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J P Schellens

Publications and source records attributed to J P Schellens.

At least 19 recordsLinked to original sources
PubMed1997

The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes.

Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenylchromone (LY294002). The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50 30 nM) and LY294002 (IC50 10 microM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids. 3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action. It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.

Adenine↗
PubMed1996

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections.

In the present study, we demonstrate the activity of several phosphatases ultrastructurally in long-term (up to 24 months) cold-stored (-80 degrees C) rat tissues. Phosphatase activity was histochemically studied with the use of unfixed cryostat sections in combination with low temperature (4 degrees C) incubation conditions in order to prevent inactivation of enzyme activity and to limit the loss of ultrastructure. 5'-Nucleotidase activity was observed at plasma membranes, mainly at bile canalicular membranes of hepatocytes in liver. Thiamine pyrophosphatase activity was detected not only in trans side cisternae but also in medial and cis side cisternae of Golgi complexes in the parotid gland. Glucose-6-phosphatase activity was localized in endoplasmic reticulum as well as at the outer membrane of the nuclear envelope. Acid phosphatase reaction product was found in lysosomes. Furthermore, the localization patterns of 5'-nucleotidase and thiamine pyrophosphatase activity were compared with those obtained after different fixation procedures such as immediate chemical fixation of tissues or fixation of tissues after freezing and thawing. The results showed similar localization patterns of these enzymes after the different pretreatments. However, with respect to the ultrastructural morphology, some damage was observed in unfixed material after incubation. It can be concluded that the procedure described here enables ultrastructural localization of activity of phosphatases in long-term cold-stored tissues. This procedure will be useful for a retrospective study on archival material when histochemical parameters are needed.

5'-Nucleotidase↗
PubMed1996

Early effects of high doses of retinol (vitamin A) on the in situ cellular metabolism in rat liver.

Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.

Animals↗
PubMed1995

Combined enzyme histochemical and ultrastructural study on cryostat sections of pig heart to detect early reperfusion damage after ischaemia.

In cardiac surgery, recognition of peroperative myocardial infarction may improve patient selection for prolonged circulatory support. The value of enzyme histochemistry to discriminate between reversible and irreversible myocardial damage at short periods of reperfusion was studied in an in vivo model of regional ischaemia in pig hearts. The left anterior descending coronary artery (LADCA) was clamped for 45 min followed by 2 h reperfusion (group 1, n = 3). Post-mortem heart tissue showed markedly decreased activities of lactate dehydrogenase (LDH) and beta-hydroxybutyrate dehydrogenase (BDH) as demonstrated in cryostat sections, accompanied by massive leakage of LDH in the venous effluent. The depleted areas showed irreversible cell damage based on the presence of flocculent densities in mitochondria. In group 2 (n = 6), LADCA flow was reduced to 40 per cent of the base-line value followed by 2 h reperfusion. Heart tissue showed normal LDH and BDH activities, except for some cells surrounding blood vessels, which activity was minimally decreased. Flocculent densities in mitochondria were never observed. We conclude that enzyme histochemistry of LDH and BDH activity on cryostat sections is a useful tool for detecting irreversible myocardial cell damage as early as 2 h reperfusion after ischaemia of the pig heart. The technique has potential applications in the detection of peroperative infarction in human biopsies.

Animals↗
PubMed1995

Electron microscopical study of a cytosolic enzyme in unfixed cryostat sections: demonstration of glycogen phosphorylase activity in rat liver and heart tissue.

Glycogen phosphorylase activity has been demonstrated at the ultrastructural level in liver and heart tissue of fasted rats. Unfixed cryostat sections were incubated by mounting them on a semipermeable membrane stretched over a gelled incubation medium. The medium contained a high concentration of glucose 1-phosphate which enables indirect detection of glycogen phosphorylase activity on the basis of the synthesis of glycogen. Tissue fixation, dehydration and embedding for electron microscopical study were performed after the incubation had been completed. The ultrastructure of both liver and heart tissue was rather well preserved. Glycogen granules resulting from glycogen phosphorylase activity were found in the cytoplasmic matrix of both hepatocytes and cardiomyocytes; no relationship with membranous structures could be detected. It is concluded that the semipermeable membrane method is well suited for localizing cytosolic enzyme activities at the ultrastructural level without prior tissue fixation; this opens further perspectives for correlations between histochemical and biochemical data.

Animals↗
PubMed1995

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver.

The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light- and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mM diaminobenzidine, 44 mM hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 microns. Catalase in rat liver showed a Km value of 2.0 mM for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mM. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.

Animals↗
PubMed1995

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat.

Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.

Animals↗
PubMed1995

Demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure.

