Biomedical subjects

E Buddecke

Publications and source records attributed to E Buddecke.

At least 37 records · Page 2Linked to original sources

PubMed1991

Different action of heparin and fucoidan on arterial smooth muscle cell proliferation and thrombospondin and fibronectin metabolism.

Fucoidan, a sulfated fucopolysaccharide of marine algae is able to inhibit the proliferation of arterial smooth muscle cells half maximally at a concentration of 80 to 100 micrograms/ml culture medium. In comparable concentrations heparin was significantly less active than the fucopolysaccharide. Sulfation of fucoidan was found to be essential for expression of antiproliferative activity. The inhibitory effect of fucoidan is a time-dependent event with highest effectiveness during the first 6 h. Fucoidan does not influence the overall rate of synthesis of cell proteins and glycoconjugates, but led to substantial alterations in the synthesis and secretion of fibronectin and thrombospondin. Immunoprecipitation and quantitation revealed that the incorporation of [35S]methionine into fibronectin is reduced whereas thrombospondin synthesis was increased. The effect on fibronectin was not shared by heparin. Desulfation of the fucopolysaccharide abolished the observed modulation. Binding experiments with [125I]fucoidan indicate a saturable binding and a maximum of 2.8 x 10(6) bound molecules per cell. Fucoidan binding sites can be only partly displaced by heparin. The results suggest that both heparin and the structurally unrelated sulfated fucopolysaccharide act as an antiproliferative agent but differ in their modulation of cell metabolism.

Animals↗

PubMed1990

Biochemical studies on human corneal proteoglycans--a comparison of normal and keratoconic eyes.

Human corneas from normal (healthy) donors and patients with keratoconus were either metabolically labelled under organ culture conditions or investigated without preincubation. The sulfated proteoglycans were isolated from a 4 M guanidinium chloride/2% Triton X 100 extract. Two predominant proteoglycans were obtained from normal cornea after digestion of total sulfated proteoglycans with chondroitin ABC-lyase or endo-beta-galactosidase. One had an overall mass of 150 kDa, two dermatan sulfate chains (Mr approximately 50 kDa) with an iduronic acid content of 24%-28% and, after chondroitin ABC-lyase digestion, a core protein of 48 kDa. The other proteoglycan had an overall mass of 110 kDa, one keratan sulfate chain of approximately 60 kDa and, following endo-beta-galactosidase (keratanase) digestion, a core protein of 46 kDa. Each proteoglycan population was further fractionated into two subpopulations by chromatography on concanavalin A-Sepharose. The dermatan sulfate- and keratan sulfate-containing proteoglycans isolated from keratan and healthy cornea had comparable Mr values and core proteins with identical molecular weights, but the ratio of dermatan sulfate/keratan sulfate proteoglycan was increased in keratoconic cornea and the keratan sulfate chains of two keratan sulfate proteoglycans from keratoconic cornea were considerably shorter (Mr 44 and 33 kDa) than those from normal corneas.

Adult↗

PubMed1990

Bovine arterial smooth muscle cells synthesize two functionally different proteoheparan sulfate species.

Cultured arterial smooth muscle cells synthesize two proteoheparan sulfate species. One is found associated with the cells, whereas the other is excreted into the medium. The two proteoheparan sulfates have similar hydrodynamic sizes but differ in the Mr of their core proteins. The cell-associated proteoheparan sulfate has a Mr of 92,000 while that of soluble proteoheparan sulfate is 38,000. The cell-associated and the soluble proteoheparan sulfate species differ in their ability to suppress the proliferation of smooth muscle cells. When added to the culture medium 2-5 micrograms/ml of the cell-associated and 20-25 micrograms/ml of the soluble proteoheparan sulfate species inhibit the growth of smooth muscle cells half maximally. The antiproliferative potency of both species resides in the heparan sulfate chains. Commercially available heparin has no antiproliferative effect and is not able to prevent the antiproliferative action of cellular heparan sulfate. In contrast to heparin, none of the heparan sulfate preparations has anticoagulant activity. Smooth muscle cells endocytose the soluble heparan sulfate at a rate three to four times higher than that of the cell-associated heparan sulfate. The data suggest that the cell-associated and the soluble proteoheparan sulfate species are separate and possibly genetically distinct molecules. Furthermore, the structural determinants for antiproliferative activity and the recognition sites for endocytotic uptake appear to be different.

