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J Tranum-Jensen

Publications and source records attributed to J Tranum-Jensen.

At least 19 recordsLinked to original sources
PubMed1995

On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.

Arteriosclerosis↗
PubMed1995

Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine.

The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 micrograms of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(alpha 1- and alpha 2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of alpha 2 was unaffected. In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of alpha 2 were decreased by 19% and 15%, respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the alpha 2-isoform of Na/K-ATPase.

3-O-Methylglucose↗
PubMed1994

Membrane topology of insulin receptors reconstituted into lipid vesicles.

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-subunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, confirming that the alpha-subunit is harbored in the cross-bar of the T-structure.

Humans↗
PubMed1994

Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation.

Chemical modification of histidine residues in staphylococcal alpha-toxin leads to loss of functional activity. Site-directed mutants of the toxin in which each of the four histidine residues was replaced by several amino acids were therefore produced. The mutant proteins were purified and characterized. Exchange of H-259 or H-144 was sometimes tolerated without reduction in hemolytic activity. These histidine residues are thus not essential for toxin function. Exchange of H-35 and H-48, however, had marked effects. H-35 mutant toxins bound with high affinity to rabbit erythrocytes but displayed faulty oligomerization and were unable to form pores. H-48 mutant toxins also had severely impaired hemolytic activity due probably to faulty hexamerization. We interpret these results to indicate that the N-terminal domain of alpha-toxin in the region of H-35 and H-48 is involved in protomer-protomer interactions that underlie the hexamerization and pore-forming process.

Animals↗
PubMed1993

Quantitation of Na+/K(+)-ATPase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling.

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.

Adipose Tissue↗
PubMed1993

Deposition of C3, the terminal complement complex and vitronectin in primary biliary cirrhosis and primary sclerosing cholangitis.

Characteristics of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are bile duct destruction and portal inflammation. Increased levels of circulating complement activation products are also present. This raises the possibility of involvement of complement-dependent cytotoxic mechanisms in the pathogenesis. Therefore, we investigated liver biopsy specimens from 21 patients with PBC, six patients with PSC and six controls for complement deposits by immunohistochemistry using polyclonal and monoclonal antibodies against C3d, the terminal complement complex (TCC) and vitronectin (S-protein). We found C3d, TCC and vitronectin deposits only in the portal tracts. C3d and TCC were present in the walls of the hepatic arteries and in the connective tissue stroma but never around the bile ducts. We found vitronectin deposits throughout the connective tissue, often independent of the TCC deposits. When vitronectin and TCC were co-localized, the staining patterns were inverse; that is, intense staining for TCC accompanied weak staining for vitronectin and vice versa. Occasionally complete dissociation between TCC and vitronectin staining was observed. Deposits of TCC and vitronectin showed a focal distribution leaving many portal tracts free of TCC. Our results question whether complement-dependent cytotoxic mechanisms take part in the bile duct destruction in PBC and PSC.

Adult↗
PubMed1992

The human placental transferrin receptor: reconstitution into liposomes and electron microscopy.

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.

Cell Membrane↗
PubMed1992

Kinetics of glucose transport in rat skeletal muscle membrane vesicles: effects of insulin and contractions.

To study the mechanism of acceleration of glucose transport in skeletal muscle after stimulation with insulin and contractions, we isolated a subcellular vesicular membrane fraction, highly enriched in the plasma membrane enzyme K(+)-stimulated p-nitrophenylphosphatase and also enriched in some intracellular membranes. Protein recovery, morphology, lipid content, marker enzyme activities, total intravesicular volume, Western blot quantitation of GLUT-1, and glucose-inhibitable cytochalasin B binding were identical in membrane fractions from control, insulin-stimulated, contraction-stimulated, and insulin- and contraction-stimulated muscle. Time course of D-[3H]glucose entry in membrane vesicles at equilibrium exchange conditions showed that initial rate of transport at 30 mM of glucose was increased 19-fold and that equilibrium distribution space was increased 4-fold in vesicles from maximum stimulated muscle. The effects of insulin and contractions on initial rate of transport as well as on equilibrium distribution space were additive, and stimulation increased the substrate saturability of glucose transport. Furthermore, cytochalasin B binding to membranes prepared by using less centrifugation time than usual showed that, after stimulation with insulin and contractions, at least 35% of the total number of glucose transporters were redistributed from one kind of vesicles to a more slowly sedimenting kind of vesicles, probably reflecting translocation within the membrane preparation from intracellular vesicles to the plasma membrane upon stimulation. In the present membrane preparation the effects of insulin and/or contractions on glucose transport resemble those seen in intact muscle, and the effects are thus not dependent on cellular integrity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A model for the quaternary structure of human placental insulin receptor deduced from electron microscopy.

Electrophoretically pure and functionally intact human placental insulin receptor was studied by electron microscopy with negative-staining techniques. The quaternary structure of the detergent-solubilized receptor was determined. The receptor had the shape of a letter T approximately 24 nm in height and 18 nm in width with a thickness of the stem and the crossbar of 3-4 nm. No consistent change in ultrastructure of the receptor could be detected after the addition of insulin alone or insulin and Mn2+/Mg2+/ATP. After partial reduction of the alpha 2 beta 2 heterotetrameric receptor into alpha beta heterodimers, the electron micrographs showed a clear reduction in average size of the molecule with disappearance of the T profiles characteristic of the alpha 2 beta 2 heterotetramers. By incubation of the heterodimers in a phosphorylation medium containing insulin, a reassociation to molecules with molecular weights of the alpha 2 beta 2 heterotetramer took place judged from SDS/PAGE. Electron microscopy showed that the molecule formed larger aggregates, and only a few solitary T-shaped copies were seen.

