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Biomedical subjects

P Vischer

Publications and source records attributed to P Vischer.

33 records · Page 2Linked to original sources
PubMed1988

Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks.

Different biochemical and cytochemical techniques were applied to characterize the sites of localization of thrombospondin in cultured endothelial cells. The results obtained by [35S]methionine labeling, immunoblotting, immunoprecipitation, fluorescence microscopy, ultracytochemistry, immunogold labeling, and silver enhancement experiments revealed that thrombospondin secreted by endothelial cells is structurally organized together with proteoheparan sulfate in spherical granules at the cell surface. These granules are about 100 to 300 nm in size. Heparin or enzymatic degradation with heparitinase, but not with ABC lyase, release thrombospondin from the cell surface. Fibronectin is expressed in the extracellular matrix of endothelial cells in a fibrillar organization, clearly distinct from the punctate pattern of thrombospondin on the cell surface. Furthermore, secreted thrombospondin is highly enriched together with fibronectin and proteoheparan sulfate in cell attachment sites and in cell migration tracks. In cell migration tracks proteoheparan sulfate more clearly resembles the fibrillar distribution pattern of fibronectin, whereas thrombospondin reveals a rather monodisperse pattern. The obtained data suggest preferential sites of interaction between thrombospondin and heparan sulfate proteoglycans on the cell surface and a participation of thrombospondin in cell adhesion and cell migration.

Animals↗
PubMed1987

A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis.

Cultured smooth muscle cells (SMC) from rat aorta and endothelial cells (EC) from pig aorta were used to study the effect of the catecholamines epinephrine and norepinephrine on cell proliferation. Both stimulated growth of SMC and EC when added to the culture medium. Besides epinephrine and norepinephrine, dopamine and some of their metabolites also stimulated proliferation of cultured endothelial cells. Smooth muscle cells originating from rats being exposed to atherosclerotic risk factors, like diabetes, hypertension and balloon-injury, exhibited an increased susceptibility to these catecholamines compared to SMC from control animals. In comparison to normotensive control animals a 10-fold elevated plasma concentration of epinephrine was found in hypertensive rats. In man plasma epinephrine and norepinephrine concentration was determined in a healthy control group and in patients suffering from diabetes mellitus and coronary artery disease. Plasma epinephrine and norepinephrine levels were similar in patients suffering from diabetes mellitus compared to the control group. But in patients with coronary artery disease significantly higher plasma concentrations for epinephrine (p less than 0.001) and norepinephrine (p less than 0.01) were observed. These data support the hypothesis that catecholamines may play a role in the development and subsequent complications of atherosclerosis.

Animals↗
PubMed1987

Arterial resistance vessels in primary hypertension--evidence for altered cellular Ca2+ metabolism.

The role of intracellular free Ca2+ in arterial smooth muscle was investigated in aortic smooth muscle cells from spontaneously hypertensive and normotensive rats, as well as in porcine aortic smooth muscle. Intracellular free Ca2+ in aortic smooth muscle from spontaneously hypertensive rats was elevated. Incubation of porcine aortic smooth muscle with plasma from essential hypertensive and normotensive subjects revealed a humoral factor in subjects with essential hypertension, which increased intracellular free Ca2+. Cross-circulation experiments between spontaneously hypertensive and normotensive rats demonstrated a transmission of hypertension to the normotensive animals as a result of a circulating hypertensive factor possibly produced in the kidneys and adrenals. The experiments suggest that vasoconstriction in primary hypertension may be caused by a humoral factor that increases intracellular free Ca2+ in arterial smooth muscle.

Animals↗
PubMed1985

Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells.

The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from 'high-mannose' to 'complex' structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A-Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.

Acetylglucosaminidase↗
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↗
PubMed1984

Glycosyl transferases of baby hamster kidney cells and ricin-resistant mutants. O-glycan biosynthesis.

