Search PubMed⌕ Search

Biomedical subjects

A K Raap

Publications and source records attributed to A K Raap.

154 records · Page 9Linked to original sources
PubMed1983

Studies on the phenazine methosulphate--tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. III. The role of superoxide in tetrazolium reduction.

A study was made of the involvement of superoxide anions in the aerobic reduction of tetrazolium salts by NAD(P)H and phenazine methosulphate (PMS). On the basis of experiments with superoxide dismutase two mechanisms of tetrazolium reduction could be distinguished--one in which fully reduced PMS (PMSH) is the reducer and one in which superoxide anion is the reducer of tetrazolium salts. It is proposed that superoxide anions is formed after a PMSH-PMS+ dismutation reaction. The relative contributions of the two distinct pathways to tetrazolium salt reduction are controlled by the PMS redox state and the oxygen tension. The consequences of the presence of superoxide anions and scavengers of superoxide anions for quantitative dehydrogenase cytochemistry are discussed.

Histocytochemistry↗
PubMed1983

Studies on the phenazine methosulphate-tetrazolium salt capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. I. Localization artefacts caused by the escape of reduced co-enzyme during cytochemical reactions for NAD(P)+-dependent dehydrogenases.

The correct localization of oxidative enzymes using cytochemical tetrazolium methods, in which low molecular weight electron carriers such as NAD(P)H and reduced phenazine methosulphate (PMSH) are used, can be endangered by the escape of the reduced intermediates before they react to form the insoluble formazan at the true enzyme-containing sites. To investigate this phenomenon, the glucose-6-phosphate dehydrogenase reaction was studied in fixed erythrocytes which, because of their microscopic dimensions, are well-suited for studying the loss of intermediates. A mixture of active and heat-inactivated fixed erythrocytes was incubated in a PMS-supplemented medium for glucose-6-phosphate dehydrogenase. The cytophotometric histograms showed that the final formazan precipitate was equally distributed over both active and inactivated cells. When bovine serum albumin was added to the medium, all the formazan was found to be bound to this protein and the erythrocytes remained essentially unstained. The false localization in this system could be explained by an unfavourable balance between the capture of electrons carried by NADPH within the erythrocyte and the diffusion of NADPH out of the erythrocyte. The rate constant of NADPH oxidation was determined, as was also the diffusion constant of NADPH in a protein matrix. Substituting the data obtained into formulae derived from the enzyme cytochemical localization theory of Holt & O'Sullivan (1958), it was calculated that the capture reaction was highly deficient and, theoretically, less than 1% of the total amount of formazan produced was localized within the erythrocyte which explains the false localization observed. The importance of these findings for the cytochemical demonstration of NAD(P)+-dependent dehydrogenases in cells and electropherograms is briefly discussed.

Coenzymes↗
PubMed1983

Studies on the phenazine methosulphate-tetrazolium capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. II. A novel hypothesis for the mode of action of PMS and a study of the properties of reduced PMS.

The results in the preceding paper have shown that the PMS-tetrazolium capture reaction as such is not sufficient to guarantee a correct localization of formazan in microscopically small dehydrogenase sites. For cytochemical reactions where the application of PMS leads to increased formazan formation, it is proposed that PMS functions not on its own, but as an efficient acceptor of NAD(P)H-oxidizing flavoproteins and thus increases the local NAD(P)H tetrazolium oxidoreductase activity. For the redox mediator vitamin K3 this type of mechanism could be proven with rat liver fractions. The relatively rapid NADPH oxidation precluded such simple experiments with PMS. An indication of such a stimulation by PMS was, however, obtained with soluble rat liver fraction. As escape of reducing equivalents from the site might also occur at the level of reduced PMS (PMSH) the solubility properties of PMSH were studied. It was found that PMSH has a low solubility in aqueous media and is hydrophobic. On basis of these findings a 'post-tetrazolium reduction' method seemed possible and could be experimentally confirmed.

Animals↗
PubMed1982

Histochemical model studies of enzyme activity after thermal damage.

The reliability of histochemical determinations of the enzyme activity after thermal damage has been studied with the aid of two model systems. Polyacrylamide films and erythrocyte ghosts containing either beta-glucuronidase or alkaline phosphatase, were submitted to heating and the activities retained were assessed both biochemically and histochemically. For the enzymes studied, the results show that tissue alterations induced by heat can influence histochemical reaction procedures, and that with these model systems, factors which are important for the histochemical quantitation of enzyme activities in thermally damaged tissues can be evaluated quantitatively. Potentialities of these model systems in the study of evaluating thermal damage through histochemical enzyme activity determinations, are discussed.

Acrylic Resins↗
PubMed1982

Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides.

The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged 'coating' on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei. The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilane-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased. Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium. In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.

Azure Stains↗
PubMed1981

Kinetics of cytochrome b reduction in submitochondrial particles.

(1) In agreement with Eisenbach and Gutman (Eisenbach, M. and Gutman, M. (1975) Eur. J. Biochem. 52, 107--116) the reduction of cytochrome b in beef-heart submitochondrial particles by succinate in the presence of antimycin was found to be biphasic, the relative amounts of fast and slow phases being dependent on the redox state of a compound located on the oxygen side of the antimycin block. (2) HQNO is a concentration sufficiently large to saturate the specific antimycin- and HQNO-binding sites can substitute for antimycin in these experiments. (3) The rate of the slow phase of the reduction of cytochrome b is decreased under anaerobic conditions and after pretreatment with 2,3-dimercaptopropanol (BAL). (4) In the presence of antimycin and cyanide, cytochrome b-562 is, to some extent, preferentially reduced in the rapid phase and b-566 in the slow phase. (5) The previously proposed regulatory effects of redox-sensitive components X and Y on the redox level and reduction kinetics, respectively, of cytochrome b are ascribed to the role of the Fe-S protein, when it is oxidized, in producing the reductant of cytochrome b by oxidation of QH2, and by the fact that when QH2 is bound to it, the reduced Fe-S protein cannot be oxidized by its natural oxidant, cytochrome c.

Animals↗
PubMed1981

Enzyme-incorporated erythrocyte ghosts: a new model system for quantitative enzyme cytochemistry.

The preparation and properties of a new microscopic model system for quantitative enzyme cytochemistry are described. The enzyme to be studied is entrapped in human erythrocyte ghosts by a simple hypotonic procedure. After fixation in suspension the ghosts can be analyzed both biochemically and cytochemically. The system has been tested with alkaline phosphatase. It is demonstrated that an azo method that uses naphthol AS-MX phosphate as substrate and 4-aminodiphenylamine diazonium salt as coupling agent can detect very low levels of enzymic activity. The biochemical activity determinations of alkaline phosphatase loaded erythrocyte ghosts were found to correlate linearly with cytophotometric activity determinations. The possible use of the erythrocyte ghost model system for other cytochemical applications is briefly discussed.

Alkaline Phosphatase↗
PubMedDate not supplied

Oestradiol, a new hapten for detecting nucleic acid sequences by FISH.

Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.

Animals↗