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R Gossrau

Publications and source records attributed to R Gossrau.

At least 55 records · Page 3Linked to original sources
PubMed1993

Enzyme histochemistry of the regressing rat decidua and metrial gland.

In order to understand more about participation of the basal placental zones in processes of regression and degradation as well as separation on the cellular level, the cell metabolism of the rat decidua and metrial gland was investigated enzyme histochemically in cryosections for activities of oxyradical-forming enzymes and hydrolyzing enzymes. Additionally, plastic sections were studied to facilitate the recognition of cell types. Decidual stromal cells and fibroblasts formed the vast majority amongst many cell types in the decidua and metrial gland. High activities of enzymes involved in purine degradation and oxyradical generation were demonstrated in decidual stromal cells and fibroblasts. Microsomal alanyl aminopeptidase and various acid hydrolases were shown to be extremely active in decidual stromal cells. The abundance of these enzyme activities in the decidua and metrial gland in contrast to other placental areas suggests, that these enzymes may have specialized functions in connection with regression and degradation processes finally contributing to placental separation.

Animals↗
PubMed1992

Is it still adequate to study the nervous system using methods of catalytic enzyme histochemistry?

The frequent use of methods different from enzyme histochemistry evokes the question, whether it is still useful to apply methods of catalytic enzyme histochemistry to study the nervous system. In this brief overview it is shown, that catalytic enzyme histochemistry can still contribute to a better understanding of nervous system function. This was enabled by methodological progress, i.e., the modification of already existing procedures or the development of new techniques for the visualization and measurement of enzymes in tissue sections using their catalytic properties. The methods for acetylcholinesterase, monoamine oxidase as well as certain exoglycosidases and phosphatases will be given as examples. The application of these procedures as well as of methods for proteases, oxyradical-generating oxidases and enzymes involved in the metabolism of amino acid and other transmitters will illustrate, that qualitative (localization) and quantitative (measurement) catalytic enzyme histochemistry can still contribute to a better understanding of nervous system function.

Animals↗
PubMed1991

Catalytic histochemistry of acid and neutral hydrolases in plant seedlings.

In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast, displayed a species-dependent activity and differences in localization. Of the glycosidases, alpha-D-galactosidase was the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.

Catalysis↗
PubMed1991

Protection against salicylate-induced hepatic injury by zinc. A histochemical and biochemical study.

Female Wistar rats received an oral dose of 700 mg salicylic acid/kg body wt., given as sodium salicylate. Some of the salicylate-treated rats received two subcutaneous injections of 100 mumol kg-1 ZnCl2 (24 h before and simultaneously with the salicylate administration). Other animals were given one subcutaneous injection of 100 mumol kg-1 ZnCl2 simultaneously with the salicylate treatment. Control rats were similarly injected with ZnCl2. Twenty four hours after salicylate treatment, serum and livers were taken for histochemical and biochemical analysis. The most remarkable effects of the treatment were enrichment of lipid droplets and iron and a reduction of glycogen, particularly in the periportal hepatocytes. The effects of salicylate were partially prevented by two ZnCl2 injections. The protective effects of ZnCl2 may be due to lower iron uptake into hepatocytes and by the induction of zinc metallothionein, which can serve as a scavenger for oxygen radicals.

Alanine Transaminase↗
PubMed1991

Light microscopical detection of H2O2-generating oxidases using cerium ions and aqueous incubation media.

Light microscopical procedures were optimized and tested for specificity for the histochemical demonstration of D-amino acid oxidase, alpha-hydroxy acid oxidase, monoamine oxidase, and xanthine oxidase using cerium ions and a visualization step originally described by Angermüller and Fahimi (1988a, b), and modified for D-amino acid oxidase by Gossrau et al. (1989). The visualization medium contained diaminobenzidine, cobalt ions, and small amounts of hydrogen peroxide. Procedures of pretreatment of cryostat sections, types of substrate, concentrations of substrates, and cerium ions were varied. Optimum procedures are reported for the detection of these oxidases in different rat tissues. The results are compared with those obtained with other methods described for the demonstration of hydrogen peroxide-generating oxidases such as the tetrazolium, Hatchett Brown, and coupled peroxidatic methods.

