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Biomedical subjects

K Hannig

Publications and source records attributed to K Hannig.

At least 19 recordsLinked to original sources
PubMed1992

Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis.

Commercial alginates consisting of variable homopolymeric regions of beta-D-mannuronic acid and alpha-L-guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross-linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10-20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross-linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.

Alginates↗
PubMed1990

Free-flow electrophoresis under microgravity: evidence for enhanced resolution of cell separation.

A mixture of fixed rabbit, guinea pig and rat erythrocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g- and microgram- conditions using a specially designed electrophoretic module. Short duration microgram conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g-conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g-conditions, clear separation of the cell mixture could be recorded after lift-off of the rocket under microgram-conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire microgram-phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under microgram-conditions in media of high ionic strength.

Animals↗
PubMed1990

Electrofused mammalian cells analyzed by free-flow electrophoresis.

Somatic cell fusion is a powerful and widely used technique. In recent years, electrofusion has become increasingly popular because it is a gentle process that can be optically controlled and carefully monitored using appropriate fusion chambers and because it permits the efficient fusion of smaller cell numbers. However, damage of the cell membrane and cell lysis occurs during application of the electrical field and is accompanied by changes in surface charge which can be detected by free-flow electrophoresis. In this study, we evaluated free-flow electrophoresis to detect changes in cell viability after application of electric-field conditions employed in mammalian cell electrofusion and to separate dead cells and cell debris from intact unfused or fused cells.

Animals↗
PubMed1988

Countercurrent centrifugal elutriation in a table-top centrifuge.

An elutriation system has been developed which fits into a small table-top centrifuge. The new CS1 rotor functioned as accurately as the JE-6 rotor, commercially available from Beckman Instruments. However, fewer cells were required for the separation experiments. Leukocytes obtained from 25 ml whole blood by Ficoll-Hypaque gradient centrifugation were sufficient for separating lymphocytes from monocytes and for purifying granulocytes. Furthermore, when lymphocytes were harvested from cell cultures 40 ml of a suspension of 1.2 x 10(6) cells/ml were enough to yield an enriched preparation of antibody producing cells.

Antibody-Producing Cells↗
PubMed1988

The negative surface charge density is a maturation marker of human B lymphocytes.

Small resting B lymphocytes were highly enriched and completely depleted of all preactivated large B lymphocytes using countercurrent centrifugal elutriation and free flow electrophoresis. They required T lymphocytes, monocytes, and a mitogen to produce antibodies after 5 days of preincubation. Large activated B lymphocytes were obtained in cell fractions which were free of resting ones. They produced antibodies even in the absence of a mitogen. Two groups were distinguished, differing in their stage of differentiation and their negative surface charge density. The cells of one group had an electrophoretic mobility (EM) like resting B lymphocytes ranging from 0.85 to 0.99 X 10(-4) (cm2 V-1 s-1). They took 2 to 3 days of preincubation before they started to secrete antibodies. Interleukin 2 and pokeweed mitogen enhanced their antibody production capability. The cells of the other group had an EM between 0.99 and 1.13 X 10(-4) (cm2 V-1 s-1). They secreted antibodies even during the first day of incubation. The quantity of the antibodies which they produced depended only on the blood donor. It could not be influenced by a mitogen or by interleukin 2. The study shows that large B lymphocytes with high negative surface charge density are in a later maturation stage than those with lower negative surface charge density.

Antibody-Producing Cells↗
PubMed1988

Cell electrophoresis of group B streptococci: separation of types Ia, Ib/c, II, III and IV before and after neuraminidase treatment.

Group B streptococcal strains of types Ia, Ib/c, II, III and IV could be characterized by distinct electrophoretic mobilities before and after neuraminidase treatment. By this method of cell electrophoresis in the type I a strain, two subpopulations could be detected; whereas in all strains the electrophoretic mobility is markedly reduced after enzymatic removal of neuraminic acid, type II remained unaffected. The method of "bacteriopheresis" offers a new approach to classification of bacteria with respect to the primary and secondary surface structures.

Electrophoresis↗
PubMed1988

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis.

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.

Animals↗
PubMed1986

Studies on the activation mechanism of human mononuclear leukocytes isolated by physical methods.

