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K J Halbhuber

Publications and source records attributed to K J Halbhuber.

At least 55 records · Page 3Linked to original sources
PubMed1990

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking.

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.

Diamide↗
PubMed1990

[Does induced Heinz body formation lead to the liberation of IgG receptors on the erythrocyte membrane?].

Immunofluorescence microscopical and biochemical studies led other authors to the conclusion that the formation of membrane-bound Heinz bodies at these parts of the plasmalemma of erythrocytes leads to clustering of band 3-protein and increased binding of IgG. We failed to detect immunocytochemically an increased IgG binding over phenylhydrazine induced membrane-bound Heinz bodies in otherwise intact erythrocytes using transmission electronmicroscopy and we also did not notice any clustering of intramembraneous particles over the numerous Heinz bodies by means of freeze etching. Especially at higher phenylhydrazine concentrations the erythrocytes show formation of vesicles and hemolysis accompanied by increased IgG binding and clustering of the intramembraneous particles. Photographs of such cells obtained by immunofluorescence microscopy are very similar to the pictures known from the literature.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1989

Topo-optical studies of gradually disintegrated erythrocyte membrane derivates: different kinds of ghosts.

Several kinds of ghosts from human erythrocytes (blood-group A1D) were investigated by the topo-optical Toluidine-Blue (TB) reaction. In comparison to intact cells, all ghosts demonstrated a decreased TB-anisotropy. These results reflect an altered glycocalyx structure of ghosts, especially conformational changes of the TB-binding N-terminal extracellular segments of the glycophorines. It was assumed that this structural glycocalyx alteration was caused by substantial losses of membrane skeleton components during the ghost preparation. Moreover, disturbed molecular interactions between the membrane skeleton and the glycocalyx may contribute to this effect. Therefore, the glycocalyx and the membrane structure of ghosts in general are significantly different from the membrane of the intact erythrocyte. The experiments show that the effects of the reconstitution procedures for ghost membranes are restricted to a reconstitution of a defined membrane function (i.e. dynamic barrier for monovalent cations) in a widely disturbed membrane structure.

Acetylcholinesterase↗
PubMed1989

[The elimination mechanism of erythrocytes in vivo].

The erythrocytes exhibit an unusual cyto-architecture. Because the cytosol is without organelles, the erythrocytes are cells, which have got only one single organelle, i.e. the plasmalemma. It reacts extremely sensitively to environmental factors with changes of the membrane structures, especially with an expression of IgG-and lectin receptor sites. This takes place during in vivo ageing, after attack of hydrolytic enzymes and after alteration of the membrane skeleton. The membrane bound IgG mediates the endocytosis of the macrophages (primary elimination). The role of the lectin receptors is unclear. The cell trapping (secondary elimination) is restricted to cells with extremely decreased deformability.

Antigens, Differentiation↗
PubMed1988

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes.

New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5-60 min at 37 degrees C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2-9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1988

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry.

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

3,3'-Diaminobenzidine↗
PubMed1988

New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity.

New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.

Acid Phosphatase↗
PubMed1987

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran.

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.

Dextrans↗
PubMed1987

Gadolinium and didymium (praseodymium/neodymium) cations as capture agents in lightmicroscopical histochemistry of acid and alkaline phosphatase.

In previous papers, cerium and lanthanum based methods for light-microscopical detection of acid and alkaline phosphatase activity were proposed. In this paper, the usefulness of other lanthanide cations such as gadolinium and praseodymium/neodymium cations as capture agents in phosphatase histochemistry is tested. It is evident that phosphate ions were sufficiently trapped by these cations. According to the lead and silver multistep procedures earlier described it is possible to visualize alkaline phosphatase activity in the brush borders of the intestine or kidney as well as acid phosphatase activity in the lysosomes. These methods can be recommended.

Acid Phosphatase↗
PubMed1987

Light microscopical localization of enzymes by means of cerium-based methods. V. Optimization of the cerium-lead (Ce-Pb)-technique for alkaline phosphatase.

A modification of the earlier published cerium-based technique for histochemical detection of alkaline phosphatase activity at light microscopical level (Halbhuber and Zimmermann 1985) is described. The reduction of the s-collidine concentration from 200 mmol to 50 mmol, increase of cerium ion concentration rom 1 mmol to 5 or 10 mmol, and sucrose concentration from 7.5% to 15% at increased from pH = 9.0 to 9.5 less than or equal to 9.9 in the incubation medium led to a high intensification of the histochemical reaction. The brush borders of the rat kidney (especially of the epithelial cells of the primary convoluted tubules) and of the enterocytes demonstrate black-brown tinged and precisely localized final reaction products. Moreover, a simplification of the histochemical procedure by employment of postfixed cryostat sections (small intestine) instead of the time consuming perfusion fixed material (kidney) is presented. Several fixatives were also tested. Nakane's periodate-lysine-paraformaldehyde (PLP) or the periodate-lysine-glutaraldehyde (GLP) fixations are superior to the classical glutaraldehyde/paraformaldehyde double fixation. The proposed optimized cerium-based techniques are recommended for a broad use.

