Heide Schmid

A rapid method to separate endosomes from lysosomal contents using differential centrifugation and hypotonic lysis of lysosomes

Here we describe a fast and efficient subcellular fractionation procedure that permits lysosomes to be separated from endosomes. Differential centrifugation is used to isolate a subcellular fraction containing both endosomes and lysosomes. Because lysosomes are sensitive to osmotic stress, hypotonic conditions destroy them, whereas endosomes, which are osmotically insensitive, stay intact. We demonstrate that hypotonic lysis of an endosome-lysosome-pool releases 85% of the lysosomes into the supernatant as measured by the activity of the lysosomal marker enzyme N-acetyl-beta-D-glucosaminidase (beta-AGA). The endosomal fraction is thoroughly characterised using a variety of subcellular markers. After pulsing cells with fluorescein isothiocyanate labelled transferrin (FITC-Tf), only about 12% of the marker is released under hypotonic conditions. A typical fractionation procedure takes about 1-2 h from initial cell homogenisation. The fractionation gives a pure lysosomal fraction (fraction L) containing high activities of lysosomal enzymes and an endosomal fraction (fraction E) reflecting different stages of endosomes.

Localization of S-adenosylhomocysteine Hydrolase in the Rat Kidney:

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 ± 0.02 mU/mg in the kidney, 0.32 ± 0.03 mU/mg in the glomeruli, and 0.50 ± 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation.

Modulation of the endosomal and lysosomal distribution of cathepsins B, L and S in human monocytes/macrophages.

Abstract Endosomal and lysosomal fractions of human monocytes/ macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferonγ treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.

Expression and Localization of S-Adenosylhomocysteine-Hydrolase in the Rat Kidney Following Carbon Monoxide Induced Hypoxia

Background/Aims: Tissue hypoxia induces a variety of functional changes including enhanced transcriptional activity associated with high transmethylation activity (e.g. mRNA cap methylation) in the nucleus. It is well known that the kidney responds to hypoxia with enhanced transcription of erythropoietin (EPO) in the interstitial cells. Since S-adenosylhomocysteine (AdoHcy)-hydrolase regulates most S-adenosylmethionine (AdoMet) dependent transmethylation reactions by hydrolyzing the potent feedback inhibitor AdoHcy to adenosine and homocysteine we studied the effect of hypoxia by carbon monoxide (CO) inhalation (1200ppm) on AdoHcy-hydrolase gene expression and its localization in rat kidneys. Results: CO lowered renal AdoHcy-hydrolase mRNA expression by 64% whereas AdoHcy-hydrolase activity was not changed during 4h of CO exposure 0.7±0.04mU/mg (control) vs. 0.75±0.06mU/mg protein. Using two-channel immunofluorescence confocal laser scanning microscope AdoHcy-hydrolase was visualized in different cells of the hypoxic rat kidney. A very bright immunofluorescence of AdoHcy-hydrolase was observed in the nuclei of single interstitial cells of renal cortex and outer medulla which respond to hypoxia with increased EPO secretion indicating translocation of AdoHcy-hydrolase from the cytosol to the nucleus. Conclusions: These data suggest that AdoHcy-hydrolase accumulation in the nucleus of adult mammalian cells is involved in maintaining efficient transmethylation reactions in transcriptionally active cells by removing the product inhibitor AdoHcy.

Increased activity of cathepsin B in fibroblasts isolated from primary melanoma in comparison to fibroblasts from normal skin

Abstract: We determined activity of cathepsin B in early‐passage fibroblasts isolated from primary melanoma and in fibroblasts from normal skin. Our results show an up to 5‐fold increase in activity of cathepsin B in the tumor‐derived fibroblasts in comparison to the fibroblasts from normal skin. We conclude that fibroblasts isolated from melanoma tissue are altered with regard to their specific activity of cathepsin B and preserve this elevated activity in early‐passage cell culture. The data support the idea that stromal cells are not passive elements of the peritumoral environment but actively participate in the production of proteolytic enzymes.

Expression of the brain and muscle isoforms of glycogen phosphorylase in rat heart.