Chromatography, Affinity↗
PubMed1991

Alpha-toxin of Staphylococcus aureus.

Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occurs in both cases after membrane-bound toxin molecules collide via lateral diffusion to form ring-structured hexamers. The latter insert spontaneously into the lipid bilayer to form discrete transmembrane pores of effective diameter 1 to 2 nm. A hypothetical model is advanced in which the pore is lined by amphiphilic beta-sheets, one surface of which interacts with lipids whereas the other repels apolar membrane constitutents to force open an aqueous passage. The detrimental effects of alpha-toxin are due not only to the death of susceptible targets, but also to the presence of secondary cellular reactions that can be triggered via Ca2+ influx through the pores. Well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction. Such processes cause profound long-range disturbances such as development of pulmonary edema and promotion of blood coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Morphology of electrophysiologically identified junctions between Purkinje fibers and ventricular muscle in rabbit and pig hearts.

Purkinje fiber-ventricular muscle (PV) junctions were identified by extracellular recording in isolated, superfused preparations from rabbit and pig hearts. Microelectrode recordings from different cell types at the PV junctions were obtained, and the cells recorded from were retrieved microscopically. To this end 26 tissue blocks were serially sectioned at 4 microns. Microscopic identification of the very cell recorded from was obtained in five of seven Purkinje, five of 16 transitional, and two of two ventricular muscle cell recordings. In addition, some tissue blocks from both junctional and nonjunctional sites identified only by extracellular recording were examined in serial sections. Transitional cells in the rabbit heart are thin, broad bandlike cells (30-35 by 3-5 microns) arranged in one or two sheets in the subendocardium between the Purkinje layer and ventricular mass. Transitional cells are coupled via short, thin strands to both Purkinje and ventricular muscle cells. A second type of PV coupling was observed frequently in the pig, but in only one of 21 cases in the rabbit. Here, a short, linear segment of small transitional cells connected large-diameter Purkinje cells to ventricular muscle cells. Distances found between Purkinje-transitional cell coupling sites and transitional cell-ventricular muscle coupling sites varied from 100 to 1,000 microns in the rabbit heart and from 50 to several hundred micrometers in the pig heart. Action potentials from transitional cells typically showed multiple components in their upstroke. Both our morphological and electrophysiological findings are compatible with the existence of a relatively high-resistance barrier between Purkinje and transitional cells and between transitional and ventricular muscle cells.

Action Potentials↗
PubMed1990

Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation.

Aorta↗
PubMed1990

Functions and relevance of the terminal complement sequence.

The terminal complement sequence is initiated upon cleavage of C5 with liberation of C5a anaphylatoxin, and involves the assembly of macromolecular C5b-9 complexes either on cell surfaces or in plasma. Cell-bound C5b-9 complexes generate transmembrane pores that can cause cell death, or they can elicit secondary cellular reactions triggered, for example, by passive flux of calcium ions into the cells. In vivo functions of the fluid-phase SC5b-9 complex have not yet been defined, but the identity of S-protein with vitronectin (serum spreading factor) provokes the anticipation that significant biological functions of this complex do exist. The terminal complement sequence may fulfil protective functions when it is triggered on alien cells that are marked for destruction. Dysregulation in the complement sequence may, however, result in detrimental attack by C5b-9 on autologous cells. Examples include not only autoimmune disease states, but also the activation of complement on dead or dying cells, and bystander attack on blood cells during cardiopulmonary bypass. Methods for detecting and quantifying C5b-9 are outlined, and the potential usefulness of such assays in clinical research is discussed.

Complement C3-C5 Convertases↗
PubMed1990

[Functions and relevance of the terminal complement sequence].

The terminal complement sequence is initiated upon cleavage of C5 with liberation of C5a anaphylatoxin, and involves the assembly of macromolecular C5b-9 complexes either on cell surfaces or in plasma. Cell-bound C5b-9 complexes generate transmembrane pores that can cause cell death, or they can elicit secondary cellular reactions triggered, for example, by passive flux of calcium ions into the cells. In vivo functions of the fluid-phase SC5b-9 complex have not yet been defined, but the identity of S-protein with vitronectin (serum spreading factor) provokes the anticipation that significant biological functions of this complex do exist. The terminal complement sequence may fulfill protective functions when it is triggered on alien cells that are marked for destruction. Dysregulation in the complement sequence may, however, result in detrimental attack by C5b-9 on autologous cells. Examples include not only autoimmune disease states, but also the activation of complement on dead or dying cells, and bystander attack on blood cells during cardiopulmonary bypass. Methods for detecting and quantifying C5b-9 are outlined, and the potential usefulness of such assays in clinical research is discussed.

Animals↗
PubMed1989

Hereditary, complete deficiency of complement factor H associated with recurrent meningococcal disease.

Complement factor H (beta-1H globulin) is an important regulatory protein which inhibits the spontaneous complement activation via the alternative pathway. We describe a 15-year-old girl without any detectable factor H in plasma. She has had two episodes of meningococcal disease, but is otherwise completely healthy. Secondary to the factor-H deficiency, the levels of factor B, properdin, C3, and C5-C9 were strongly reduced due to spontaneous in vivo activation of the alternative complement pathway. Plasma C3dg was strongly elevated in spite of the factor-H deficiency; apparently erythrocyte CR1 substitutes for factor H in C3 degradation. Neither C3 nor complement lesions were demonstrable on her erythrocytes which did, however, show increased, spontaneous haemolysis in vitro in citrate plasma, but not in serum. The patient is a single child and her parents, who are unrelated and healthy, had half-normal levels of factor H. This reduction of factor H is sufficient to cause increased, spontaneous activation of the alternative pathway.

Antibodies, Bacterial↗