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E Baumgart

Publications and source records attributed to E Baumgart.

At least 37 records · Page 2Linked to original sources
PubMed1997

Application of in situ hybridization, cytochemical and immunocytochemical techniques for the investigation of peroxisomes. A review including novel data. Robert Feulgen Prize Lecture 1997.

In situ hybridization, cytochemical and immunocytochemical techniques have contributed significantly to the understanding of the biology of peroxisomes, since they permit in situ demonstration of the sites of synthesis and distribution of peroxisomal proteins without the necessity of homogenization and subcellular fractionation of tissues or cultured cells. This article reviews the results of research on mammalian peroxisomal metabolism, biogenesis and proliferation in which morphological techniques have played a significant role in the elucidation of the biological problem. Some new data on peroxisomal heterogeneity and morphogenesis are included. The morphological methods applied have made it possible to characterize the differences in distribution of mRNAs encoding peroxisomal proteins in different tissues, as well as to monitor the marked heterogeneity in the protein composition and in the activity of specific enzymes in the peroxisomal population of single cells, or in tissues with complex organization (e.g. liver and kidney). In addition, the dynamic alterations and high plasticity of the peroxisomal compartment--partly dependent on contact of the peroxisomes to the microtubular network-are presented.

Animals↗
PubMed1997

A mouse model for Zellweger syndrome.

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Animals↗
PubMed1996

Molecular characterization of the human peroxisomal branched-chain acyl-CoA oxidase: cDNA cloning, chromosomal assignment, tissue distribution, and evidence for the absence of the protein in Zellweger syndrome.

Peroxisomes in human liver contain two distinct acyl-CoA oxidases with different substrate specificities: (i) palmitoyl-CoA oxidase, oxidizing very long straight-chain fatty acids and eicosanoids, and (ii) a branched-chain acyl-CoA oxidase (hBRCACox), involved in the degradation of long branched fatty acids and bile acid intermediates. The accumulation of branched fatty acids and bile acid intermediates leads to severe mental retardation and death of the diseased children. In this study, we report the molecular characterization of the hBRCACox, a prerequisite for studying mutations in patients with a single enzyme deficiency. The composite cDNA sequence of hBRCACox, derived from overlapping clones isolated via immunoscreening and hybridization of human liver cDNA expression libraries, consisted of 2225 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76,739 Da. The C-terminal tripeptide of the protein is SKL, a known peroxisome targeting signal. Sequence comparison with the other acyl-CoA oxidases and evolutionary analysis revealed that, despite its broader substrate specificity, the hBRCACox is the human homolog of rat trihydroxycoprostanoyl-CoA oxidase (rTHCCox) and that separate gene duplication events led to the occurrence in mammals of acyl-CoA oxidases with different substrate specificities. Northern blot analysis demonstrated that--in contrast to the rTHCCox gene--the hBRCACox gene is transcribed also in extrahepatic tissues such as heart, kidney, skeletal muscle, and pancreas. The highest levels of the 2.6-kb mRNA were found in heart, followed by liver. The enzyme is encoded by a single-copy gene, which was assigned to chromosome 3p14.3 by fluorescent in situ hybridization. It was absent from livers of Zellweger patients as shown by immunoblot analysis and immunocytochemistry.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and further characterization of rat peroxisomal trihydroxycoprostanoyl-CoA oxidase.

