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Biomedical subjects

R Gossrau

Publications and source records attributed to R Gossrau.

At least 109 records · Page 6Linked to original sources
PubMed1986

Comparative hydrolase cytochemistry of the mature guinea-pig and marmoset yolk sac with special reference to proteases.

Proteases and gamma-glutamyl transferase linked to plasma membranes, lysosomes and secretion granules were investigated cytochemically together with phosphatases, glycosidases and non-specific esterases in the mature guinea-pig and marmoset yolk sac. Species-independently, the yolk sac epithelial cells showed high activities of lysosomal proteases, glycosidases and non-specific esterases and phosphatases, whereas species-dependent patterns were found for plasma membrane proteases, gamma-glutamyl transferase and phosphatases. Furthermore, the guinea-pig yolk sac epithelium showed marked regional differences and that of both species intercellular differences, which allowed the subdivision of yolk sac epithelial cells in various types. The high activities of all lysosomal enzymes suggests their general importance for the yolk sac function in guinea-pigs and marmosets. The different equipment of the plasma membrane with proteases, gamma-glutamyl transferase and phosphatases indicates a more species-specific physiological role of this part of the yolk sac epithelial cells.

Aging↗
PubMed1986

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes.

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.

Animals↗
PubMed1985

Ultrastructure and hydrolase cytochemistry of the developing marmoset yolk sac.

Yolk sacs from Callithrix jacchus were investigated light and electron microscopically as well as by qualitative light microscopic enzyme histochemistry on days 35 to 126 of gestation. The thin yolk sac wall of the early stages (day 35-41) consists of the cuboid, endodermal epithelium, the mesothelium of the exocoelom and some interposed blood vessels. The inner endodermal surface is rather smooth. At later stages, the epithelium becomes highly prismatic and forms folds which are lined by a mesenchyme and blood vessels. Microvilli and a small number of endocytotic vesicles are observed at the apices of the epithelial cells, which are interconnected by gap junctions, desmosomes and interdigitations. The cytoplasm of the epithelial cells is characterized by a well-developed rough endoplasmic reticulum, a large Golgi apparatus and glycogen deposits. Four different membrane-bordered types of inclusions can be distinguished in the cytoplasm of the epithelial cells: The type I and II inclusions are considered as secretion granules. Their increase and their localization in the cavities of the endoplasmic reticulum at later stages are ascribed to an inhibition of the intracellular transport at the onset of involution. The type III and IV inclusions may represent lysosomes and related organelles. Bile capillary-like spaces exist between the epithelial cells. The basement membrane is incomplete below the epithelium and absent around the capillaries, the endothelium of which is porous in certain areas. Aminopeptidase M is highly active in the plasmalemma and the bile capillary-like structures of the epithelium, dipeptidylpeptidase IV in the mesothelium and alkaline phosphatase in the blood vessel endothelium. Other membrane hydrolases are absent. Acid proteases, glycosidases, non-specific phosphatases and non-specific esterases can be detected stage-dependently with moderate to high activities in the yolk sac epithelium. Compared with other organs, the yolk sac structure and hydrolase equipment are similar to those of the liver and may, therefore, have similar functions, e.g. synthesis and secretion of proteins. In addition, however, the yolk sac epithelium might also be involved in resorptive processes of material from the lumen followed by lysosomal digestion. The Callithrix jacchus yolk sac starts involution on day 80 of gestation by disintegration of the cells. On day 100, this process is completed. The stage of involution which is late in comparison with other primates, e.g. man and Rhesus monkey, is ascribed to the strongly delayed development of Callithrix jacchus.

Animals↗
PubMed1985

Enzyme cytochemistry combined with electron microscopy, pharmacokinetics, and clinical chemistry for the evaluation of the effects of steady-state valproic acid concentrations on the mouse.