In the present study a technique was developed to demonstrate 5'-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4 degrees C rather than at 37 degrees C. The optimized incubation medium contained 300 mM Tris-maleate buffer, pH 7.2, 5 mM adenosine monophosphate as substrate, 30 mM cerium chloride as capturing agent for liberated phosphate, 10 mM magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5'-nucleotidase activity were obtained when incubations were performed at 37 degrees C and 4 degrees C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37 degrees C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8 micrograms. 5'-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37 degrees C than at 4 degrees C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37 degrees C, in comparison with that at 4 degrees C, were the appearance of multi-lamellar structures at bile canaliculi at 37 degrees C. We conclude that the present method is valid for the demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.

5'-Nucleotidase↗
PubMed1994

Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver.

Because of controversial data in the literature, we studied the localization of uric acid oxidase (UAOX) activity in rat liver by light microscopy (LM) and electron microscopy (EM). UAOX is partially inactivated by aldehyde fixation and therefore we developed a technique that permits the use of unfixed cryostat sections for both LM and EM studies. Sections of rat liver were mounted on a semipermeable membrane stretched over a gelled incubation medium containing urate as specific substrate for UAOX and cerium ions to capture H2O2 produced by oxidase activity. The specificity of the reaction was checked by comparing incubations in the presence of substrate with incubations either in the absence of substrate or in the presence of substrate and 2,6,8-trichloropurine, a competitive inhibitor of UAOX. After incubation the sections were either fixed immediately for EM or visualized for LM with a second-step incubation. At the LM level, final reaction product was found in a granular form, homogeneously distributed throughout the hepatocytes. EM revealed excellent subcellular morphology and electron-dense reaction product in both the core and the matrix of peroxisomes, but not in other organelles or the cytoplasmic matrix. After incubations without substrate or with substrate and inhibitor, hardly any reaction product was found. We conclude that, because of the use of unfixed tissue, UAOX is not inactivated, which results in localization of UAOX activity not only in the core of peroxisomes but also in the peroxisomal matrix.

Animals↗
PubMed1992

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.

Animals↗
PubMed1991

Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes.

In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.

Adenosine Triphosphate↗
PubMed1991

The effect of internal biliary drainage on bile pigment accumulation and acid phosphatase activity in human liver during obstructive jaundice.

The effect of internal drainage after biliary obstruction due to primary cholangiocarcinoma has been studied in seven human liver biopsies with respect to bile pigment accumulation and acid phosphatase activity. Enzyme activity was demonstrated at the light-microscopic level in unfixed cryostat sections using an incubation medium containing naphthol AS-BI phosphate as substrate, and hexazotised pararosaniline as simultaneous coupling agent, and at the ultrastructural level in fixed tissue blocks and chopped tissue sections using sodium beta-glycerophosphate as substrate and lead or cerium ions as capture reagent. Large amounts of bilirubin were found in cryostat sections of non-drained cholestatic livers, especially in pericentral areas. At these sites a high acid phosphatase activity was found. At the ultrastructural level, acid phosphatase activity was found only in the lysosomal compartment--possibly due to the procedure necessary for tissue processing. After internal biliary drainage, the amount of bilirubin diminished, with a concomitant decrease in acid phosphatase activity. The co-localization of accumulations of bile compounds and acid phosphatase activity indicates that lysosomes play a role in the breakdown of bile compounds.

Acid Phosphatase↗
PubMed1990

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes.

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.

Acid Phosphatase↗
PubMed1988

Hepatic autophagy and intracellular ATP. A morphometric study.

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.

Adenosine Triphosphate↗
PubMed1987

Gap junction ultrastructure in rat liver parenchymal cells after in vivo ischemia.

The ultrastructure of gap junctions between rat liver parenchymal cells has been studied after in vivo ischemia, with and without subsequent blood reflow. Freeze fracture replicas were analysed by electron microscopic observation, optical diffraction and morphometric analysis. In control specimens gap junction connexons were widely dispersed and arranged in nearly random fashion over nearly the whole junctional area, with only minute spots of hexagonal connexon arrangement. An ischemic period of 30 min, from which the vast majority of cells are capable of recovery after restoration of the blood supply, usually entails only a slight enlargement of the areas of hexagonally arranged connexons. After 120 min of ischemia without reflow, which results in necrosis of most parenchymal cells, all gap junctions showed a completely hexagonal arrangement of connexons. The numerical density of connexons after 30 and 120 min of ischemia without reflow was significantly higher than in controls, whereas after 30 min of ischemia followed by 2 h of reflow the numerical density had returned to control levels. A fully hexagonal arrangement of gap junction connexons, as occurs after longer periods of ischemia, seems to be related to irreversible cell damage and presumably to metabolic uncoupling of cells. This was preceded by an increase in the numerical density of connexons, which is probably a reversible phenomenon.

Animals↗