The composite trihydroxycoprostanoyl-CoA oxidase cDNA sequence, derived from overlapping clones isolated via screening of two different rat liver expression libraries, consisted of 2509 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76711 Da. The reading frame and identity of the trihydroxycoprostanoyl-CoA oxidase cDNA were confirmed by the location of various tryptic peptides, obtained from the purified enzyme, in the deduced amino acid sequence. The C-terminus (His-Lys-Met) of trihydroxycoprostanoyl-CoA oxidase did not seem to interact with the C-terminal peroxisomal targeting signal 1 (PTS1) import receptor, although the tripeptide fits the rule of conserved PTS1 variants for targeting of proteins to glycosomes of Trypanosomatidae. At the protein level, trihydroxycoprostanoyl-CoA oxidase showed 45% identical amino acids with rat palmitoyl-CoA oxidase, whereas the identity with pristanoyl-CoA oxidase was much lower (22%). Northern analysis of multiple rat tissues revealed a signal (approx. 2.6 kb) only in liver and (although much weaker) in kidney. Dot-blot analysis of total liver RNA revealed that the mRNA for trihydroxy-coprostanoyl-CoA oxidase is not induced after treatment of rats with structurally unrelated peroxisome proliferators and indicates that highly similar mRNAs are present in other mammals, including man. Immunocytochemistry showed a decrease in trihydroxycoprostanoyl-CoA oxidase protein in individual liver peroxisomes ('diluting-out effect') after treatment of rats with bezafibrate, whereas the palmitoyl-CoA oxidase labelling was significantly increased.

Amino Acid Sequence↗
PubMed1996

Further characterization of the peroxisomal 3-hydroxyacyl-CoA dehydrogenases from rat liver. Relationship between the different dehydrogenases and evidence that fatty acids and the C27 bile acids di- and tri-hydroxycoprostanic acids are metabolized by separate multifunctional proteins.

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L- or D-hydroxyacyl-CoA) stereo-specificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme III revealed the following picture. Rat liver peroxisomes contain two multifunctional beta-oxidation proteins: (a) multifunctional protein 1 (the classical multifunctional protein; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and delta 3, delta 2-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme III). Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring beta-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the beta-oxidation of fatty acids and bile acids intermediates, respectively. The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17 beta-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J. Biochem. 222, 221-227]. In agreement, MFP-2 also displayed estradiol 17 beta-dehydrogenase activity, indicating that MFP-2 and the steroid dehydrogenase are identical enzymes. MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17 beta-dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase II is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

Amino Acid Sequence↗
PubMed1996

Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes.

The suitability of Iodixanol {5,5'-[(2-hydroxy-1, 3-propanediyl)-bis(acetylamino)] bis-[N,N'-bis(2, 3-dihydroxypreopyl-2,4,6-triiodo-1,3-benzenecarboxamide)]}, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) Iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient. Peroxisomes banded around 30% (w/v) Iodixanol (d approximately 1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of Iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.

Animals↗
PubMed1996

L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation.

The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral.

Animals↗
PubMed1996

Contributions of the immunogold technique to investigation of the biology of peroxisomes.

The immunogold labeling technique has been extremely useful in investigation of the structure and function of peroxisomes. In this report a few examples of the application of this technique with significant implications in the field are briefly reviewed. The problem of extra-peroxisomal catalase, the subject of long controversy between the biochemists and cytochemists, was settled with the immunogold technique, which unequivocally revealed the presence of that enzyme not only in the cytoplasm, but also in the euchromatin region of nucleus, in addition to peroxisomes. On the other hand, lactate dehydrogenase, a typical cytoplasmic protein, has also been shown recently to be present in peroxisomes and to be involved in the reoxidation of NADH produced by the peroxisomal beta-oxidation system. The immunogold technique has revealed several distinct compartments in the matrix of mammalian peroxisomes: urate oxidase in the crystalline cores, alpha-hydroxy acid oxidase B in the marginal plates and D-amino acid oxidase in a non-crystalline condensed region of matrix. The specific alterations of peroxisomal proteins are reflected in their immunolabeling density with gold particles. Quantitation of gold-label by automatic image analysis has revealed that the induction of lipid beta-oxidation enzyme proteins by diverse hypolipidemic drugs is initiated and more pronounced in the pericentral regions of the liver lobule. Finally, immunogold labeling with an antibody to 70 kDa peroxisomal membrane protein has identified a novel class of small peroxisomes that initially incorporate radioactive amino acids more efficiently than regular peroxisomes and thus may represent early stages in the biogenesis of peroxisomes.

Animals↗
PubMed1996

Nonradioactive in situ hybridization for detection of mRNAs encoding for peroxisomal proteins: heterogeneous hepatic lobular distribution after treatment with a single dose of bezafibrate.

