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K J Halbhuber

Publications and source records attributed to K J Halbhuber.

At least 73 records · Page 4Linked to original sources
PubMed1986

Light microscopical localization of acid and alkaline phosphatase activity by lanthanum-lead-(La-Pb)-methods.

This paper reports on the application of lanthanum cations instead of cerium ions as captures to detect acid and alkaline phosphatase activity in the rat kidney and small intestine. The basis of the proposed histochemical reactions is a three step procedure in analogy to the earlier published cerium-based techniques: the lanthanum phosphate was converted into lead phosphate and finally into the brown coloured lead sulfide [lanthanum-lead-(La-Pb)-reaction]. The results reveal that the La-Pb-reactions are more sensitive in comparison to the Ce-Pb-reactions. Methodological experiments involving lanthanide specific blocking reactions are discussed. Lanthanum cations are recommended as a useful tool in histochemical capture techniques of phosphatases.

Acid Phosphatase↗
PubMed1986

Light microscopical localization of enzymes by means of cerium-based methods. I.V. Optimization procedures for acid phosphatase.

The earlier described cerium based histochemical reaction for acid phosphatase [Ce-Pb-reaction, Zimmermann and Halbhuber (1985)] was optimized. The target tissues (kidney, intestine) were fixed by perfusion with glutaraldehyde in cacodylate or piperazine buffer in anesthetized animals. Postfixation of prefixed sections is not advantageous because of the detectable repressing of the enzyme activity. Moreover, the employment of unfixed cryostat sections, which were postfixed, was always connected with a complete abolition of the acid phosphatase activity. The optimal concentration of the primary capture cerium III chloride in the incubation medium is about 1 mmol. Lower concentrations lead to an incomplete histochemical detection of phosphatase activity in lysosomes. The treatment of cryostat sections of perfusion fixed tissue with borohydride (diminution of aldehyde induced cross links) or with dimethylsulfoxide (extraction of lysosomal materials or the well known vehicle property) brought about an improvement of the penetration capacity for cerium-III-cations into the target structures. After conversion of the cerium phosphate (primary specific reaction product) into cerium perhydroxide, oxalate or fluoride, the Ce-Pb-reaction was negative. Therefore, these blocking reactions represent specific inhibition controls, which indicates the formation and presence of cerium phosphate. On the basis of these reactions it is possible to check the specificity of the histochemical Ce-Pb-reaction for phosphatase activity in sections.

Acid Phosphatase↗
PubMed1986

Topo-optical investigations of the conformational change of the erythrocyte glycocalyx in dependence on extracellular pH and presence of dextran.

The erythrocyte (RBC) glycocalyx reacts very sensitively to environmental influences. But few is known about the effects of certain factors, which can change the membrane structure. Therefore we have tried to investigate in model experiments by means of the topo-optical toluidine blue reaction the conformational changes of the RBC glycocalyx in dependence on pH (range from 7.5 to 4.5) and presence of dextran. The findings, which are presented here, demonstrate a high sensitivity of the glycocalyx to pH influences. A distinct drop of the membrane anisotropy can be observed in the pH range from 6.5 to 4.5. Also dextran molecules evidently cause structural changes of the RBC glycocalyx via interaction with H-bonds. On the basis of a hypothetical structural model of the glycophorins outer segment the conformational effects are discussed.

Dextrans↗
PubMed1986

Cytochemical and cellbiological investigations of the signal function of the erythrocyte plasmalemma--the membrane structure as code for cell life span.

The erythrocyte membrane was characterized with regard to its alteration of different degrees. A very important process of the membrane alteration is the expression of IgG receptor sites, which is connected with the binding of autologous IgG at the membrane. Several detection methods for membrane bound IgG were described: immunocytochemical techniques (Anti IgG--Anti Ferritin-sandwich technique, Anti IgG-Protein A-Gold- and the Silver enhancement technique), Antiglobulin agglutination test (Coombs test) and erythrocyte-macrophage test. Moreover, the topo-optical toluidine blue reaction and the ultra-histochemical NAD(H) oxidase reaction were checked to characterize the erythrocyte-macrophage interaction. Finally, the microvesiculation is understood as special form of the erythrocyte membrane disintegration, which is connected with a remodelling of an intact plasmalemma. The findings presented here summarize the fact that the membrane associated IgG regulates an immune-signal for the elimination of the erythrocytes in the Reticulo-Histiocytic-System in vivo and in vitro. Therefore, the structural integrity of the erythrocyte plasmalemma is the code for the cell life span.

Animals↗
PubMed1986

Topo-optical investigation of the erythrocyte membrane alteration after prolonged retention in the splenal reticulum.

