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R Gossrau

Publications and source records attributed to R Gossrau.

At least 145 records · Page 8Linked to original sources
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↗
PubMed1978

[Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)].

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.

Acetylglucosaminidase↗
PubMed1978

[Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)].

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.

Animals↗
PubMed1978

[Distribution of enzymes in the stereocilia of the ductus epididymis of rats (author's transl)].

Using unfixed or cryostate sections from aldehyde fixed epididymides of adult rats gamma-glutamyl naphthylamides are hydrolysed in the stereocilia of the principal cells in zone 1A-4B and glycyl-prolyl naphthylamides in those of zone 1B-4A (nomenclature of Reid and Cleland, 1957); this is not the case when other amino acid and peptide naphthylamides are employed. Adenosine triphosphate (ATP) is metabolized by the stereocilia in zone 1a, 2, 3 and 4A and adenosine monophosphate (AMP) in the stereocilia of zone 3-6. With thiamine pyrophosphate (TPP) especially the Golgi apparatus of the principal cells in zone 1B and C, 2 and 3 reacts. In addition ATP, AMP and TPP stain the stereocilia of single epithelial cells outside the zones where all stereocilia react. The AMP pattern allows a further subdivision of zone 3 into 3 A and B. Substrates for the demonstration of alpha- and beta-D-glycosidases, non-specific esterases and alkaline phosphatases are either not hydrolyzed by the stereocilia or the rate of hydrolysis is low.

Adenosine Triphosphatases↗
PubMed1978

[Azoindoxyl methods for the investigation of hydrolases. III. Histochemical studies of beta-D-N-acetylglucosaminidase (author's transl)].

The suitability of various azoindoxyl procedures for the light microscopical demonstration of beta-N-acetylglucosaminidase is described. The incubation media tried consist of 0.5 mg N-Acetyl-(5-bromindol-3-yl)-beta-D-glucosaminide (5-Br-3-indolyl-beta-D-N-acetylglucosaminide; 1 mg dissolved in 0.05 ml N,N-dimethylformamide) in 1 ml 0.1 M citric acid phosphate buffer, pH 4.5 or 5. 0.02 ml hexazotized p-rosaniline or new fuchsine/ml or tetrazotized BAXD or 0.5 mg Fast Blue B or Garnet GBC/ml were employed as a coupling reagent. Hexazotized new fuchsine yields the best results independent on the pretreatment of the tissue and the organ investigated followed by hexazonium-p-rosaniline. Compared with the azo dye method using naphthol AS-BI beta-D-N-acetyl-glucosaminide as a substrate and hexazotized p-rosaniline or new fuchsine or tetrazotized BAXD for simultaneous coupling especially the azoindoxyl technique with the new fuchsine is equvialent or superior. When the indolyl glucosaminide is used in the indigogenic, tetrazolium or metal precipitation method the results are mostly inferior with the exception of the tetrazolium reaction using BSPT. However, the main advantage of the azoindoxyl procedure is that at least the azoindoxyl dye deriving from hexazotized p-rosaniline can be osmificated and withstands treatment with organic solvents and resins. Therefore, the reaction product seems to be suitable for the electron microscopic demonstration of glucosaminidase. Among the other reaction principles this can reliably be achieved only with BSPT as a tetrazolium salt followed by osmification of its formazan. After fixation of blocks of tissue in form- or glutaraldehyde beta-D-N-acetylglucosaminidase can be localized with 5-Br-3-indoxyl-beta-D-N-acetylglucosaminide as a substrate and hexazotized new fuchsine for simultaneous coupling in the lysosomes of many rat organs.

Acetylglucosaminidase↗
PubMed1978

[Postnatal development of the epithelium of the small intestine of guinea-pigs (author's transl)].