We present a nonradioactive in siru hybridization (ISH) protocol for detection of mRNAs in rat liver encoding for three peroxisomal proteins: catalase and urate oxidase as representatives of high-level abundance mRNAs and trifunctional protein (PH) as that of low-level abundance mRNAs. In addition to normal rats, animals treated for 24 hr with a single dose of bezafibrate were studied. The use of perfusion-fixation with 4% depolymerized paraformaldehyde/0.05% glutaraldehyde combined with paraffin embedding and the application of digoxigenin-labeled cRNA probes provided optimal cytological resolution and high sensitivity comparable to that of radioactive ISH. In parallel experiments, the same digoxigenin-labeled cRNA probes were used for Northern and semiquantitative dot-blot analysis of isolated RNAs. In control animals, the mRNAs for catalase and urate oxidase were uniformly distributed across the liver lobule and were confined to liver parenchymal cells. The bile duct epithelial and the sinusoidal cells remained negative. The specificity and the high resolution of our protocol were further substantiated by reciprocal localization of transcripts for albumin and glyceraldehyde-3-phosphate dehydrogenase in different regions of the liver lobule and for catalase in the proximal tubules of the renal cortex. Whereas in control livers the transcripts for PH were barely detectable, a strong signal was found in pericentral hepatocytes of bezafibratetreated animals, corresponding to an 8-10-fold increase of mRNA detected in dot-blots. In contrast, the urate oxidase mRNA was reduced by more than 50%, with diminution of staining in pericentral regions of the liver lobule. The mRNA encoding for catalase was only slightly affected. Further applications of this protocol should be helpful in elucidation of the cell-specific transcriptional regulation of peroxisomal proteins in various organs under normal and pathological conditions.

Animals↗
PubMed1996

Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs.

We have studied the interaction of microtubules with peroxisomes and the influence of changes in the microtubular network on the peroxisomal compartment. From the several cell lines analyzed for this purpose, HepG2 cells proved to be the best candidate exhibiting both a well-developed cytoskeleton and a peroxisomal compartment with great plasticity. Three distinct types of peroxisomes: small spherical (0.1-0.3 micron), rod-shaped (0.5 micron) and elongated tubular (up to 5 microns) ones were identified in this cell line. A shift of the elongated tubular forms to spherical particles was noted by increasing the density of cells in culture, whereas no correlation between the distinct peroxisomal forms and the cellular proliferation could be observed. At time points when the elongated tubular peroxisomes were disappearing, many spherical peroxisomes arranged like 'chains of beads on a string' were observed, suggesting that the fission of elongated tubular forms may give rise to newly developing spherical peroxisomes. A clear association of spherical peroxisomes with microtubules was visualized by double immunofluorescence in combination with confocal laser scanning microscopy (CLSM). Treatment with a variety of microtubule-depolymerizing drugs (colcemid, nocodazole, vinblastine) induced a significant increase in the frequency of tubular peroxisomes and led to the formation of peroxisomal clusters. These effects were reversible since already 1 to 2 h after removal of the drugs from the culture medium, a uniform distribution of spherical peroxisomes was reestablished. Taxol, a microtubule-stabilizing drug, on the other hand exerted no significant effects on the peroxisomal compartment. The direct interaction of microtubules with peroxisomes in vitro was demonstrated using highly purified rat liver peroxisomes and taxol-stabilized microtubules from bovine or pig brain. The binding of peroxisomes to microtubules was visualized by video-enhanced contrast microscopy (VECM) and was abolished by pretreatment of peroxisomes with 100 mM KCl ('stripping'), proteinase K or trypsin. Incubation with cytosol restored the binding capacity of KCl-treated peroxisomes, but did not complement the protease treatment. The data presented provide for the first time evidence for a direct interaction of microtubules with the peroxisomal compartment indicating that this cytoskeletal system plays an important role in the morphogenesis and intracellular distribution of peroxisomes.

Actin Cytoskeleton↗