Alkaline Phosphatase↗
PubMed1987

Preservation of red cell concentrates. Dependence on storage time of IgG binding, osmotic fragility, MCV, and surface area index.

In a comparative study of red cells from concentrates preserved in SAG medium without and with 30 mM sucrose, mannitol or sorbitol, resp., we determined the variation of IgG binding, osmotic fragility, MCV and the surface area index with storage time. IgG binding gave no conclusive results. Osmotic fragility turned out to be increased in simple SAG in comparison to the sugar-supplemented media. From measurements of microhematocrit and pH, the mean cellular volume (MCV), standardized to the initial pH value, turned out to decrease in all the media tested by not more than about 5 per cent after 3 weeks, and 10 per cent after 6 weeks. This is in advantageous contrast to the strong decrease in microhematocrit formerly observed in red cell concentrates in CDS-AG medium. Cells resuspended in simple SAG medium exhibited the smallest decrease in MCV. However, as inferred from data on hemolysis and vesiculation (D. Stibenz, accompanying paper), in these cells the loss of surface area proved to be maximal.

Blood Preservation↗
PubMed1987

Detection of IgG bound at the erythrocyte membrane by means of an immunohistochemical gold-silver technique.

The binding of IgG at the erythrocyte membrane during aging plays an important role in the elimination process of the cells by the reticulohistiocytic system. By means of an indirect protein A gold- and protein A gold-silver-method bound IgG was detected immunocytochemically on physiologically aged, pronase and neuraminidase treated red blood cells (RBC). At the light and electron microscopic level an increased binding of IgG at "old" as well as enzymatic treated cells in comparison to "young" and normal RBC was noted. The silver enhancement of gold particles visualized the immunostaining and permitted the semiquantitative analysis of the RBC by a scanning microdensitometer.

Erythrocyte Aging↗
PubMed1987

Morphometrical studies of erythrophagocytosis in the spleen.

The elimination of altered RBC was investigated morphometrically in the rat spleen by determination of the percentage of erythrophagosomes in macrophages. PHZ and diamide treated RBC led to a time dependent increase of the percentage of erythrophagosomes in comparison to normal spleens. The deformability of the target RBC was lowered as revealed by measurements with glass micropipettes. RBC-microvesicles and diamide treated RBC are primarily loaded with IgG and undergo a rapid elimination. PHZ-RBC and cells with a prolonged stay in the spleen are sequestrated by secondary mechanisms. Details of primary and secondary elimination are discussed.

Animals↗
PubMed1987

Membrane skeleton alteration--a factor promoting IgG receptor expression in the erythrocyte membrane.

IgG receptor expression after selective cross-linking of spectrin by means of diamide was investigated. A diamide concentration dependent IgG loading of erythrocytes was observed. Furthermore, diamide causes disturbance of lipid asymmetry, decrease of the anisotropy after topooptical staining of the glycocalyx, increase of the phagocytosis index and aggregation of the IMP's. Our findings support the hypothesis that the arrangement of the membrane skeleton at the inner aspect of the membrane is decisive not only for the lipid asymmetry but also for the spatial structure of the glycocalyx at the outer aspect of the membrane and thus for the degree of exposure and arrangement of IgG-receptors, which are thought to be localized at an extracellular portion of band 3 protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Red blood cell aging--membrane skeleton alteration and IgG receptor expression.

Investigations were performed on aging of erythrocytes. It has been assumed that structural changes of the membrane result after exposer of the cells to certain environmental influences in vivo or in vitro. Cell aging can be connected with varying combinations of membrane structure disturbances. It is postulated that the messenger which signals membrane structure lesion is involved in a mechanism given by the expression of immunoglobulin G (IgG) receptor sites which bind autologous IgG1 and IgG3. This antibodies are cytophilic for macrophages. The performed studies demonstrated that an intact molecular arrangement of the membrane skeleton is not only a supposition for stabilization of the membrane asymmetry but also for IgG receptor masking to prevent an early elimination of the red blood cells from the organism.

Erythrocyte Aging↗
PubMed1986

The rate of lateral diffusion of phospholipids in erythrocyte microvesicles.

31P-NMR spectra of phospholipids in membranes of erythrocyte microvesicles isolated from outdated blood units were recorded in the temperature range 5 to 55 degrees C. Within that range the lineshape is strongly influenced by an increasing rate of lateral diffusion of phospholipids. At 36 degrees C a diffusion constant, D, of (2 +/- 1) X 10(-12) m2/s was obtained. The diffusion rate is by a factor of 3 to 10 greater than in erythrocyte membranes measured by the photobleaching technique and is comparable with values obtained for several lipid model membranes. The differences in lateral diffusion rates are probably connected with the depletion of microvesicle membranes in membrane proteins.

Blood Viscosity↗