This investigation deals with the histochemical, biochemical and electron microscopical development of the postnatal epithelium in the small intestine of guinea-pigs. Immediately after birth the enterocytes of the whole small intestine are rich in glycogen. Within 48 hours the glycogen in broken down by intralysosomal digestion and glycogenolysis or disappears from the villus epithelium by extrusion of the absorptive cells. The first loss of glycogen occurs in the jejunum; at the latest it leaves the lining epithelium of the ileum so that a proxoim-distal gradient exists. Afterwards for maximal 14 days fat ist taken up from the mother's milk only by the enterocytes of the jejunum without any signs of endocytosis; the bigger part of the fat leaves the cells by exocytosis and enters the intercellular space. Most of the lipid reaches the lymphatics or is absorbed by marcophages; only small amounts are found in the blood capillaries. The number of the enterocytes engaged in the absorption and passage of fat depends on its quantity in the lumen of the small intestine. During the first days of life everywhere in the small intestine the ultrastructure of the enterocytes is characterized by 2 types of mitochondria (large and small ones with different internal structure). Furthermore in the ileum the absorptive cells contain more lysosomes and a more extensive inframicrovillous membrane system than in jejunum. The membrane system consists of aggregated tubules, vacuoles and vesicles; they are situated below the microvilli and sometimes communicate with the lumen of the gut. The big mitochondria are broken down in the lysosomes or appear in the lumen of the small bowel following extrusion of the enterocytes. The lysosomes are involved in autophagic (digestion of glycogen and cell organelles) as well as in autophagic processes (hydrolysis of molecules from the mother's milk and foreign food). These substances are probably incorporated by means of the inframicrovillous membrane system. With respect to the microvillous hydrolases (lactase, alpha-D-glucosidases, peptidases, alkaline phosphatase) histochemical and biochemical assays were carried out with the same artificial substrate. The results depend on the method employed and the enzyme investigated. Using histochemical techniques and indolyl, naphthly, naphthol AS or naphthylamine derivatives as substrates the activity of peptidases and alkaline phosphatase correspond to that in adult guinea-pigs already at the time of birth; alpha-D-glucosidases (glucoamylase, saccharaseisomaltase) become mature at the end of the first, and the development of lactase is finished after the second week of life. For the biochemical investigations (fluorometric measurements of naphthol and naphthylamine) of microvillous enzymes with naphthyl and naphthylamine substrates a new technique of homogenisation using freeze-dried cryostate sections is successfully employed...

Animals↗
PubMed1977

[Peptidases. I. Histochemical investigations with 2-naphthylamides and hexazonium-p-rosanilin (author's transl)].

Using fresh frozen (with and without semipermeable membranes), freeze-dried or sections from aldehyde fixed material and hexazotized p-rosaniline for simultaneous coupling more than 20 different unsubstituted or substituted L-amino acid naphthylamides are split especially in the microvilli and/or stereocilia of the small intestine, kidney and epididymis from rats. Further sites of positive reactions can be revealed by L-alanyl, L-leucyl, L-lysyl, alpha,L-glutamyl, gamma,L-glutamyl, L-asparaginyl, N-benzoyl-L-arginyl, N-carbobenzoxy-L-arginyl and N-benzoyl-L-phenylalanyl 2-naphthylamide. Among the substituted and unsubstituted peptide 2-naphthylamides L-prolyl-L-arginyl 2-naphthylamide is not hydrolysed in visible amounts; L-arginyl-L-arginyl, L-alanyl-L-arginyl-L-arginyl, L-alanyl-L-leucyl-L-tyrosyl, L-histidyl-L-seryl, L-seryl-L-tyrosyl and L-glycy-L-phenylalanyl 2-naphthylamide are metabolized in the renal and intestinal brush border; the reaction pattern obtained with N-carbobenzoxy-L-glycyl-L-glycyl-L-arginyl 2-naphthylamide differs from that of N-carbobenzoxy-L-arginyl 2-naphthylamide. In addition L-glycyl-L-prolyl, L-leucyl-L-alanyl, L-lysyl-L-alanyl and L-alanyl-L-phenylalanyl-L-prolyl 2-naphthylamide are also split in the lysosomes of many organs and the secretion granules of gland cells.

Amides↗
PubMed1977

[Suitability of naphthyl-alpha-L-fucosides for the investigation of alpha-L-fucosidases (author's transl)].

In comparison with 1- and 2-naphthyl beta-D-glucoside, beta-D-galactoside, beta-D-glucuronide, beta-D-N-acetylglucosaminide, alpha-D-glucoside, alpha-D-galactoside and alpha-D-mannoside 1- and 2-naphthyl alpha-L-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freeze-dried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of alpha-L-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosies deliver positive results. The reasons for these discrepancies are the marked inhibition of alpha-L-fucosidase by aldehyde fixation and diazonium salts. Then, alpha-L-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, alpha-D-mannosidase, beta-D-glucuronidase and beta-D-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl alpha-L-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated. For biochemical measurements, however, especially 1-naphthyl alpha-L-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl alpha-L-fucoside used for the photometric evaluation of alpha-L-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of alpha-L-fucosidase.

Animals↗
PubMed1977

[Microchemical investigation of alpha D-glucosidases using 4-methylumnelliferyl- and 2-naphthyl-alpha-d-glucoside (author's transl)].

In crude homogenates prepared from freeze-dried cryostate sections of various rat organs the Km and Vmax of acid and neutral alpha-glucosidase as well as the effect of the pH, substrate and enzyme concentration and the incubation time on the activity were determined fluorometrically with 4-methylumbelliferyl- and 2-naphthyl alpha-d-glucoside as substrates. On the basis of the biochemical data 2 assays were developed for the microchemical measurement of both alpha-glucosidases in groups of epithelial cells isolated from freeze dried cryostate sections of the epididymis, jejunum, ilium, liver and kidney of suckling and adult rats. The rate of hydrolysis of 2-naphthyl and 4-methylumbelliferyl alpha-d-glucoside differs moderately. However, due to the higher sensitivity of 4-methylumbelliferone the methylumbelliferyl derivative is preferable especially for the evaluation of alpha-d-glucosidases in cells with low enzyme activity.