Responder lymphocytes, accessory lymphocytes and monocytes were isolated using countercurrent centrifugal elutriation and free flow electrophoresis. The Concanavalin A (Con A) binding behaviour of the isolated cell populations was studied. Responder lymphocytes and accessory cells bound Con A by different mechanisms. Cells in all the isolated populations were able to interact directly with Con A. Con A binding to responder lymphocytes was inhibited by alpha-methylmannoside (alpha MM) and by a distinct plasma protein. Accessory lymphocytes and monocytes bound Con A even in the presence of 10% human plasma or 50 mM alpha MM. The plasma protein which inhibited interaction of responder lymphocytes with Con A also reduced lymphocyte proliferation, when resting monocytes were used as accessory cells. If, however, monocytes were used after activation by lipopolysaccharide no inhibition effect was observed. From the results we conclude that there is a distinct plasma protein which protects the organism against lymphocyte stimulation not controlled by accessory cells. This inhibitory plasma protein appears to be a euglobulin.

Blood Proteins↗
PubMed1982

Antigen-specific electrophoretic cell separation (ASECS): isolation by human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies.

The electrophoretic mobility of human lymphocytes can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders subpopulations of human peripheral blood lymphocytes accessible to separation by free-flow electrophoresis. After reaction of lymphocyte preparations with anti-IgM antibody and a fluorescent second antibody, B lymphocytes showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell subpopulation reactive with the monoclonal antibody T811 could be isolated, even though only small amounts of this antibody were bound, by using a double-sandwich method. Non-specific antibody uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow cytometric analysis showed that cells were separated according to their antigen density. Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,000 cells/sec this method has a much higher separation capacity than fluorescence-activated cell sorting. The described method should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.

Animals↗
PubMed1981

Lymphocyte surface proteins recognized by an anti-thymocyte-globulin.

Rat lymphocyte surface proteins were labelled by lactoperoxidase-catalysed iodination and immunoprecipitated by an anti-thymocyte globulin (ATG). ATG-Reactive proteins from cortical and medullary thymocytes, peripheral T cells and B cells were examined by gel electrophoresis. Several surface molecules were identified which showed differences in their distribution in the four cell populations. A 105 000-Da component was found in relatively high density on thymocytes and peripheral T cells only. Two high-molecular mass components of 205 000 Da and 190 000 Da were observed which were highly specific for peripheral T cells. These molecules appeared to share antigenic determinants with two other proteins found on the surface of both peripheral T cells and thymocytes. Limited proteolysis with Staphylococcus aureus protease showed these four proteins to have a similar peptide composition indicating the existence of a family of related surface molecules with varying expression on different cells. One of these T cell-specific proteins (P 190) was present at a comparatively high density, making up about 4% of the membrane protein. The main protein recognised by ATG was a component with a molecular mass of 175 000 Da found on all lymphocytes which made up about 7% of membrane protein. The other ATG-reactive proteins were all minor components of the cell surface, constituting less than 1% of membrane protein.

Animals↗
PubMed1980

Radio-iodinated surface proteins of electrophoretically separated rat lymphocytes.

Rat thymocytes and lymph node cells were separated into three T and one B subpopulation by means of free flow electrophoresis. The surface proteins of the separated cells were labeled by lactoperoxidase catalysed radioiodination. Most of the label was demonstrated to be at the cell surface. The labelled cells were either lysed in sodium dodecyl sulphate or treated with Nonidet P-40 to extract cell proteins. The radioiodinated proteins were analysed on sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by autoradiography. In contrast to sodium dodecyl sulphate Nonidet P-40 did not extract cell proteins completely. Furthermore the degree of extraction varied considerably in the different cell populations. 58% of protein bound radioactivity was extracted from thymocytes, 67% from peripheral T cells and 81% from B cells. Gel electrophoresis revealed that four proteins were not extracted at all, whereas five components were better soluble in Nonidet P-40 than all other proteins. One protein was extracted from B cells only although it was present in all cells. Although the surface protein patterns of the four lymphocyte subpopulations were rather similar, distinctive differences could be found. B cells had six labelled proteins which seemed to be absent in the other cells, In the T cell group, three protein bands were identified, each with specificity for peripheral T cells, thymocytes and all T cells respectively. Four other proteins were found which showed quantitative differences between the four cell groups.

Animals↗
PubMed1979

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.

Animals↗