[Without the bag my life would no longer be a life].
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Bandages↗
Biomedical subjects
E Baumgart
Publications and source records attributed to E Baumgart.
Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor.
To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.
Amino Acid Sequence↗
[Elevation of 7-dehydrocholesterol concentrations in serum and liver and pericentral peroxisome proliferation in hepatocytes of rats after inhibition of cholesterol biosynthesis by BM 15,766].
Sprague-Dawley rats of both sexes were treated for three months with BM 15,766, an inhibitor of cholesterol biosynthesis in conjunction with standard or high-fat and high-cholesterol diets. In serum and livers of all drug-treated rats lowered cholesterol concentration associated with an increase of 7-dehydrocholesterol (7-DHC) was found. Electron microscopy of the liver showed a distinct proliferation of peroxisomes and an increase of dumb-bell shaped mitochondria in the pericentral zone 3. Abnormal-shaped peroxisomes with DAB-negative loops attached to their membranes were found in the intermediate zone 2. These alterations were more accentuated in drug-treated rats fed standard diet, then in treated rats receiving a high-fat and high-cholesterol diet. The observations demonstrate, that the increase of 7-DHC is due to the inhibition of 7-DHC-delta 7-reductase by BM 15.766 and emphasize the zonal heterogeneity of hepatocytes. The relevance of these observations for the investigation of the human Smith-Lemli-Opitz syndrome, in which also decreased plasma-cholesterol levels and an increase of 7-DHC were reported, is discussed.
Animals↗
Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system.
The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 microns) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (-20 degrees C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.
Acetone↗
Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2. Evidence of distinct subpopulations.
Peroxisomes in human hepatoblastoma cell line HepG2 have been studied using immunocytochemical, ultrastructural and biochemical techniques. By immunofluorescence, small spherical peroxisomes were seen next to rod-shaped and elongated forms. By electron microscopy and catalase cytochemistry, small particles with a diameter of 90 to 100 nm were found next to larger ones measuring up to 300 nm and some exhibiting tail-like extensions. Both the intensity of DAB-staining and the immunolabeling density for catalase were heterogenous in different peroxisomes. Contrary to a recent biochemical study, the enzyme alanine-glyoxylate-aminotransferase was found by double immunofluorescence and immunoelectron microscopy to be localized exclusively in peroxisomes of HepG2 cells. By Metrizamide density gradient centrifugation two populations of peroxisomes were isolated: a regular fraction banding at 1.20 g/cm3 with a mean diameter of 222 nm and a lighter peroxisome fraction banding at 1.14 g/cm3. The ratio of lipid beta-oxidation enzymes to catalase was significantly higher in peroxisomes with lower density than in the regular ones. These observations show clearly the existence of heterogenous populations of peroxisomes in HepG2 cells which may provide a useful model system for the investigation of biogenesis and metabolism of peroxisomes of human origin.
Centrifugation, Density Gradient↗
Ultrastructural aspects of the biogenesis of peroxisomes in rat liver.
A model summarizing our current concepts on the ultrastructural basis of the biogenesis of peroxisomes is presented. Accordingly, the initial stage of de novo build-up of peroxisomes is characterized by the formation of myelin-like figures and membranous attachments onto the surface of pre-existing peroxisomes. Such membranous structures may provide the appropriate lipid environment for the incorporation of peroxisomal membrane proteins and subsequently become the preferential sites for import of newly synthesized matrix proteins. After the import the membranous structures develop into small peroxisomes which may remain attached briefly to the larger particles but eventually separate to become new peroxisomes. Whereas some matrix proteins such as catalase are distributed in all newly formed peroxisomes, other ones like urate oxidase and D-amino acid oxidase are compartmentalized only in some of them, giving rise to heterogeneity of peroxisomes.
Animals↗
Proliferation of peroxisomes without simultaneous induction of the peroxisomal fatty acid beta-oxidation.
Marked proliferation of rat hepatic peroxisomes is observed after treatment with a new potent hypolipidemic drug BM 15766, as well as after bezafibrate. Whereas the relative specific activity of the peroxisomal fatty acid beta-oxidation system is not affected by BM 15766 it is significantly increased after bezafibrate. This is also confirmed by immunoblot analysis of individual beta-oxidation enzymes in highly purified peroxisome fractions. These observations suggest that peroxisome proliferation and the induction of the fatty acid beta-oxidation are regulated separately.
Animals↗
Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.
Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.