Animals↗

PubMed1990

Two different proteoheparan sulfate species are synthesized by bovine corneal endothelial cells.

Cultured bovine corneal endothelial cells were labeled with either [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The medium proteoheparan sulfate (HS-PG) and cell-associated HS-PG were isolated and identified as heparan sulfate-containing proteoglycans by the susceptibility of their glycosaminoglycan component to specific enzymatic and chemical degradation. The native medium HS-PG has an apparent Mr of about 300,000 and an Mr of its protein core of 64,000, while the native cell-associated HS-PG had a lower molecular weight (approx. 100,000) and an Mr of its protein core of 33,000. The protein core of both HS-PG types consisted of a single polypeptide chain. After beta-elimination reaction of the native HS-PGs the resulting heparan sulfate side chains of medium HS-PG had an Mr of about 60,000, and those of cell-associated HS-PG an Mr of about 30,000. The different molecular parameters of medium HS-PG and cell-associated HS-PG refer to different functional roles. The medium HS-PG secreted by the endothelial cells is regarded as a metabolic precursor of the Descemet's membrane, while the cell-associated HS-PG might represent a cell membrane-integrated proteoglycan.

Animals↗

PubMed1990

Mapping of proteoglycans in atherosclerotic lesions.

The involvement of sulphated glycosaminoglycans in atherosclerotic changes have been studied in human and rat arteries, and biochemical experiments have revealed that a significant increase in the contents of chondroitin sulphate/dermatan sulphate and cholesterol, but loss of heparan sulphate, occurs in human atherosclerotic arterial tissues. Electron micrographs have revealed that extracellular deposits of lipid are predominantly present in areas rich in chondroitin sulphate proteoglycans but not in areas rich in collagen bundles and dermatan sulphate proteoglycans. The different types of proteoglycans have been distinguished in situ by the cuprolinic blue staining method and enzymatic degradation experiments, and their topohistochemical distribution patterns analysed by morphometry of proteoglycan/cuprolinic blue precipitates. The ultracytochemical investigations indicate changes in size and pattern of chondroitin sulphate-rich proteoglycan-cuprolinic blue precipitates in human atherosclerosis. In plaque tissue, these precipitates are significantly enlarged. In addition, they accumulate around smooth muscle cells in the medial tissue. An increase in the size of proteoglycan-cuprolinic blue precipitates has also been observed in balloon catheter-induced lesions in rat carotid arteries. The large chondroitin sulphate as well as the small dermatan sulphate proteoglycan-cuprolinic blue precipitates show this alteration 2 weeks after balloon injury. We suggest that quantitative and qualitative alterations in the arterial proteoglycans occur in the pathogenesis of atherosclerosis in addition to the cell proliferation and lipid accumulation.

Animals↗

PubMed1990

Changes in heparan sulfate structure during transition from the proliferating to the non-dividing state of cultured arterial smooth muscle cells.

Cultured arterial smooth muscle cells synthesize a cell-associated heparan sulfate proteoglycan which consists of a 92 kDa core protein with 3 to 4 heparan sulfate side chains covalently attached. Biosynthesis of the cell-associated heparan sulfate proteoglycan was compared in proliferating and in non-dividing vascular smooth muscle cells which are preincubated in the presence of [35]sulfate or a combination of [35S]methionine and [3H]glucosamine. The Mr of the core protein was identical in either growth state, but changes in the structure of the heparan sulfate side chains were observed. Non-dividing (postconfluent) arterial smooth muscle cells form longer heparan sulfate chains with a higher proportion of hydrophobic (N-acetyl) groups than proliferating (preconfluent) cells as judged from gel filtration experiments, hydrophobic interaction chromatography and heparitinase degradation. An enzyme preparation from proliferating cells catalyzes deacetylation and N-sulfation of heparan sulfate at a 5-fold higher activity than from non-dividing cells. Cell density-dependent structural differences of heparan sulfate are related to the finding that heparan sulfate isolated from non-dividing cells has a 10-fold higher antiproliferative potency than heparan sulfate from proliferating (preconfluent) cells.

Animals↗

PubMed1989

Cytochemical changes in a human arterial proteoglycan related to atherosclerosis.

