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K J Halbhuber

Publications and source records attributed to K J Halbhuber.

At least 19 recordsLinked to original sources
PubMed1992

Modified cerium-based and Gomori-based cerium methods for light microscopic phosphatase histochemistry: the cerium-perhydroxide-diaminobenzidine-nickel (Ce-H2O2-DAB-Ni and Ce/Ce-H2O2-DAB-Ni) two-step procedures.

Some modified cerium-based and Gomori-based cerium methods for the demonstration of phosphatase activity in cryostat sections were described. Dextrane as stabilizing agent was added to the incubation media for ATPase, 5'-Nase, and TPPase. The oxidation of the CeIII-phosphate primary reaction product in a separate step by H2O2 before the DAB incubation yielded an increase of the intensity of the DAB-based visualization reaction (Ce-H2O2-DAB-Ni two step method). The sensitivity of the histochemical enzyme reaction was remarkably increased if CeIII-ions were employed as amplifying agent (Ce/Ce-H2O2-DAB-Ni two-step method). A new suitable DAB medium consisting of 0.015% DAB, 2.0% Ni-sulphate, 15% methanol, and 0.005% H2O2 in 0.1 mol/l acetate buffer, pH = 5.2, was used. The disadvantage of diffuse background staining has been overcome by addition of 15% methanol to the DAB solution. Electrovalently bound CeIII (cerophilia) was removed by treatment of the incubated sections with CeIII-citrate (CeIII-complexation). In addition, a novel membrane floating incubation for sections is proposed. At present, the modified procedures are some of the most sensitive modes for the demonstration of phosphatases and improve the earlier described cerium-DAB one-step technique (Halbhuber et al. 1988b).

Animals↗
PubMed1992

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.

Alkaline Phosphatase↗
PubMed1991

Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: a photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy.

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.

Diamide↗
PubMed1991

The perinatal development of glucose-6-phosphatase activity distribution pattern in rat liver. A microdensitometrical study.

The ontogenetic development of the intralobular distribution pattern of glucose-6-phosphatase activity in the rat liver is described in terms of histochemical changes determined with microdensitometry. A newly developed cerium-lead technique was employed and compared with the common lead technique optimized by Teutsch (1978a). The cerium technique has advantages, meets the prerequisites for quantitative determinations and yields results comparable to biochemically obtained data from microdissected tissue. The first signs of a heterogeneous distribution pattern of glucose-6-phosphatase activity are observed on the 3rd d after birth, and differences between periportal and centrolobular areas are largest around 10th and 15th d. At 30th d after birth, the adult pattern is complete with a centrolobular glucose-6-phosphatase activity of 67% of the periportal value.

Aging↗
PubMed1991

Improved light microscopic demonstration of D-amino acid oxidase activity in cryotome sections using cerium ions as capturing and amplifying agent--the Ce/Ce-H2O2-DAB procedure.

The light microscopical demonstration of D-amino acid oxidase (AAOX) activity with cerium (Ce III) as the capturing agent was improved. The incubation medium was stabilized by the employment of triethanolamine and detrane complexed cerium. A considerable increase in intensity of the reaction was accomplished by treatment of the AAOX-incubated sections with Ce III which reacted with the primary reaction product Ce IV-perhydroxide to form Ce IV-hydroxide. In this way the primary reaction product was reduced and enlarged concomitantly. The Ce IV-hydroxide was converted into Ce IV-perhydroxide by H2O2, which was visualized by blue-black stained Ni-DAB complexes. Thus, Ce III is used as capturing agent as well as amplifier (Ce/Ce-H2O2-DAB method). The primary reaction product Ce III-phosphate formed by coreacting phosphatases was selectively extracted by citrate containing glycine-NaOH buffer while Ce IV-perhydroxide remained in the sections. In model experiments it was proven that the perhydroxide groups in the Ce IV-perhydroxide compound initiate predominantly the DAB polymerization while the contribution of Ce III and Ce IV is small.

3,3'-Diaminobenzidine↗
PubMed1991

A new visualization principle of cerium phosphate reaction product of phosphatases in cryotome sections for light microscopy--the cerium-oxalate-osmium-silver (Ce-Ox-Os-Ag) method.