Animals↗
PubMed1977

[Azoindoxyl methods for the investigation of hydrolases. II. Biochemical and histochemical studies of acid beta-galactosidase (author's transl)].

The determination of various reaction constants yields the following assay for the photometric evaluation of acid beta-galactosidase (measurement of the azoindoxyl dye at 540 nm after extraction with dimethylformamide or -acetamide): 1.5 mM 5-Br-4-Cl-3-indolyl-beta-D-galactoside (1 mg dissolved in 0.05 ml dimethylformamide) and 0.01-0.015 ml hexazotized p-rosaniline/ml in 0.1 M citric acid-phosphate buffer, pH 4. By means of this procedure it becomes evident that the activity of the enzyme differs considerably in various rat organs; NaCl does not influence acid beta-galactosidase. -- Similar results were obtained with the indigogenic method; indigo can be dissolved and measured photometrically as the azoindoxyl dye. The enzyme is suppressed by high concentrations of hexazotized p-roaniline to 50%; low concentrations do not inhibit; the same is true for ferricyanide-ferrocyanide employed in the indigogenic media. -- The effect of glutar- and formaldehyde on acid beta-galactosidase cannot be investigated with the azoindoxyl reaction since the azoindoxyl dye partially withstands extraction from fixed blocks of tissue. On the basis of the biochemical findings the azoindoxyl technique can be recommended for the histochemical demonstration of acid beta-galactosidase: 7.5 mg (1.5 mM) 5-Br-4-Cl-3-indolyl-beta-D-galactoside (dissolved in 0.25 ml dimethylformamide) and 0.05-0.15 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid-phosphate buffer, pH 4. After incubation the sections can be treated with osmium tetroxide followed by dehydration and mounting in resins or can be mounted without prior osmification of the azoindoxyl dye in glycerin jelly. The osmium chelate resists treatment with organic solvents; the stability of the chelate depends on the concentration of hexazotized p-rosaniline. After fixation in glutaraldehyde or in a mixture of form- and glutaraldehyde acid beta-galactosidase can be exactly localized in the lysosomes of many rat organs. In comparison with the indigogenic, the metal precipitation and the simultaneous azocoupling reactions for the in situ detection of acid beta-galactosidase the azoindoxyl procedure is superior if fixed material is used; it is equivalent or inferior in connection with membrane technique. The biochemical azoindoxyl assay represents a useful method for combined qualitative and quantitative studies of acid beta-galactosidase.

Aldehydes↗
PubMed1976

[Histochemical and biochemical investigation of alpha-glucosidases by means of 2-naphthyl-alpha-D-glucoside (author's transl)].

Histochemical and biochemical studies yield the following method of choice for the in situ detection of neutral (microvillous) and acid (lysosomal) alpha-glucosidases: 12 mg 2-naphthyl-alpha-D-glucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.6--0.8 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid phosphate buffer for aqueous or 5 ml buffer mixed with equal parts of 2% agar for incubation with semipermeable membranes, pH 5 or 6.5. With this method neutral alpha-glucosidases can be exactly demonstrated in the brush border of the small intestine (glycoamylase, sucrase-isomaltase) and kidney of mammals, birds,fishes, amphibia and reptiles; localization of acid alpha-glucosidases is achieved at the cellular level in many organs and tissues. Fluorometric and photometric measurements prove that 2-naphthyl-alpha-D-glucoside is superior to 6-brom-2-naphthyl-alpha-D-glucoside for the demonstration of alpha-glucosidases in situ due to the lower Michaelis constant and higher maximal reaction velocity of the naphthol derivative.--Among the coupling reagents tested neutral alpha-glucosidases can be localized correctly with hexazotized p-rosaniline (with and without semipermeable membranes) for simultaneous coupling. Fast Blue B delivers false positive results in the suczedaneous and simultaneous coupling procedure using aqueous incubation media; in combination with the membrane technique azo dye can not be observed in the sections. Hexazonium-p-rosaniline inhibits neutral and acid alpha-glucosidases to nearly the same extent as Fast Blue B. Fixation of blocks of tissue in formaldehyde and glutaraldehyde suppresses alpha-glucosidases in the intestine and epididymis. The inhibition rates amount to 50 and 70% respectively. Washing in sugar solution rises enzyme activity to 65 and 50%. Species and organ dependent activity differences of neutral and acid alpha-glucosidases and changes of enzyme activity in the intestine and kidney after castration as well as in the course of pregnancy can be detected by means of biochemistry but not with the histochemical assay including minimal incubation. In comparison with p-nitrophenyl-alpha-D-glucoside the 2-naphthyl derivative is also the substrate of choice for the biochemical determination of alpha-glucosidases.--Agar gel electrophoresis reveals one band in the neutral and acid pH range.

Animals↗