteoglycan populations eluted at Kav 0.15 (fraction M-A) and 0.48 (fraction M-B). On further fractionation M-A dissociated into hyaluronate (Mr 1.6 X 10(6)) and a proteoglycan monomer (M-PG A, Mr 180 000), which contained chondroitin 4-sulfate (Mr 21 000) as the main glycosaminoglycan moiety. The proteoglycan isolated from M-B (M-PG B) was identified as a proteoglycan monomer (Mr 200 000) containing mainly chondroitin sulfate/dermatan sulfate hybrid side chains (Mr 34 000). [3H]Mannose labelling and binding to ConA Sepharose of both M-PG A and B indicated the presence of oligosaccharides of the glycoprotein type. An analogous fractionation of proteoglycans associated with the cell layer yielded two hyaluronate-proteoglycan complexes (C- PreA and C-A). The proteoglycan monomers of these complexes (C-PG PreA and C-PG A) had Mr values of 420 000 and 130 000. A non-complexed proteoglycan monomer C-PG B (Mr 90 000) was also found. All cell layer bound proteoglycans had glycosaminoglycan side chains with Mr approximately 36 000 but the predominant glycosaminoglycan component was either heparan sulfate, chondroitin sulfate or dermatan sulfate. All cell layer bound proteoglycans contained [3H]mannose radioactivity, about 15% of which was bound to ConA Sepharose.

Animals↗
PubMed1984

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. I. Endocytosis and intracellular translocation of proteoglycan-gold conjugates.

Proteoglycans (Mr approximately 200 000) isolated from bovine arterial tissue were decorated with 17 nm diameter gold particles for tracing in electron microscopic thin sections and surface replicas. Lysine and arginine residues of their proteoglycan protein core are assumed to be essential for gold conjugation. The resulting proteoglycan-gold conjugates, which appear as pearl string-like gold strands of about 170 nm in length were used to visualize binding, endocytosis and intracellular translocation of proteoglycans by homologous cultured arterial smooth muscle cells. The proteoglycan-gold conjugates bind to coated as well as to non-coated cell surface membrane areas at 4 degrees C. This is followed by the formation of membrane invaginations. Postincubation at 37 degrees C leads to a time-dependent uptake of proteoglycan-gold conjugates via non-coated and coated vesicles which after fusion are translocated to multivesicular bodies and to large sized vesicles within 1 h. After conversion of these vesicles to lysosomal compartments the gold particles are uncoupled from the proteoglycans and are concentrated within residual vacuoles. From these vacuoles the gold particles are extruded. In contrast to the surface-bound proteoglycan-gold conjugates the released gold particles are condensed to bulky aggregates. The results, which include competition, inhibition and pulse chase experiments, extend biochemical data on endocytosis and degradation of proteoglycans.

Animals↗
PubMed1982

[35S]proteoglycan metabolism of arterial smooth muscle cells cultured from normotensive and hypertensive rats.

Arterial smooth muscle cells cultured from normotensive and hypertensive rats incorporated [35S]sulfate into the extracellular and pericellular sulfated proteoglycans and endocytose extracellular [35S]proteoglycans at a significantly higher rate in the phase of logarithmic growth than did nondividing cells. 35S incorporation into proteoglycans was positively correlated with [3H]thymidine incorporation into the cellular TCA-precipitable material. The rates of [35S]proteoglycan synthesis and endocytosis per cell pr day were higher in smooth muscle cells from hypertensive than from normotensive animals, the observed differences being related to a higher average protein content of smooth muscle cells cultured from hypertensive rats as compared with cells of normotensive animals. Gel filtration under dissociative conditions separated the [35S]proteoglycans into high and low molecular weight fractions (A, B) differing in glycosaminoglycan composition and their ability to be endocytosed by smooth muscle cells. The relative proportion of the high molecular weight proteoglycan fraction A decreased continuously from sparse to confluent cell cultures.

Animals↗