Inge Clemmensen

Attachment of staphylococci to silicone catheters in vitro

The adherence of radiolabeled staphylococci to silicone catheters was investigated in vitro. Staphylococcus aureus and Staphylococcus epidermidis strains bound to the same extent to the catheters. Also, S. epidermidis strains isolated from patients with plastic‐related infections showed binding similar to that of other S. epidermidis strains. By preincubation of catheters the influence of purified staphylococcal cell surface components on the binding was evaluated. The most potent inhibitors of the binding of S. aureus were the two surface proteins, clumping factor and protein A, and the cytoplasmic membrane. Surface proteins and the cell membrane of S. epidermidis also blocked the binding. Only protein‐containing surface proteins inhibited the binding. The production of slime correlated with the degree of S. epidermidis binding. Human plasma and serum, as well as purified albumin and IgG, inhibited the binding of both staphylococcal species. Fibrinogen, and to a certain extent fibronectin, inhibited the binding of S. epidermidis, while both these purified plasma proteins enhanced the binding of S. aureus.

Enzymic properties of the neo-plasmin-Val-442 (miniplasmin)

Abstract The enzymic properties of the activated plasminogen fragment (Val-442-Arg-560; S-S bridged to Val-561-Asn-790) also called miniplasmin or neo-plasmin-Val-442, were studied. This neo-plasmin was prepared by urokinase catalysed conversion of the corresponding fragment of plasminogen (Val-442-Asn-790) produced by specific limited proteolysis of native plasminogen by porcine pancreatic elastase and purified by chromatography on l -lysine-Sepharose 4B. The kinetic parameters of hydrolysis of a number of synthetic substrates by plasmin and by ‘miniplasmin’, respectively, were found to be alike. Furthermore the inhibition by 6-aminohexanoic acid and the pH-dependence of the hydrolysis of Bz-Arg-OEt were identical for the two enzymes. It is concluded that the catalytic site of ‘miniplasmin’ is very similar to that of plasmin. The interaction of ‘miniplasmin’ with α2-antiplasmin was studied in the presence and in the absence of 6-aminohexanoic acid. The reaction scheme which accounts satisfactorily for the reaction of plasmin with the inhibitor also fits the reaction of ‘miniplasmin’ with the inhibitor. However, it was found that ‘miniplasmin’ initially reacts with the inhibitor less readily than does plasmin, but that the rate of the second reaction step is equal to that of the corresponding step in the inhibitor-plasmin reaction. The hypothesis that site(s) other than the catalytic site of plasmin located at the NH2-terminal part of the heavy chain (residues 77–441) primarily determine the association rate of plasmin and α2-antiplasmin is supported.

Inhibition of urokinase by complex formation with human α1-antitrypsin

Human α1-antitrypsin was prepared from fresh human plasma by (NH4)2SO4-precipitation, gel filtration, affinity chromatography on concanavalin A, ion exchange chromatography and isotachophoresis. Human urokinase (EC 3.4.99.26) (plasminogen activator from urine) with Mr 46 000 and 36 000 was further purified from Urokinase Leo reagent preparation by gel filtration on Sephadex G-100 Superfine. The hydrolytic activity of urokinase on acetyl-glycyl-l-lysine methyl ester acetate (Ac-Gly-Lys-OMeAc) was inhibited in a strong timedependent manner by α1-antitrypsin. Complex formation between enzyme and inhibitor could be demonstrated in crossed immunoelectrophoresis against antiα1-antitrypsin and anti-urokinase serum as well as by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The latter method revealed the formation of 1:1 and 2:1 molar enzyme-inhibitor complexes.

Purification of a Plasminogen Activator Inhibitor Indistinguishable from α1-Antitrypsin and an Urokinase Inhibitor in Pregnancy Plasma

An inhibitor of urokinase present in increased concentrations in plasma during pregnancy was purified. The inhibitor was identified by its pronounced effect on urokinase-induced fibrinolysis as opposed to the absence of effect on fibrinolysis induced by a tissue plasminogen activator. The inhibitor progressively inactivated urokinase upon incubation and was found to be indistinguishable from alpha1-antitrypsin.

The Immunohistochemical Distribution of Laminin and Fibronectin in Human Female Breast Cancer

THE DEVELOPMENT OF METHODS for the immunohistochemical demonstration of laminin and fibronectin has resulted in an increased knowledge of tumor biology due to the prominent role of these noncollagenous glycoproteins in tissue organization and structure.1–6

Fibronectin-Binding Glycoprotein From Human Platelet Membranes

Fibronectin (cold-insoluble globulin) has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.