Extracts of baby hamster kidney (BHK) cells catalyzed the incorporation of N-acetylgalactosamine from UDP-N-acetyl[14C]galactosamine into myelin basic protein and an acylated tetrapeptide, N-acetylthreonyl-triproline, based on the threonine residue 98, glycosylated in myelin basic protein. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to the peptide moieties. Several ricin-resistant BHK cell lines contained enhanced (approximately twofold) levels of the transferase activity. Apomucins obtained from bovine submaxillary gland mucin by chemical or enzymic degradation were relatively poor acceptors. Using asialomucin as acceptor, galactosyl, transferase activities and a weak sialyl transferase activity were detected in BHK cell extracts. Galactose transfer occurred at two sites: to peptide-linked N-acetylgalactosamine residues to form the linkage, galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine and to terminally linked N-acetylglucosamine residues that exist as a minor constituent in bovine submaxillary mucin O-glycans, to form a galactosyl N-acetylglucosamine linkage. This reaction was not inhibited by ovalbumin, an efficient acceptor of the beta 1 leads to 4 galactosyl transferase involved in N-glycan assembly. Incorporation of galactose and N-acetylgalactosamine into endogenous proteins of BHK cell extracts was also detected. Sialic acid, fucose and N-acetylglucosamine residues were not incorporated. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to polypeptide, and galactose incorporation represented synthesis of the galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine sequence linked to polypeptide. The major endogenous protein labelled by either sugar had a molecular weight of approximately 80 000. A BHK-cell-associated glycoprotein, analogous to the urinary Tamm-Horsfall glycoprotein of molecular weight similar to the major endogenous acceptor of glycosylation, was not glycosylated in the experiments in vitro.

Animals↗
PubMed1983

Influence of prostacyclin and indomethacin on castor oil-induced gastrointestinal effects in rats.

The effects of castor oil, alone, as well as in combination with PGI2 and indomethacin on gastrointestinal functions have been examined in rats. Oral administration of the oil to fasted rats induced severe diarrhoea, with increased intestinal motility and fluid volume. Pretreatment with PGI2 (s.c.) inhibited the effect of the oil on intestinal fluid accumulation and decreased intestinal motility below control values, but only delayed the occurrence of mucoid diarrhoea. Indomethacin (i.p.) reduced the accumulation in intestinal fluid after castor oil administration to a much smaller extent (47%) than PGI2 and depressed the increased intestinal motility to control values. In contrast to PGI2, indomethacin inhibited the occurrence of diarrhoea after administration of castor oil. The present results do not definitely confirm the general opinion that the diarrhoeal action of laxative agents is due only to an altered intestinal electrolyte and water transport or an increase of intestinal motility.

Animals↗
PubMed1982

Pharmacological properties of Ciloprost, a stable prostacyclin analogue, on the gastrointestinal tract.

The effect of Ciloprost on the gastrointestinal tract was studied in rats and compared with prostacyclin (PGI2) and/or PGE2. In the anaesthetized rat continuous i.v. infusion of Ciloprost decreased pentagastrin stimulated gastric acid secretion with the same potency [ED 50 -35 micrograms/(kg x h)]as PGI2 [ED 50 -42 micrograms/(kg x h)]. In the pylorus-ligated rat Ciloprost reduced acid secretion with an ED50 of about 6.4 mg/kg p.o. whereas PGE2 and PGI2 were ineffective during submaximal stimulation of acid secretion by pentagastrin. In the same experimental design continuous i.v. infusion of Ciloprost and PGI2 caused 50% reduction in gastric acid output with doses of 80 and 24 micrograms/(kg x h), respectively. Ciloprost prevented indomethacin-induced gastric erosions at an ED50 of 60 micrograms/kg p.o., compared to 10 micrograms/kg p.o. of PGE2, whereas PGI2 showed only weak cytoprotective activity. The stable prostacyclin analogue inhibited castor oil induced enteropooling with a 4-5 fold higher potency than PGI2 and delayed castor oil induced diarrhoea more effectively than its natural counterpart. Intestinal motility was strongly reduced by both PGI2 and Ciloprost. Gastric emptying was also reduced after PGI2 and Ciloprost administration, with a 4-5 fold higher activity of the stable analogue. The present results demonstrate, that Ciloprost shows a biological profile similar to that of PGI2.

Animals↗
PubMed1981

Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin.

A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [14C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655-671) for the presence of high mannose N-glycans and N-acetylgalactosamine-rich O-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.

Aedes↗
PubMed1981

Glycosyl transferases of baby-hamster-kidney (BHK) cells and ricin-resistant mutants. N-glycan biosynthesis.

Five cell lines of ricin-resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N-glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N-acetylglucosamine as acceptors and N-acetylglucosaminyl transferases using ovalbumin and glycopeptides as acceptors. Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N-acetylglucosamine in cellular glycoproteins and lacks almost completely N-acetylglucosamine transferase I, an essential enzyme in assembly of ricin-binding carbohydrate sequences of N-glycans. These cells also contain reduced levels of N-acetylglucosamine transferase II active on a product of N-acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose-(alpha 1 leads to 6)-N-acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N-acetylglucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells. None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.

