Werner Linss

Hemolysis of human erythrocytes with saponin affects the membrane structure

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.

Magnetic nanoparticles for selective heating of magnetically labelled cells in culture: preliminary investigation

The minimally invasive elimination of tumours using heating as a therapeutic agent is an emerging technology in medical applications. Particularly, the intratumoural application of magnetic nanoparticles as potential heating sources when exposed to an alternating magnetic field has been demonstrated. The present work deals with the estimation of the basic relationships when the magnetic material has access and binds to structures on cell membranes of target cells at the tumour region, particularly as a consequence of administration through tumour supplying vessels. Therefore, using mouse endothelial cells in culture, the binding of dextran coated magnetic nanoparticles (mean hydrodynamic particle diameter 65 nm) was modelled using the periodate method. The efficacy of cell labelling was demonstrated by magnetorelaxometry (MRX)—a selective method for the detection of only those magnetic nanoparticles that were immobilized—as well as by electron microscopy and iron staining. The amount of iron immobilized on cells was found to be 153 ± 56 µg Fe per 1 × 107 cells as determined by atomic absorption spectrometry. Moreover, after exposure of those 1 × 107 labelled cells to an alternating magnetic field (frequency 410 kHz, amplitude 11 kA m−1) for 5 min, temperature increases of 2 °C were achieved. The consequences of particle immobilization are reflected by the results of the measurements related to the specific heating power (SHP) of the magnetic material. Basically, the heating potential is explained by the superposition of Brown and Neel relaxation while for immobilized nanoparticles the Brown contribution is absent. In the long term the data could open the door to targeted magnetic heating after further optimization of the heating potential of magnetic material as well as after functionalization with biomolecules which recognize specific structures on the surface of cells at the target region.

Flow Cytometry as a Diagnostic Tool for Hereditary Spherocytosis

Flow cytometric analysis of eosin-5′-maleimide-labeled red blood cells has been proposed as a new method of identifying hereditary spherocytosis (HS). The aim of the present study was to analyze sensitivity and specificity of this method. Red blood cells from patients with HS (n = 58) revealed significantly lower mean channel fluorescence values than red blood cells from normal subjects (n = 110), unaffected HS family members (n = 8), and patients with other anemias (n = 44). Taking a mean channel fluorescence of 400.0 units as the threshold value identified by logistic regression, sensitivity and specificity of the test for HS were 96.6 and 99.1%, respectively. Flow cytometric analysis is a valuable screening test for the diagnosis of HS.

Flow cytometric analysis of band 3 protein of human erythrocytes

Flow cytometry was used to quantify the transmembrane anion exchanger (band 3 protein) of human erythrocytes by covalently bound eosin-5-maleimide. In vitro and in vivo vesiculated red blood cells were investigated. The fluorescence and light scatter signals of cells after heat induced vesiculation, in vivo ageing, and in patients with hereditary spherocytosis were decreased. These results reflect a deficiency of band 3 protein which is presumably caused by membrane surface area loss. It was possible to distinguish control erythrocytes, erythrocytes from patients with hereditary spherocytosis, and from other forms of haemolytic anaemias on the basis of their light scatter and fluorescence signals characteristics.

Problematik des Aufbaus einer Datenbank dreidimensional erfasster menschlicher Schädel als Voraussetzung für eine präoperative Anfertigung passgenauer Implantate zum Ausgleich von Knochendefekten

Zusammenfassung Mit einem Laserscanner wurden menschliche Schadel vermessen. Um die Daten fur eine dreidimensionale Rekonstruktion im gleichen Koordinatensystem vergleichbar abzulegen, wurden markante Schadelmesspunkte festgelegt und erprobt. Als besonders geeignet erwiesen sich fur die Ausrichtung in der Medianebene das Rhinion, das Nasion, die Spina nasalis anterior, das Prosthion und das Opisthokranion. Die Frontalebene wurde durch das Rhinion und die Spina nasalis anterior festgelegt. Fur die Einstellung in die Horizontalebene wurde zuruckgegriffen auf die Mastoidealia, die Zygomaxillaria und die Orbitalia. Anhand von zwei klinischen Beispielen wird gezeigt, dass die Datenbank als Grundlage fur den Vergleich von Schadeln geeignet ist und zum Auffinden von Ahnlichkeiten genutzt werden kann. Daraus leitet sich die Moglichkeit zur passgenauen Anfertigung von Implantaten zur Deckung groser seitenubergreifender Knochendefekte ab.