A number of organs from adult female mice were investigated after continuous application of the anticonvulsant drug valproic acid (VPA) by enzyme cytochemistry, light and electron microscopy, pharmacokinetics and clinical chemistry. VPA plasma levels were maintained between 55 micrograms/ml and 67 micrograms/ml for three days following subcutaneous implantation of drug reservoirs. Effects detectable by enzyme cytochemical or electron microscopical means were mainly observed in liver, kidney, thymus and spleen. A strict concentration-dependency of drug effects could not be found. In the liver, the activities of some surface-membrane hydrolases were increased at the biliary pole; the activities of other hydrolases were decreased or unchanged. Electron microscopically, number and length of microvilli of hepatocytes were increased and many of them showed fat inclusions, mitochondrial swellings and autophagic vacuoles. In some of the proximal convoluted tubules of the kidney, the reaction product originating from microvillous and lysosomal hydrolases was diffusely distributed and its amount lowered. This was paralleled by tubular cells with an increased number of fat droplets and swollen mitochondria or destroyed tubular cells, as demonstrated by electron microscopy. Additionally, peritubular endothelial cells were arranged in a garland-like pattern. Alkaline phosphatase was activated in the straight portion of the proximal tubules. Increased glucose, creatinine and total protein concentrations and increased gamma-glutamyl transpeptidase and alkaline phosphatase activities in the urine reflected well the damage of the proximal renal tubules. Cortical and medullary morphology varied considerably in the thymus. In extreme cases, the cortical zone was either reduced in size or the medulla showed a cortex-like structure or vice versa (inverted type of thymus). The thymic cortical reticular cells showed increased aminopeptidase A activity accompanied by a generalized aminopeptidase M and alkaline phosphatase reaction. Our data indicate that--in addition to the liver--also the kidney, thymus and spleen are target organs of VPA-induced toxicity in the mouse.

Alkaline Phosphatase↗
PubMed1985

Cytochemistry of membrane proteases.

Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), gamma-glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible exopeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined. The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.

Animals↗
PubMed1984

Recent advances in protease research using synthetic substrates.

2-Naphthylamine, 4-methoxy-2-naphthylamine, 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin and X peptides have become a useful tool to study proteases by a battery of complementary methods; they now include biochemistry, section biochemistry, location cytochemistry, ultracytochemistry and isoelectric focusing. By the use of these procedures we hope that more insight will be possible in the functional role of proteases in health and disease in the near future.

Animals↗
PubMed1984

Fluorescence detection of proteases with AFC, AMC and MNA peptides using isoelectric focusing.

The fluorescence detection of proteases is not only possible with MNA but also with AFC and AMC peptides which were used up till now only for biochemical protease investigations. The MNA method is more sensitive due to the coupling capability of MNA with NSA than the AMC and AFC procedure. In comparison with the simultaneous azo-dye method the fluorescence technique may be inferior as far as the sharpness of the bands is concerned. The fluorescence staining is superior, however, if proteinases have to be investigated that are inhibited by diazonium salts, or where interference of diazonium salts and activators or inhibitors occurs; finally, it is a simple procedure to elucidate the value of MNA, AFC and AMC peptides for one and the same protease. In the field of applied isoelectric focusing the azo-dye and fluorescence procedure are now used together by us to detect possible differences in protease isoenzyme patterns in normal and diseased human biopsies, e.g. from liver, kidney and small intestine.

Animals↗
PubMed1984

Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF).

A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and gamma-glutamyltranspeptidase (GGT) show striking heterogeneous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.

Animals↗
PubMed1984

Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates.

In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited by diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methyl-coumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.

Aminopeptidases↗
PubMed1984

Enzyme histochemistry of malignant T cell lymphoma due to chronic magnesium deficiency in rats.

The lymphocytes of the rat thymus can be grossly differentiated by their cell membrane-bound proteinases. Subcapsular thymocytes lack aminopeptidase A (APA) and AMP and gamma-glutamyltranspeptidase (GGT). Cortical thymocytes show a high activity of APA but no APM and no GGT. Medullar thymocytes possess a high GGT and APM activity but are free of APA. Under Mg deficiency, the APA-negative subcapsular thymocytes are reduced. In lymphoma and beginning lymphoma, APA, APM and GGT are absent. In lymphoma, the alkaline phosphatase activity is increased. Differences are found for dipeptidylpeptidase IV (DPP IV). In some lymphoma, its activity is reduced, in others the DPP IV activity is increased.

Alkaline Phosphatase↗