The cuprolinic blue (CB) staining method has been used to visualize and characterize proteoglycans (PG) in the extracellular matrix (ECM) of normal and atherosclerotic human arteries. Arterial tissues of 13 individuals (1-83 years of age) were obtained by autopsy. For electron microscopic visualization of PGs staining with CB was performed in the presence of a critical electrolyte concentration of 0.3 M MgCl2. Under these conditions CB selectively interacts with the polysulfated glycosaminoglycan (GAG) side chains of the molecules. Removal of PG side chains by GAG-degrading enzymes prior to CB staining selectively prevented the formation of chondroitin sulfate (CS)-rich and dermatan sulfate (DS)-rich PG-CB precipitates. The DS-rich type of PG is mainly associated with collagen fibrils, the CS-rich type of PG is preferentially localized in nonfibrous areas of the ECM (soluble matrix). When normal arterial tissues are compared with those affected by atherosclerosis quantitative and qualitative changes of PG-CB precipitates are detected. In fibrous plaques a strong accumulation of a large CS-rich type of precipitate close by smooth muscle cells (SMC) and foam cells is observed. In addition, these precipitates are significantly longer in fibrous plaques than in adjacent normal media (116 nm vs. 100 nm; P less than 0.001). This alteration is independent of the age of the donor. Small DS-rich PG-CB precipitates associated with collagen fibrils show strong variations in their length, but not a significant tendency towards elongated precipitates in atherosclerosis. The present results demonstrate that ultracytochemical and morphometric analysis are useful in providing information on the diverse types, locations, interactions, and possibly of molecular changes of PGs in normal and atherosclerotic human arteries.

Arteries↗

PubMed1989

[Biochemistry and pathobiochemistry of proteoglycans of the eye].

Proteoglycans are a family of protein polysaccharide conjugates that fulfill various functions in the macromolecular organization of the human eye. The high hydrodynamic volume of the proteoglycans, the large polyanionic domain of their polysaccharide chains, the specific primary structure of their protein moiety and their localization both on the cell surface and in the extracellular matrix enable the proteinglycans to regulate cell growth and development, assembly of the extracellular matrix components and cell-matrix and cell-cell interactions. Proteoglycans containing chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate have been found to be structural and functional elements of the eye. This is exemplified by the corneal stroma, corneal endothelial cells, vitrous body, photoreceptor cells, and retinal pigment epithelium.

Animals↗

PubMed1988

Calmodulin antagonists increase the amount of mRNA for the low-density-lipoprotein receptor in skin fibroblasts.

The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a 'Northern' blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA.

Actins↗

PubMed1988

Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue.

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion-exchange chromatography on DEAE-cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high-molecular-mass (P-HS 1) and a low-molecular-mass (P-HS 2) fraction by gel filtration on Sephacryl S-400. P-HS 1 has a Mr of 175,000 and possesses four heparan sulfate side-chains (Mr 32,000) covalently bound to the protein core via a galactose- and xylose-containing polysaccharide-protein binding region. The protein core (Mr 38,000), which was obtained after deglycosylation of PG-HS 1 with trifluormethane sulfonic acid, contained in addition a few N-glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P-HS 2 is a single-chain peptidoheparan sulfate of Mr of 38,000 containing one heparan sulfate chain (Mr 32,000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P-HS 1 and P-HS 2 was 1:0.66.

Amino Acids↗

PubMed1988

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells.

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The purified proteoheparan sulfate had an apparent Mr of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (Mr approximately 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent Mr of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.

Animals↗

PubMed1988

Tyrosine O-sulfate ester in proteoglycans.

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.

Animals↗

PubMed1987

Calmodulin antagonists suppress cholesterol synthesis by inhibiting sterol delta 24 reductase.

Preincubation of hepatoma cells and human skin fibroblasts in the presence of the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide resulted in a dose-dependent suppression of [14C]mevalonolactone incorporation into cholesterol. At a calmodulin antagonist concentration of 25 mumol, the incorporation of [14C]mevalonolactone into cellular cholesterol was suppressed to about 30% (hepatoma cells) and 10% (human skin fibroblasts) of control values. When the total nonsaponifiable [14C]lipids were separated and analyzed by two-dimensional thin layer chromatography, an accumulation of [14C]desmosterol was observed along with reduced formation of [14C]cholesterol. However, when cells were preincubated in the presence of [14C]dihydrolanosterol, [14C]cholesterol formation was not inhibited by the calmodulin antagonists. About 25% of the cell-associated dihydrolanosterol radioactivity was converted to cholesterol in both control and calmodulin antagonist-pretreated cells. The data suggest that calmodulin antagonists prevent the conversion of desmosterol into cholesterol by inhibiting sterol delta 24 reductase and that the enzymes catalyzing sterol ring modifications are not affected by the inhibitors.

