Biomedical subjects

A Nykjaer

Publications and source records attributed to A Nykjaer.

39 records · Page 3Linked to original sources

PubMed1990

Cellular targets and receptors for interleukin-6. II. Characterization of IL-6 binding and receptors in peripheral blood cells and macrophages.

Within 15 min, approximately 2.5% of 125I-labelled interleukin-6 (IL-6) injected intravenously into rats was taken up by the spleen. As determined by light microscopic autoradiography, uptake was mainly (60%) accounted for by macrophages in the red pulp. 125I-IL-6 binding in rat peritoneal macrophages was quantitatively similar to that in cultured human monocytes and T-cells. By comparison, IL-6 binding to polymorphonuclear granulocytes and freshly isolated monocytes was low. Stimulation with antigen, but not with mitogen (PWM), induced receptor presentation in B-cells, whereas antigen and mitogen downregulated the binding in T-cells. At 4 degrees C, labelled IL-6 bound to cells with a half-time of about 1.5 h. Binding appeared reversible, but dissociation was slow and incomplete. The apparent Kd for IL-6 binding was about 30 pmol l-1 in most cell types, however, values of approximately 120 pmol l-1 were obtained in polymorphonuclear granulocytes. At 37 degrees C, 125I-IL-6 was rapidly internalized by T-cells and monocyte-macrophages, and after a lag time, TCA-soluble radioactivity was released from the cells following a sigmoidal curve. Polyacrylamide gel electrophoresis of radiolabelled IL-6 cross-linked to its binding sites in T-cells, yielded receptor-ligand complexes with molar masses of 70-80 and 120-140 kg mol-1. This would agree with a dimeric conformation of the IL-6 receptor.

Animals↗

PubMed1989

Urokinase binds to a plasminogen activator inhibitor type-2-like molecule in placental microvillous membranes.

Placental microvillous membranes exhibited saturable binding of urokinase-type plasminogen activator with plateau achieved by 30 min at 4 degrees C and 10 min at 37 degrees C. The binding was essentially irreversible. The capacity was about 8 pmol urokinase per mg membrane protein. Half-maximal displacement of 125I-labelled urokinase was achieved with about 1.0 nM unlabelled urokinase when using 75 micrograms membrane protein/ml. 125I-labelled urokinase did not bind when treated with diisopropylfluorophosphate to block the catalytic activity. Single-chain urokinase (prourokinase), devoid of catalytic activity, did not bind. Catalytically active tissue-type plasminogen activator did compete with 125I-labelled urokinase for binding although less efficiently than urokinase. Binding activity remained in the 100,000 x g pellet after treatment of the membranes with 3 M KCl, alkaline stripping at pH 12 or extraction by the detergent Triton X-100. The binding was essentially blocked by antibodies against plasminogen activator inhibitor-type-2 (PAI-2). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized membranes with bound 125I-labelled urokinase showed that the urokinase-PAI-2 complexes largely migrated in fractions corresponding to a very large Mr although no clearly defined peaks were observed. It is suggested that PAI-2 occurs in a form anchored to syncytiotrophoblast microvilli, possibly to the cytoskeleton.

Chorionic Villi↗

PubMed1989

Bioactive human recombinant tumor necrosis factor-alpha: an unstable dimer?

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-alpha was approximately 35 kDa. When incubated at low concentrations (less than 1 nM) 125I-labeled TNF-alpha and unlabeled TNF-alpha rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-alpha demonstrated bands of multi- and trimeric TNF-alpha in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-alpha were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%-15% was obtained with cross-linked TNF-alpha dimers, whereas none was recovered from preparations of trimeric TNF-alpha. Multimeric and monomeric TNF-alpha exhibited little or no binding activity, and cell-bound, cross-linked TNF-alpha which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-alpha bound to lymphokine-activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 x 10(3). Cross-linking of TNF-alpha to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 degrees C, 125I-labeled TNF-alpha was rapidly internalized and degraded in L-929, U-937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF-alpha-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-alpha uptake, indicating that L-929 lysis was mediated by receptors.

Cell Survival↗