The extent of the splenal transit time is an important factor which influences the immunological behaviour of the red blood cells. An increased transit time of the RBC in the splenal reticulum leads to conformational effects on the erythrocyte glycocalyx. They consist in spatial disorder of the oligosaccharide side chains of the glycophorins with their sialic acid residues. These spatial disorders of the sialic acid residues correspond with the expression of IgG receptors, which initiate the process of the secondary elimination of the red cells.

Animals↗
PubMed1986

Immunocytochemical investigations of the membrane of experimentally altered and physiologically aged erythrocytes.

The expression of IgG receptor sites during aging of red blood cells plays an important role in the elimination process of these cells by the Reticulo Histiocytic System. By means of an indirect protein A gold-method, membrane bound IgG was detected immunocytochemically on pronase and neuraminidase treated red blood cells as well as on physiologically aged red cells. The silver enhancement of the gold particles led to an improved labeling of the bound IgG favouring its quantification at both electron and light microscopic level. The result of this gold-silver-technique was analyzed semiquantitatively at the light microscopic level by use of a Vickers scanning microdensitometer. It was shown that the aging of erythrocytes as well as the enzymatic alteration of the erythrocyte glycocalyx by pronase and neuraminidase are accompanied with an unmasking of IgG receptor sites followed by an IgG loading of the cells in presence of autologous serum or plasma. The experimental results, which are presented here indicate that a silver enhancement of gold particles is sufficient to signalize changes in membrane bound ligands at low concentrations.

Animals↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. II. A new cerium-lead-technique for alkaline phosphatase.

The use of cerium ions as a primary capture reagent in phosphatase histochemistry on the light and the electron microscopic level is a progress in the field of enzyme localization. Many influences of other captures (as of lead ions), e.g. enzyme inhibition, diffusion and other artefacts, are restricted when cerium-based methods are used. But the broader use of cerium is difficult, because cerium ions are at alkaline pH converted to the insoluble cerium hydroxide, which intensively precipitates in the incubation medium. This is an important disadvantage for a successful histochemical detection of alkaline phosphatase. The aim of this paper is to describe a new cerium-based method for the light microscopical detection of alkaline phosphatase, which is free from all these above mentioned problems. It is proposed a collidine buffer-sucrose containing medium, which holds cerium ions at pH = 9.0 in solution. The histochemical results of this method are excellent. The method is compared with a strontium based technique, the coupling azo dye technique for alkaline phosphatase as well as with in vitro and histochemical experiments with several chelator agents. The cerium-based collidine-sucrose technique is superior to all other procedures tested here and is recommended for a broader use.

Alkaline Phosphatase↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. I. Detection of acid phosphatase by a new cerium-lead-technique (Ce-Pb-method).

Cerium-III-ions are more and more used as capturing reagent and opaque marker for the electron microscopic localization of a number of H2O2-generating enzymes as well as phosphohydrolases. Contrary to its advantages over common lead methods in the histochemical detection of enzyme activities at the electron microscopic level, cerium-based methods proved to be a failure for light microscopic investigations. Therefore, our cerium-based method for the ultrahistochemical detection of acid phosphatase was developed for further observations at the light microscopic level. The principle of that new Ce-Pb-method is the conversion of light microscopic not visible cerium phosphate into lead phosphate by the secondary capture reagent alkaline lead citrate. Finally, the lead phosphate can be visualized as lead sulfide in the section. The Ce-Pb-method in its finally proposed manner was compared with a common lead method and showed a range of advantages. Because of that fact, the new Ce-Pb-method is recommended for a broader use in histochemistry, e.g. for the light microscopic enzyme investigation parallel to ultrahistochemical preparations.

Acid Phosphatase↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. III. Visualization techniques for cerium phosphate.

Cerium-based methods are more and more used for the electron microscopic localization of phosphohydrolases. By means of the earlier described Ce-Pb-technique, it is possible to localize these enzymes on the light microscopical level. The final product of this reaction is lead sulfide. In addition to this technique, other visualization methods for the light microscopically not visible cerium phosphate are proposed. 3 successful techniques are described in the report: The cerium perhydroxide reaction. By means of H2O2 cerium phosphate is converted into cerium perhydroxide which has an orange-yellowish colour. The manganese dioxide reaction with the conversion of cerium phosphate into cerium oxalate, which is able to reduce permanganate into the hardly soluble brown coloured manganese dioxide. A silver technique (Ce-Pb-AgS-method), which is characterized by the conversion of cerium phosphate into lead phosphate and in a second step to lead sulfide. At the active sites of the lead sulfide, the reduction of silver ions takes place. The reduced silver is converted in a final step into silver sulfide. The enzyme activity is represented by a brown or black coloured staining.

Acid Phosphatase↗
PubMed1985

Evidence for erythrocyte-microvesiculation in vivo.