Acid Phosphatase↗
Localization of urate oxidase in the crystalline cores of rat liver peroxisomes by immunocytochemistry and immunoblotting.
We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.
Animals↗
Proliferation of peroxisomes in pericentral hepatocytes of rat liver after administration of a new hypocholesterolemic agent (BM 15766). Sex-dependent ultrastructural differences.
The effects of a new piperazine derivative BM 15766, which inhibits the biosynthesis of cholesterol at the 7-dehydrocholesterol-delta 7-reductase step, upon the ultrastructure of rat liver and the serum lipids, have been investigated. Treated animals showed a marked reduction in total sterol content in serum with simultaneous reduction of triglycerides. The catalase activity in liver homogenates was unchanged, carnitine acetyltransferase increased only slightly, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase was augmented by a factor of 2. In sections stained with alkaline 3,3'-diaminobenzidine for catalase, distinct proliferation of peroxisomes (PO) in perivenous regions of the hepatic lobules was noted in rats of both sexes. In male animals many PO showed loop-like extensions and invaginations of their limiting membranes into the matrix. Such alterations were seen less frequently in female animals; instead, females exhibited in the same regions of the hepatic lobules, large aggregates of PO, smooth endoplasmic reticulum, and mitochondria with longitudinal cristae. Close contacts of PO and fenestrated segments of smooth endoplasmic reticulum were noted in both sexes. These observations demonstrate the marked adaptive response of rat hepatocyte organelles to severe hypocholesterolemia induced by BM 15766. The alterations of PO may reflect attempts to increase their surface membrane, which plays a crucial role in the exchange of substrates between the cytoplasm and the peroxisomal matrix. Moreover, the close association of PO and smooth endoplasmic reticulum could facilitate the shuttle of lipid intermediates between these two intracellular compartments involved in the biosynthesis of complex lipids.
Animals↗
[Effects of adrenalectomy and cortisone treatment on the course of malaria (Plasmodium berghei) in NMRI mice].
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Adrenalectomy↗
Detection of mRNAs encoding peroxisomal proteins by non-radioactive in situ hybridization with digoxigenin-labelled cRNAs.
We have used a non-radioactive in situ hybridization (ISH) protocol for the detection of mRNAs encoding proteins localized in peroxisomes. In this presentation the literature on detection of "peroxisomal mRNAs" is reviewed and the results obtained by application of the non-radioactive method are compared with those obtained by hybridization with radioactive probes. Moreover, the special processing conditions and the application of the method for the specific visualization of mRNAs coding for several peroxisomal proteins with different abundance levels and distinct tissue distributions are presented. The combination of the following technical details in the ISH procedure were found to be essential for obtaining optimal sensitivity and good histological quality of the preparations: (a) perfusion-fixation with a fixative containing 4% depolymerized paraformaldehyde/0.05% glutaraldehyde, (b) the use of paraffin embedding instead of frozen sections, (c) specific proteinase K-digestion time for each tissue, and (d) the use of digoxigenin-labelled cRNA probes (hydrolyzed to a length of about 200 bases) for detection. By using this technique, we were able to localize several peroxisome-specific mRNAs with different degrees of abundance: (1) high-level (catalase and urate oxidase) and (2) low-level (all beta-oxidation enzymes and the 70-kDa peroxisomal membrane protein) in rat liver and kidney. The specificity of the method was confirmed by the negative results obtained with corresponding sense controls and the distinct positive staining patterns obtained for albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. All transcripts for mRNAs encoding peroxisomal proteins were localized to the cytoplasm of hepatocytes, with all nuclei as well as epithelial cells of bile ducts and sinusoidal cells remaining negative. In rat kidney, the catalase transcripts were confined to proximal tubular epithelial cells, which is consistent with the high abundance of peroxisomes in this part of the nephron. In contrast, no transcripts for urate oxidase were present in the kidney, corresponding to the absence of that protein in this organ. The transcripts for GAPDH on the other hand were localized in proximal and distal tubular epithelial cells as well as in collecting ducts. The application of this technique to the rat adrenal gland and testis in recent unpublished studies have revealed exclusive localization of catalase transcripts to the adrenal cortex and to interstitial cells of Leydig, which are known to be rich in microperoxisomes. These observations demonstrate the suitability of this technique for accurate localization of mRNAs encoding peroxisomal proteins and for the analysis of alterations in the expression of the corresponding genes under different experimental conditions.
Animals↗