A new visualization principle for the detection of cerium phosphate reaction product of phosphatases in light microscopy is described. The new mode is based on the conversion of cerium phosphate into cerium oxalate. The latter is able to react with OsO4. In this way osmium black is formed, staining enzymatic activity sites grey or greyish-black. Utilizing the argyrophilia of osmium black, the staining contrast could be remarkably intensified by posttreatment with Ag-proteinate (Ce-Ox-Os-Ag procedure). This procedure is of similar sensitiveness in comparison to the DAB-Ni method, proposed earlier. The advantages are a very pale background staining and a complete suppression of the cerophilia. Moreover, it substitutes to DAB, a probable potent carcinogen. The method is time-saving when the processing of the reactions was stimulated in a microwave oven.

Animals↗
PubMed1991

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

3,3'-Diaminobenzidine↗
PubMed1990

The binding of a polyclonal antibody against human band 3 to in vitro aged erythrocytes.

In vitro aged human erythrocytes were checked for their anti-band-3-antibody loading by means of a protein A-gold technique. The in vitro-ageing procedures are introduced as an alternative to the questionable preparation of in vivo aged cells. They include the influences of NaF in various concentrations, of heat (49 degrees C) and urea. After the treatment with 20 mmol/l NaF and more, the IgG loading increases significantly. In contrast to that, however, exovesiculation induced by heat and urea lower the IgG loading. Possible mechanisms for the growing accessibility of the epitopes concerned are discussed in connection with changes occurring during the physiological ageing processes.

Anion Exchange Protein 1, Erythrocyte↗
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↗
PubMed1979

Rat endo- and mesothelium lack Fc receptors.

The present study is concerned with the interaction of rat endo- and mesothelium with homologous erythrocytes under various conditions of pretreatment and incubation. Its findings show rat endo- and mesothelial cells a) nonadhesive to native or pretreated erythrocytes irrespective of the presence of gamma-globulin in the medium, b) devoid of primary or cryptic receptors sensitive to the Fc segment of the IgG molecule, and c) provided with binding sites at the oxidized glycocalyx which together with receptor groups of modified erythrocytes share the same class of IgG.

Agglutination↗
PubMed1979

[Electron microscopic autoradiography on semithin sections (author's transl)].

The present study applies semithin sections for electron microscopic autoradiography. Due to (a) the decrease of exposition time, and (b) lower intensity of isotope labelling, low power electron microscopic autoradiography on semithin sections is favoured in comparison with conventional (ultrathin section high power) electron microscopic autoradiography, and due to (c) more precise localization of silver granules, and (d) ease of quantitative evaluation of autoradiography, it is superior to ordinary (thick section) light microscopic autoradiography.

Animals↗
PubMed1979

[Sialic acid content and IgG binding of the glycocalyx of preserved erythrocytes].

Fractions of light and heavy erythrocytes were separated by centrifugation from blood samples banked in ACD-AG medium at 4 degrees C for periods up to 6 weeks. Both light and heavy erythrocytes have lost about 4,9% of their content of sialic acids during banking for 6 weeks. This reduction is in accord with a 6%-decrease of their agglutination by means of alcian blue. It is, however, a variance with the inhibition of agglutination by anti-IgG sera. The present findings provide evidence for the role alterations of the red cell membrane may play in the process of recognition and phagocytosis of banked erythrocytes. With regard to these alterations we suggest two types of rapid elimination of transfused banked erythrocytes: a) Primary elimination refers to cells primarily loaded with immunoglobulins such as to get recognized and phagocytized by macrophages. b) Secondary elimination accounts for rigid erythrocytes suffering from additional degradation while retained in the spleen prior to their loading with immunoglobulins and ensuing phagocytosis. Secondary elimination is considered a process more relevant to reutilisation of banked blood.

Binding Sites, Antibody↗
PubMed1978

[Accumulation of phosphate ions in biological materials, phosphate ion diffusion and electron microscopical investigations of phosphohydrolases (author's transl)].

