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R Gossrau

Publications and source records attributed to R Gossrau.

At least 19 recordsLinked to original sources
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

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

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

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

Light microscopical detection of D-amino acid oxidase activity in unfixed cryostat sections of rat kidney and liver using the cerium-DAB-cobalt-H2O2 procedure and a semipermeable membrane.

The two-step method for the light microscopical detection of oxidase activity using cerium ions and a visualization procedure with diaminobenzidine and nickel or cobalt ions as introduced by Anger-müller and Fahimi, has been improved. Inactivation by fixation and leakage of D-amino acid oxidase molecules were avoided by the application of unfixed cryostat sections adhered to a semipermeable membrane separating the sections from a gelled incubation medium. Optimum activity in rat kidney and liver was obtained by increasing the cerium concentration from 3 to 30 mM and the Tris-maleate concentration from 100 to 200 mM in the incubation medium for the first step. D-proline was used as substrate and a concentration of 20 mM gave maximum activity. The second visualization step was performed in a medium consisting of DAB, cobalt and hydrogen peroxide. Final reaction product was found in a granular form in the basal part of the epithelial cells of the proximal tubules and in liver parenchymal cells. The activity in periportal areas was much higher than in pericentral areas. Cytophotometric analysis revealed that a 30 times higher activity could be detected when using the membrane technique in comparison with incubations in aqueous media. The specificity of the reaction was proven by a nearly complete inhibition of the reaction by beta-hydroxybutyrate.

Animals↗
PubMed1990

Enhancement of immunoreactivity of von Willebrand factor in vascular endothelial cells of rat organs after glucocorticoid administration.

Our study showed that synthetic glucocorticoids are potent mediators of vWF-reactivity in rat fetal placental, liver and lung vascular elements. However, it is not yet possible to draw final conclusions from these results obtained only by immunohistochemical studies using antibody against vWF, since the mechanisms in factor VIII/vWF are highly complex. Further investigations for example with anti-vW AgII and measurements of blood vWF levels after treatment with synthetic glucocorticoids may deliver more insight into the mechanisms of action of these drugs. Investigations after treatment of pregnant rats with methylprednisolone might help, in addition, to find out whether the increased vWF-reactivity in caused by special substituents in the steran ring of the glucocorticoids.

Animals↗
PubMed1990

Indoxyl alfa-D-galactoside as the temporarily last substrate for glycosidase histochemistry. The present state of the art in histochemical glycosidase research using indoxyl glycosidas.

Initiated by the recently published histochemical method for the investigation of alfa-D-galactosidas with an indoxyl substrate, the current state of this group of synthetic compounds in light and electron microscopic histochemical glycosidase research is evaluated whereby historical, functional, methodological and applied aspects are considered. Beginning with the introduction of indoxyl acetate for non-specific esterase in 1951 and 1952 numerous other indoxyl substrates and mostly substituted in the 5- and 4-position of the indol ring by Br and Cl were developed to study histochemically non-specific phosphatases and glycosidases and frequently used in indigogenic, azoidoxyl, tetrazolium salts and metal salt techniques for catalytic (activity) histochemical and less often for immunohistochemical, affinity histochemical and hybridohistochemical purposes. The last substrate which became available and was validated for activity histochemistry was 5-Br-4-Cl-3-indoxyl alfa-1-galactoside for alfa-1-galactosidase. At present, the indoxyl glycosides are more widely used than 5-Br-4-Cl-3-indoxyl acetates and phosphates when compared with the alternative synthetic (artificial) naphthol, 6-Br-2-naphthol or ternative synthetic (artificial) naphthol, 6-Br-2-naphthol AS substrates, and among the indoxyl glycosides those for the oxoglycosidases lactase, maltase-glucoamylase, glucoamylase, acid beta-D-galactosidase, neuroaminidase and alfa-D-galactosidase are superior to other artificial compounds. When one considers in addition, electron microscopic catalytic glicosidase histochemistry (ultracytochemistry, 5-Br-4-Cl-3-indoxyl is the only suitable moiety for this purpose. These glycosidase can mostly be localized in plasma membranes or lysosomes and also measured there in tissue sections but are also found in secretion granules, endoplasmic reticulum and organ lumina.

Animals↗
PubMed1979

[Peptidases II. Localization of dipeptidylpeptidase IV (DPP IV). Histochemical and biochemical study].

Fresh frozen, unfixed, chloroforme-acetone treated or freeze-dried cryostat sections or sections from aldehyde-fixed blocks of tissue were tried for the histochemical investigation of dipeptidylpeptidase IV (DPP IV) with L-glycyl-L-prolyl(gly-pro)-naphthylamides as substrates and stable or unstable diazonium salts for simultaneous coupling and various buffers, pH 5--7.5 in rats, mice, guinea-pigs, cats, rabbits, hamsters and human enterobiopsies. The best results are obtained with 1.7--3.4 mM gly-pro-4-methoxy-2-naphthylamide and 1 mg Fast Blue B/ml or (with some limitations) 0.025 ml hexazotized new fuchsine/ml in 0.1 M cacodylate or phosphate buffer, pH 7.5 and unfixed sections for the demonstration of the total activity of DPP IV and freeze-dried celloidin-mounted cryostat sections for the precise localization of the enzyme or the detection of lysosomes, Golgi apparatus and secretion granules sections from aldehyde fixed tissue blocks are only suitable to study the lysosomal hydrolysis of gly-pro-naphthylamides between pH 5 and 7 when hexazotized p-rosaniline or new fuchsine are employed. DPP IV is firmly bound to strutures and shows species- and organ-dependent differences. In general, the enzyme occurs in the capillary endothelium, sinusoidal cells, perineurium, epithelial cells of intercalated and striated ducts, microvillous zone of intestinal crypts and villi, uterus, Fallopian tubes, ductus epididymis and proximal renal tubules, hepatocyte and lymphocyte membrane, plasmalemma of pseudostratified and transient epithelia and in the capsules and interstitium of many organs. These sites of activity can be completely inhibited by diisopropyl fluorophosphate and partially by Pb2+; Mg2+, Mn2+, Co2+ EDTA are without any influence. Phenantrolin may activate DPP IV. The biochemical assay works with 10 mM gly-pro-2-naphthylamide in 0.1 M cacodylate buffer, pH 7; the enzyme activity is determined fluorometrically in guinea-pig and rat organs; the data confirm and enlarge the species- and organ-dependent differences revealed by histochemistry. Compared with other dipeptide as well as tripeptide and amino acid naphthylamides the results obtained for DPP IV suggest a peptidylpeptidase which seems to be involved in other metabolic processes beside the degradation of collagen.

Animals↗