Animals↗
PubMed1980

Biogenesis of hepatocyte plasma-membrane domains. Incorporation of (3H)fucose into plasma-membrane and golgi-apparatus glycoproteins.

1. Rats were injected intracaudally with [3H]fucose and its rate of incorporation into the fucoproteins of serum, Golgi and plasma-membrane subfractions was followed for up tp 2h. 2. Incorporation into the Golgi dictyosome and secretory-vesicular fractions reached a maximum at 15 min or less, but most of the radioactivity was associated with classes of secretory glycoproteins. Incorporation into sinusoidal plasma-membrane fractions reached a maximum at 30 min, coinciding with the maximum release of fucoproteins into the serum. Contiguous and canalicular plasma-membrane fractions were labelled slightly later and at a lower rate and specific radioactivity. 3. Fluorography of fucoproteins separated by polyacrylamide-gel electrophoresis helped to distinguish between the major secretory and membrane-bound glycoproteins. The results show that a major biogenetic sequence is probably from Golgi dictyosomes to Golgi secretory elements to a sinusoidal plasma membrane. 4. The kinetics of incorporation make it unlikely that there is rapid and direct insertion of glycoproteins into the bile-canalicular plasma membrane. A route involving direct transfer of glycoproteins via a membrane-mediated intracellular path from the blood sinusoidal to the bile-canalicular plasma membranes is proposed.

Animals↗
PubMed1980

Different turnover of fucose residues in plasma membranes of rat liver and Morris hepatoma 7777.

Half-lives and rate constants of degradation of protein-bound fucose have been determined in plasma membranes and total cell homogenates of rat liver and Morris hepatoma 7777. The existence of at least two dynamically different classes of fucose-containing glycoproteins could be demonstrated in both liver and hepatoma plasma membranes. The apparent half-lives were 8.4 and 24.5 h (host liver) and 11.5 and 33.9 h (Morris hepatoma). Since this biphasic loss of fucose residues was not observed for sialic acid [Harms & Reutter (1974) Cancer Res. 34, 3165--3172], the differences are possibly related to specific functions of fucosylated glycoproteins of the plasma membrane.

Animals↗
PubMed1977

Glycosyltransferases and glycosidases in Morris hepatomas.

The following three parameters were studied in Morris hepatomas of different growth rates: (a) the specific activity of guanosine dephosphate (GDP)-fucose:glycoprotein fucosyltransferase and cytidine monophosphate (CMP)-N-acetylneuraminic acid:glycoprotein sialyltransferase, (B) the content of GDP-fucosee and CMP-N-acetylneuraminic acid, and (c) the activity of alpha-L-fucosidase and neuraminidase. Fucosyltrasferase activities were significantly elevated in all hepatomas investigated. Especially high levels of enzyme were measured in the rapidly growing tumors 7777, 66, and 3924A. The increase varied between 2- and 3-fold when compared with the corresponding host liver. Conversely, the activity of the sialytransferase was greatly decreased in all hepatoma lines with a rapid or intermediate growth rate. In the fast-growing tumor 9618A2, the activity was reduced to 8%. GDP-fucose and CMP-N-acetylneuraminic acid were determined by the isotope dilution technique. In normal rat liver from Buffalo or ACl rats, the concentration of GDP-fucose was 6.5+/-0.9 and 9.5+/-1.1nmoles/g, wet weight, respectively. In the fast-growing hepatomas 3924A and 9121, levels up to 21.5 nmoles/g, wet weight, were found, However, the content of CMP-N-acetylneuraminic acid in hepatomas was indluenced to a lesser extent by the degree of differentiation of the tumor. In the most rapidly growing tumor, 9618A2, a level of alpha-L- fucosidase seven times higher than in host liver was determined. Moreover, there existed a correlation bewteen the age of the hepatoma and enzyme activity. Within the 2nd week after inoculation, fucosidase activity increased from 130 to 343 nmoles/hr/mg of protein. Neuraminidase was measured in a new linked assay system. The activity of this enzyme was lowered by 50% or was at least unchanged when compared to the activity in host liver. Our results indicate that specific alterations of fucose metabolism are a characteristic feature of Morris hepatomas.

Animals↗