Isolated unfixed nuclei of mouse liver accumulate different amounts of phosphate ions dependent on pH, temperature and concentration of phosphate ions. At 37 degrees C and pH = 7.2, 8.5 X 10(9) binding places and a stability constant of 2.0 +/- 0,5 . 10(4) l/mol were calculated, the data at 30 degrees C and pH = 6.5 are 4 X 10(9) Nucleus-1 and 6 X 10(3) l/mol. Nuclei fixed by formaldehyde-ethanol or glutaraldehyde do not accumulated phosphate ions. Under conditions of nearly undisturbed diffusion phosphate ions taken up by the nuclei and precipitated by the help of lead ions are detected electron microscopically in the nuclear envelope, preferently in intracisternal space. Nucleoprotein structures do not show enrichments of crystals. These structures are also not stained after precipitation of phosphate ions under conditions of diminuation of diffusion. A possible mode of phosphate binding in cell nuclei and the influence of adsorbed phosphate ions on localization of enzymatic activities are discussed.

Animals↗
PubMed1978

[Environmentally dependent erythrocyte membrane permeability as a source of error in the determination of hematocrit].

The volume of human red blood cells (RBC) was evaluated by means of the centrifugation method (hematocrit) and 131-J-labelled human serum albumin, respectively. Both of the methods yielded an identical volume of about 107 micron3 of the single RBC, provided the evaluation was performed in autologous plasma. Contrary to the 131-J-albumin method the results of which were found independent of various pretreatments of RBC, the centrifugation hematocrits of RBC previously washed with PBS and resuspended in PBS or saline protein media resulted in a mean cell volume of about 86 micron3. The decrease of the cell volume was associated with an efflux of K+ ions. If the RBC are centrifuged at 800 g instead of 15000 g, their volume will remain unchanged. The assessment of cytodeformability has shown, that RBC in PBS by loss of cell volume could enter a 2.3 micron micropipette completely. RBC in plasma, though traversing a 2.9 micron micropipette were incapable of entering a 2.3 micron channel completely. With pressures ranging from 300 to 350 mm H2O the processes of these cells undergo microspherulation.

Cell Membrane Permeability↗
PubMed1978

Glutaraldehyde mediated echinocyte/discocyte transformation is Ca2+ dependent.

Echinocytes, which were produced from freshly banked blood by repeated washes in phosphate buffered saline, undergo a transformation to the discoid shape within less than 30 seconds of incubation in isotonic 0.05% glutaraldehyde pH 7.4. This echinocyte/discocyte transformation is not associated with a change of cell volume or critical hemolysis volume although a slight decrease of cellular deformability and a 4-8 fold increase of K+ efflux within 1 hour after glutaraldehyde incubation provide evidence of the fixative's attack on the cell membrane. Trypsination prior to the incubation in isotonic glutaraldehyde could not inhibit the shape change. Hypertonic glutaraldehyde solutions partially prevent the E/D transformation with regard to both the osmolarity of the medium and the permeability of the cell membrane. The glutaraldehyde stimulated transformation is entirely inhibited in the presence of a chelating agent the efficiency of which is overcome by addition of a more-than-equivalent amount of Ca2+. The mutual action of either agent is discussed, however, the mechanism of the phenomenon remains unclear.

Aldehydes↗
PubMed1977

Reversible conformational changes of plasmalemmal glycoproteins.

Repeated incubations of human red blood cells in low ionic isotonic sucrose result in an instantaneous agglutination. In the same medium which had caused the agglutination, erythrocytes completely disagglutinate within 60 to 90 min. Disagglutination is accompanied by the efflux of cellular ions, which causes a 500-fold increase of extracellular K+. Decomposition of agglutinates occurs at once upon addition to the medium of about 3 mM KCL. It will be inhibited for hours, if the medium is renewed twice an hour. Erythrocytes washed successively with phosphate buffered saline and isotonic sucrose are devoid of adhering blood plasma proteins. If these cells were fixed with glutaraldehyde in isotonic sucrose they had lost a) their anisotropic staining with toluidine blue, and b) most of their colloidal iron binding capacity. The staining with ruthenium red and the electrophoretic velocity of these erythrocytes apparently were identical with the controls. The findings are considered evidence of the reversible unfolding of glycocalyx glycoproteins in the low ionic medium.

Animals↗