Heart glycogen represents a store of glucosyl residues which are mobilized by the catalysis of glycogen phosphorylase (GP) and are mainly destined to serve as substrates for the generation of ATP. The brain isoform of GP (GP BB) was studied in rat heart in comparison with the muscle isoform (GP MM) to find functional analogies to the brain. Western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed that at the protein level, but not at the mRNA level, the content of GP BB is similar in heart and brain. In contrast, GP MM is more abundant in the heart than in the brain. Immunocytochemically GP BB was colocalized with GP MM in cardiomyocytes. GP MM was also detected in interstitial cells identified as fibroblasts. The physiological role of co-expression of GP BB and GP MM in cardiomyocytes and in brain astrocytes is discussed in a comparative way.

Renal Expression of the Brain and Muscle Isoforms of Glycogen Phosphorylase in Different Cell Types

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5′-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.

Nephrotoxicity of Cyclosporine A in the Rat

The intranephronal distribution pattern of the activity of succinate dehydrogenase, a marker enzyme of mitochondrial inner membranes, was examined by histochemical investigation in the kidneys of 27 male Sprague-Dawley rats. The animals received cyclosporine A per os (15 30 or 50 mg/kg day) for 12 or 24 days. Six animals of the latter group remained untreated for a further 24 days. Five rats treated orally with 30 mg/kg day olive oil served as controls. The kidneys of 2 normal male Sprague-Dawley rats were examined for comparison. Cyclosporine A reversibly induced a characteristic pattern of nephron segments with various degrees of reduced succinate dehydrogenase activity: in the proximal and distal tubules of subcapsular areas of the cortical labyrinth, of the medullary rays, and of the outer stripe of the outer medulla. The remaining areas contained tubules with normal succinate dehydrogenase activity, as confirmed by microphotometrical measurement. The number of these tubules appeared to be decreased under higher doses of cyclosporine A, irrespective of the duration of treatment. The finding of heterogeneous affection of tubular mitochondrial cristae membranes reflects direct tubulotoxicity of cyclosporine A.

Human fibroblasts in culture exhibit a low sensitivity against cyclosporin A treatment.

The nephrotoxicity of cyclosporin A (CSA) after chronic treatment is well known and includes in later stages tubular atrophy associated with interstitial fibrosis. In order to examine whether interstitial fibrosis due to CSA treatment in vivo is related to a hyperproliferative activity of fibroblasts, the effects of CSA on the growth characteristics of cultured human skin fibroblasts (HUSF) were investigated at CSA concentrations ranging from 10 ng/ml to 50 micrograms/ml. We found that CSA at concentrations higher than 7.5 micrograms/ml inhibited cell proliferation (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) and cloning efficiency (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) in a dose-dependent manner and caused a promotion of cell attachment at concentrations above 10 micrograms/ml (p < 0.05; n = 4), but did not influence cell spreading. At lower concentrations CSA-treated HUSF did not differ in their growth characteristics from the corresponding controls. A 50% inhibition of proliferation was calculated by extrapolation for a CSA concentration of 70 micrograms/ml for HUSF. The inhibition of HUSF proliferation was reversible even at the highest CSA concentration of 50 micrograms/ml. Under the same experimental conditions, a 50% inhibition of proliferation was observed for Madin-Darby canine kidney (MDCK) cells to be 5.5 micrograms/ml, e.g. at a 15-fold lower CSA concentration. Moreover, CSA caused a dose-dependent and reversible cell elongation of HUSF and a significant increase in the average cell diameter from 19.2 +/- 0.3 microns (control; mean +/- SEM, n = 4) to 22.2 +/- 0.2 microns for 50 micrograms/ml CSA (mean +/- SEM, n = 4) and in median cell volume from 4,210 +/- 160 fl (control; mean +/- SEM, n = 4) to 7,020 +/- 190 fl for 50 micrograms/ml CSA (mean +/- SEM; n = 4). These alterations described above were not correlated with a cytotoxic effect as checked by a fluorescent staining for cell vitality. Alterations in the organization of cytoskeletal components such as stress fibers, intermediate-sized filaments and microtubules directly due to CSA treatment were not observed. In contrast, the amount of fibronectin present on the cell surface was considerably increased by CSA. Although HUSF in culture do not respond to CSA treatment by an increased proliferative activity, they are much less affected by CSA than other cell types (i.e MDCK cells).(ABSTRACT TRUNCATED AT 400 WORDS)