Alcohol Oxidoreductases↗
PubMed1991

Histochemical and biochemical studies of dipeptidyl peptidase I (DPP I) in laboratory rodents.

Different from other proteases the halide- and thiol-dependent lysosomal dipeptidyl peptidase I (DPP I, cathepsin C, EC 3.4.14.1.) was not thoroughly analysed up to now. Therefore, using complementary methods of catalytic histochemistry and biochemistry, DPP I was studied in many rat and mouse organs and compared with DPP II, another lysosomal protease, whose biochemical and histochemical behaviour is known. Continuous (kinetic) fluorometric measurements revealed Gly-Arg naphthylamine (NA) as the substrate with the highest rate of hydrolysis in which, however, also microsomal alanyl aminopeptidase may participate. A more specific substrate appeared to be Pro-Arg-NA; Gly-Phe-NA, and Ser-Tyr-NA were inferior substrates. The Km values were 0.1 mmol/l and 0.2 mmol/l for Gly-Arg-NA and Pro-Arg-NA. The optimal substrate concentration was between 1 and 2.5 mmol/l and the optimal pH value between 5 less than or equal to pH less than or equal to 5.5. The highest reaction velocity was measured with cacodylate or phosphate buffer. Chloride ions and thiol reagents increased the rate of hydrolysis, but an absolute chloride or thiol dependence was not found. Formaldehyde or glutaraldehyde inhibited DPP I depending on the type and concentration of aldehyde. The DPP I activities in rat and mouse organs varied species-dependently and were highest in the extraorbital gland, liver, jejunum, and kidney. In many of the organs, DPP I activity differed considerably from that of DPP II. For catalytic DPP I histochemistry and localization of the enzyme in lysosomes and secretion granules freeze-dried celloidin-mounted cryosections incubated with Gly-Arg-4-methoxy-2-naphthylamine in the presence of Fast Garnet GBC or Fast Blue B were the method of choice in comparison with other tissue pretreatments, Pro-Arg-MNA as substrate and other coupling agents. Use of thiol reagents and chloride ions did not improve the histochemical results. Using this method DPP I was visualized in many cells of rat and mouse organs where its existence was not yet known before. At many sites, DPP I and DPP II distribution patterns differed considerably. In conclusion, a histochemical chromogenic method is now available, which allows the reliable detection of DPP I as was already possible for DPP II using an MNA substrate.

Animals↗
PubMed1991

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.

Allopurinol↗
PubMed1991

A new approach to intravital videomicroscopy of rat spinotrapezius muscle.

A modified preparation of the rat spinotrapezius muscle is described in which optimal conditions for intravital microscopy can be achieved while the supplying blood vessels are left fully intact, and mechanical stress to the muscle during preparation is reduced to an unavoidable minimum. The viability of the preparation is demonstrated using the response of arterial microvessels to endothelium-dependent and -independent dilators and to changes of ambient PO2, the presence of spontaneous vasomotion, and histochemical analysis of pertinent enzyme systems. The preparation is viable for much longer experimental time periods (up to 10 hours) than reported previously, provided the intensity of illumination is kept at a very low level. If the latter prerequisite is met, tissue edema, maximal vasodilation, and the associated loss of responsiveness to vasoactive stimuli of arterioles is reliably avoided.

Animals↗
PubMed1990

Histochemical response of mice to mistletoe lectin I (ML I).

The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgi-associated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1990

Histochemistry of reactive oxygen-species (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase.

Cutaneous and mucous epithelia of various organs of laboratory rodents were analysed histochemically for reactive oxygen species (ROS)-generating oxidases using cerium methods. High activities of xanthine oxidase and also superoxide dismutase were present in orthokeratotic stratified squamous epithelia of skin, lips, esophagus and forestomach and parakeratotic keratinizing stratified epithelia of vagina, tongue and penis. Moreover, activity was found in simple epithelium of the uterus and intestine of rats, mice and guinea-pigs. Moderate activities of monoamine oxidase and D-amino acid oxidase were only seen in enterocytes of large and small intestine, whereas alpha-hydroxy acid oxidase could not be detected at all. With the use of specific inhibitors for superoxide anions-producing xanthine oxidase and H2O2-generating superoxide dismutase it was shown that epithelial cells of all studied external and internal surface epithelia contain a highly effective xanthine oxidase-superoxide dismutase system. It is hypothesized that this system might have a general microbicidal function and might play a special role in tumor promotion of the skin.

