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Biomedical subjects

J Tranum-Jensen

Publications and source records attributed to J Tranum-Jensen.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1986

Pig intestinal microvillar maltase-glucoamylase. Structure and membrane insertion.

The NH2-terminal sequence (25 residues) of amphiphilic single polypeptide chain maltase-glucoamylase (EC 3.2.1.20) was determined by gas-phase sequencing. The result indicates that the NH2-terminal segment anchors the enzyme to the microvillar membrane. The single-chain form and the proteolytically processed two-chain form have two distinct active sites differing in heat stability. However, both sites are sensitive to chonduritol B-epoxide and have similar substrate specificity. The amphiphilic single-chain maltase-glucoamylase and the amphiphilic proteolytically processed form were inserted into liposomes and studied by electron microscopy. The results showed that the enzyme is predominantly present as a homodimeric complex in the membrane.

Amino Acid Sequence↗
PubMed1986

Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase.

Pig sucrase/isomaltase (EC 3.2.1.48/10) was purified from intestinal microvillar vesicles prepared from animals with and without pancreatic-duct ligation to obtain the single-chain pro form and the proteolytically cleaved final form respectively. The purified enzymes were re-incorporated into phosphatidylcholine vesicles and analysed by electron microscopy after negative staining. The two forms of the enzyme were observed as identical series of characteristic projected views that could be unified in a single dimeric model, containing two sucrase and two isomaltase units. This shows a homodimeric functional organization similar to that of other microvillar hydrolases. The bulk of the dimer was separated from the membrane by a maximal gap of 3.5 nm, representing a junctional segment connecting the intramembrane section of the anchor to the catalytically active domain of sucrase/isomaltase. The enzyme complex protrudes from the membrane for a distance of up to 17 nm. From charge-shift immunoelectrophoresic studies of hydrophilic prosucrase/isomaltase and from electron microscopy of reconstituted pro-sucrase/isomaltase, there was no evidence to suggest the presence of anchoring sequences between the sucrase and isomaltase subunits.

Cell Membrane↗
PubMed1986

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore.

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.

Animals↗
PubMed1986

Specialized membrane areas in non-activated and thrombin-activated platelets.

Filipin, a complex of polyene antibiotics, forms morphologically distinctive complexes with cholesterol in cell membranes under proper experimental conditions. When applied to non-activated, discoid platelets, filipin-induced lesions (FIL) occurred in rows at the platelet equator, suggesting a specialized membrane organization at the platelets' largest circumference. In some thrombin-activated platelets we observed surface membrane blebbing and release of lipid vesicles that predominantly originated from the plasma membrane proper, but some originated from (unidentified) platelet granules. FIL were initially present in high numbers over the entire bleb, they accumulated later at the neck of blebs, while the released vesicle was free of FIL. Absence of intramembrane protein particles (IMP) from the membranes of blebs and vesicles suggests that released vesicles are essentially without cholesterol and intrinsic membrane proteins and may consist predominantly of phospholipids. Membrane blebbing and vesicle release may represent unmasking and release of procoagulant platelet factor 3 activity.

Blood Platelets↗
PubMed1986

Production of listeriolysin by beta-hemolytic strains of Listeria monocytogenes.

Listeriolysin was isolated from target rabbit erythrocyte membranes after lysis of the cells with partially purified toxin derived from a culture supernatant of Listeria ivanovii. The membrane form of the toxin exhibited properties similar to those previously found for streptolysin O. Detergent-solubilized, delipidated listeriolysin was found to comprise a heterogeneous population of partially and fully circularized, amphiphilic oligomers whose embedment within the lipid bilayer generated large transmembrane pores. The molecular weight of the toxin monomer was estimated to be 55,000 to 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological cross-reactions between the toxin and streptolysin O were demonstrable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. An immunoblot assay for detecting listeriolysin in agar-incorporated, lysed erythrocyte membranes was developed, and 28 defined, clinical isolates of Listeria monocytogenes were examined for toxin production. These isolates caused beta-hemolysis on the agar plates and had previously been regarded as listeriolysin producers. However, we found that only two isolates produced genuine listeriolysin, since the sensitive immunoblot assay entirely failed to detect the toxin in all other cases. We excluded that this finding derived from proteolytic degradation of membrane-bound toxin. Thus, the great majority of human pathogenic Listeria strains appear to produce one or several hemolysins that are immunologically and, by inference, molecularly distinct from the streptolysin O-related listeriolysin. We propose that the streptolysin O-related toxin be designated alpha-listeriolysin and that the other hemolysin(s) be termed beta-listeriolysin.

Animals↗