During physiologic aging, erythrocytes (RBC) mainly loose membrane as shown by biochemical investigations. The most likely reason for a such loss would be the pinch off of RBC-microvesicles. The detection of RBC-microvesicles generated in vivo is rendered more difficult because of the relatively small amount of vesiculation processes under physiologic conditions accompanied by the fast phagocytosis of RBC-microvesicles in the reticulo-histiocytic system. The latter has been shown after the intravenous injection of heat-induced RBC-microvesicles. Investigations of spleens resulted in the case of PHZ-treatment, up till now in a clear demonstration of RBC-microvesicles. The result of microvesiculation in the PHZ-model led to the conclusion, that also rigid, naturally aged RBC could pinch off microvesicles in physiologic narrows of the blood flow like in the spleen.

Animals↗
PubMed1984

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes.

The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the glycophorin A.

Blood Preservation↗
PubMed1984

Activation of acetylcholine esterase (ACHE) as a sign for erythrocyte membrane alteration.

In vitro activity determination of membrane bound acetylcholine esterase (ACHE) of intact erythrocytes and differently altered RBC forms by means of the ELLMAN method yielded increase in activity in erythrocyte/vesicle mixtures and ghosts. Alteration of the cells by treatment with periodate, neuraminidase and trypsin leads to no, weak or clear decrease in the activity of this enzyme. Old cells also show a clear decrease in activity. When cells were treated with diamide ACHE is at first functionally inhibited, finally, however, it reaches supernormal values of activity. Possible causes for the different behaviour of ACHE in dependence on the degree of alteration of the erythrocytes are discussed. The results of the measurements were compared with the ultrahistochemical ACHE reactions according to KARNOVSKY (thiocholine--copper ferricyanide method) and IWAYAMA (lead thiocholine method). The influence of several components of the KARNOVSKY'S histochemical reaction mixture on ACHE activity was tested. The investigations suggest that disintegration of the erythrocyte membrane is accompanied with activation of the ACHE molecules which are nearly inactive in the intact membrane. This activation contributes to the positive histochemical ACHE test, which is an indicator reaction of membrane disintegration. Nearly intact membranes show a negative reaction. On the basis of these results we could show that physiological enucleation of normoblasts is connected with the formation of disintegrated membrane areas and their discharge from the future reticulocyte membrane.

Acetylcholinesterase↗
PubMed1984

IgG receptor expression during in vitro ageing of erythrocytes.

The IgG receptor expression on the membrane of the erythrocytes (RBC) during in vitro ageing was tested by means of a macrophage-RBC-endocytosis technique. The objects were: NaF-treated, glucose-free incubated and vesiculating RBC. The experiments show that unmasking of IgG receptor sites can be attributed to membrane conformational changes caused by ATP depletion of the cells. Vesiculation processes and (endogenous) enzymatically produced breakdown of glycoocalyx components are probably not related to increased IgG binding of aged RBC.

Adenosine Triphosphate↗
PubMed1984

[Possibilities for the use of cerium for the ultrahistochemical demonstration of enzymes].

The activity pattern of NAD(H)- or NADP(H)-oxidase was studied ultrahistochemically by means of the Cer-III-perhydroxide technique according in Briggs et al. (1979) in erythrophagocytosis. In contrast to many other findings in activated polymorph nucleated neutrophils the activity of this enzyme is restricted to rER and the intracellular vacuoles in peritoneal macrophages. Plasmalemma and the membranes of phagosomes are negative. The results allow the conclusion that oxidases do not directly participate in erythrocyte catabolism. Their role is still unclear. Additionally an ultrahistochemical procedure which used Cer-III-=ions as capture agent for detection of acid phosphatase is presented. The results are compared with the classical lead methods. Cer-IV-ions selectively enhance the electronmicroscopic contrast of biomembranes.

Acid Phosphatase↗
PubMed1984

The histochemical detection of acetylcholine esterase--an indicator of the disintegration of membranes.

The presence of ACHE in erythrocytes membrane in vitro but not detectable ultrahistochemically has been explored as a sign of membranes disintegration. The final results of the investigations have shown, that the increase in ACHE activity of the erythrocyte membrane is a qualitative sign and a semiquantitative measure of serious disturbances in the membrane structure.

Acetylcholinesterase↗
PubMed1983

Acceleration of coagulation by spectrin-free erythrocytic vesicles. Evidence for lipid flip-flop during vesicle formation.

Spectrin-free erythrocytic vesicles isolated from outdated liquid-preserved blood samples show a distinct increase of the clot-promoting activity compared with membrane phospholipid equivalents of intact erythrocytes. We suppose that, among other remodelling processes, local spectrin detachment from the inner membrane surface may trigger a flip-flop-mediated disturbance of the membrane lipid asymmetry. The interactions of spectrin with the cytoplasmic membrane surface seem to be essential for the structural membrane integrity including the lipid asymmetry.

Blood Coagulation↗