Alcohol Oxidoreductases↗
PubMed1990

Calcium-binding proteins 33 kDa, 35 kDa, and 65/67 kDa in normal rat and Morris hepatoma tissues. A biochemical and immunohistochemical study.

Polyclonal antibodies were raised against membrane-associated calcium-binding proteins (apparent molecular masses 65000 and 67000 (CBP 65/67) and 33000 and 35000 (CBP 33 and CBP 35)), which were isolated from rat liver and Morris hepatoma. Using immunoblotting, various amounts of CBP 33 and CBP 35 as well as CBP 65/67 were detected in most rat organs. Using alkaline phosphatase and monoclonal-anti-alkaline phosphatase antibodies (APAAP), all the calcium-binding proteins were detected by immunohistochemical techniques in the plasma membranes of many cells, such as vascular endothelial cells, lymphocytes, epididymal principal cells, secretory and excretory duct cells of certain exocrine glands, straight distal tubular cells of the kidney, and in the cytoplasm of muscle cells and fibres as well as nerve cells and chondrocytes, and in connective tissue elements. Immunohistochemical analysis also showed that in polarized epithelial cells, e.g., renal tubular cells, epididymal principal cells or excretory duct cells, these calcium-binding proteins are present exclusively or mostly in the luminal plasma membrane.

Animals↗
PubMed1990

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry.

Application of cryostat sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.

Animals↗
PubMed1990

Pitfalls in the light microscopical detection of NADH oxidase.

NADH oxidase activity has been detected at the ultrastructural level using cerium ions to trap H2O2 generated by the enzyme (via intermediate reactive oxygen species). In an attempt to localize NADH oxidase activity at the light microscope level using the cerium-diaminobenzidine (DAB)-nickel-H2O2, the cerium-DAB-cobalt-H2O2 or the cerium-alkaline lead procedures, the distribution patterns of the revealed enzyme were found to be identical to those for non-specific alkaline phosphatase and especially 5'-nucleotidase activity. With the cerium-DAB-cobalt-H2O2 visualization procedure, the distribution pattern of the final reaction product was similar to that obtained with the other two techniques but much less final reaction product was formed. Incubations for NADH oxidase activity performed in the presence of exogenous catalase or in the absence of catalase or peroxidase inhibitors did not affect the staining intensity, whereas inhibitors of 5'-nucleotidase (EDTA) and non-specific alkaline phosphatase (levamisole) always did. Therefore, phosphatases contribute to the formation of the final reaction product. Since NADH initially cannot be hydrolysed by either of these two phosphatases, then presumably nucleotide pyrophosphatase (E.C.3.6.1.9) cleaves NADH into 5'-AMP and nicotinamide mononucleotide in a first step. Both nucleotides can be hydrolysed further by the two monophosphatases. These then generate cerium phosphate which is detected by the DAB-nickel-H2O2, DAB-cobalt-H2O2 or lead visualization methods.

5'-Nucleotidase↗
PubMed1990

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development.

The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.

Aging↗
PubMed1990

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Alkaline Phosphatase↗
PubMed1990

Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates.

Although many synthetic substrates and methods are available for the histochemical detection of non-specific esterases and non-specific acid phosphatase, there are still further possibilities to investigate these hydrolases histochemically. This was shown for menadiol diacetate and menadiol diphosphate using tetrazolium salt, simultaneous azo-dye as well as metal salt methods in many rat tissues. In comparison, the azo-dye procedure with various Fast salts or hexazonium Pararosaniline or New Fuchsin delivered less satisfactory results; precisely localized stain in sufficient amounts was obtained for non-specific esterases using nitro BT, tetranitro BT or benzothiazolystyrylphthalhydrazidyl tetrazolium (BSPT) and for non-specific acid phosphatase with BSPT in the tetrazolium salt method or using cerium ions for phosphate trapping in the diaminobenzidine-nickel-hydrogen peroxide procedure.

Acid Phosphatase↗
PubMed1990

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method.

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.

3,3'-Diaminobenzidine↗