Calmodulin↗

PubMed1987

Proliferation-dependent changes of proteoglycan metabolism in arterial smooth muscle cells.

Cultured arterial smooth muscle cells synthesize and secrete two types of sulfated proteoglycans designated as proteoglycan A and proteoglycan B. Proteoglycan A has been characterized as chondroitin sulfate-rich, whereas proteoglycan B was found to be dermatan sulfate-rich [Schmidt, A. & Buddecke, E. (1985) Eur. J. Biochem. 153, 260-273]. During the logarithmic growth phase, arterial smooth muscle cells incorporated about 3 times more [35S]sulfate into the total proteoglycans secreted into the culture medium than did non-dividing cells. When arterial smooth muscle cells stopped proliferating the ratio of [35S]proteoglycan A/B increased. No differences were detected in the respective molecular and chemical characteristics of purified proteoglycans A and B isolated from both proliferating and non-dividing cells. Regardless of the growth phase proteoglycan A had a molecular mass of about 280 kDa and contained 8-9 chondroitin sulfate-rich side chains. Proteoglycan B had a molecular mass of about 180 kDa and contained 6-7 dermatan sulfate-rich side chains. The [35S]methionine-labelled protein cores of proteoglycan A and B had a molecular mass of about 48 kDa, but were distinguishable by their specific reactions to monospecific antibodies. Proliferating cells endocytosed proteoglycan B at a rate up to 100% higher than that of non-dividing cells. In all growth phases proteoglycan A was endocytosed at a 10-fold lower rate than proteoglycan B.

Animals↗

PubMed1987

Mapping of proteoglycans in human arterial tissue.

The major families of proteoglycans in human arterial tissue have been localized and characterized by electron microscopy. After staining with the polycationic dye cuprolinic blue in the presence of a critical electrolyte concentration, three differently sized populations of proteoglycan-cuprolinic blue precipitates are found. The precipitates are distinguished of the basis of their morphology, topographical distribution and susceptibility to specific glycosaminoglycan-degrading enzymes. Each type of proteoglycan is preferentially associated with one connective tissue component: (a) a dermatan sulfate proteoglycan interacts with collagenous fibers, (b) a heparan sulfate proteoglycan is associated with elastic fibers and with the exterior surface of the basement membrane-like layer surrounding smooth muscle cells, and (c) a chondroitin sulfate proteoglycan forms aggregates with hyaluronate in the soluble matrix. Information about the pattern of proteoglycans in normal human arterial tissue should constitute a useful basis for evaluating perturbations in proteoglycan distribution in arteriosclerotic plaques.

Adult↗

PubMed1987

Biochemical events during stapling of the proximal tibial epiphyseal plate in pigs.

Epiphyseal stapling is considered a safe, effective method of correcting angular deformities of the knee in growing children. A study of the underlying biochemical events would contribute to a better understanding of the control of longitudinal growth rate in epiphyseal growth plates and of the molecular pathology of exaggerated physiologic bowleg or knock-knee deformities. Blount stainless staples were applied to the medial side of the proximal tibia epiphyseal growth plate of domestic pigs. The resulting biochemical changes were investigated by incubation of the isolated growth plate cartilage under organ culture conditions. On the fourth to the eighth day after stapling, a significant decrease of [14C]glucose uptake and lactate formation and a significantly lower specific radioactivity of the isolated glycosaminoglycans were observed in relation to the controls. During the second to 12th week, the incorporation of [3H]thymidine and the specific radioactivity of the glycosaminoglycans following incubation with [14C]glucose showed statistically significant decreases. The glycosaminoglycan content of the cartilage was not altered, but the chondroitin sulfate/keratan sulfate ratio was lower in the stapled than in the nonstapled areas. In this period, glucose uptake and lactate production were found to be normalized.

Animals↗

PubMed1986

Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts.

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50-100 microM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [35S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [125I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca2+ levels modulate LDL receptor metabolism in human skin fibroblasts.

Calcium Channel Blockers↗

PubMed1986

Calmodulin antagonists stimulate LDL receptor synthesis in human skin fibroblasts.

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.

1-Methyl-3-isobutylxanthine↗