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Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.

Peroxisomes are key metabolic organelles, which contribute to cellular lipid metabolism, e.g. the β-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as cellular redox balance. Peroxisomal dysfunction has been linked to severe metabolic disorders in man, but peroxisomes are now also recognized as protective organelles with a wider significance in human health and potential impact on a large number of globally important human diseases such as neurodegeneration, obesity, cancer, and age-related disorders. Therefore, the interest in peroxisomes and their physiological functions has significantly increased in recent years. In this review, we intend to highlight recent discoveries, advancements and trends in peroxisome research, and present an update as well as a continuation of two former review articles addressing the unsolved mysteries of this astonishing organelle. We summarize novel findings on the biological functions of peroxisomes, their biogenesis, formation, membrane dynamics and division, as well as on peroxisome-organelle contacts and cooperation. Furthermore, novel peroxisomal proteins and machineries at the peroxisomal membrane are discussed. Finally, we address recent findings on the role of peroxisomes in the brain, in neurological disorders, and in the development of cancer.

Peroxisomes are ubiquitous dynamic and multifunctional organelles that contribute to numerous anabolic and catabolic pathways, being essential for human health and development. Their best known functions include the oxidation of fatty acids and metabolism of hydrogen peroxide with catalase as a marker enzyme. Indeed, historically, it was the cytochemical staining of catalase in many different cells and tissues that revealed the ubiquitous presence of peroxisomes in almost all animal and plant cells. In this chapter, the method for cytochemical staining of catalase with the alkaline 3, 3'-diaminobenzidine (DAB) is described. Since aldehyde fixation is a prerequisite for staining of catalase with DAB, a method for perfusion fixation of rat liver with glutaraldehyde is presented prior to the cytochemical staining method and the subsequent tissue processing for light and electron microscopy.

Peroxisomes are remarkably plastic and dynamic organelles, which fulfil important functions in hydrogen peroxide and lipid metabolism rendering them essential for human health and development. Despite great advances in the identification and characterization of essential components and molecular mechanisms associated with the biogenesis and function of peroxisomes, our understanding of how peroxisomes are incorporated into metabolic pathways and cellular communication networks is just beginning to emerge. Here we address the interaction of peroxisomes with other subcellular compartments including the relationship with the endoplasmic reticulum, the peroxisome-mitochondria connection and the association with lipid droplets. We highlight metabolic cooperations and potential cross-talk and summarize recent findings on peroxisome-peroxisome interactions and the interaction of peroxisomes with microtubules in mammalian cells.

Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.

Peroxisomes contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, which render them indispensable to human health and development. Peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. In recent years, the interest in peroxisomes and their physiological functions has significantly increased. This review intends to highlight recent discoveries and trends in peroxisome research, and represents an update as well as a continuation of a former review article. Novel exciting findings on the biological functions, biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross-talk of peroxisomes with other subcellular compartments are addressed. Furthermore, recent findings on the role of peroxisomes in the brain are discussed.

Reactive oxygen species (ROS) can surely be considered as multifunctional biofactors within the cell. They are known to participate in regular cell functions, for example, as signal mediators, but overproduction under oxidative stress conditions leads to deleterious cellular effects, cell death and diverse pathological conditions. Peroxisomal function has long been linked to oxygen metabolism due to the high concentration of H(2)O(2)-generating oxidases in peroxisomes and their set of antioxidant enzymes, especially catalase. Still, mitochondria have been very much placed in the centre of ROS metabolism and oxidative stress. This review discusses novel findings concerning the relationship between ROS and peroxisomes, as they revealed to be a key player in the dynamic spin of ROS metabolism and oxidative injury. An overview of ROS generating enzymes as well as their antioxidant counterparts will be given, exemplifying the precise fine-tuning between the opposing systems. Various conditions in which the balance between generation and scavenging of ROS in peroxisomes is perturbed, for example, exogenous manipulation, ageing and peroxisomal disorders, are addressed. Furthermore, peroxisome-derived oxidative stress and its effect on mitochondria (and vice versa) are discussed, highlighting the close interrelationship of both organelles.

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as beta-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed.

Peroxisomes (PO) are essential and ubiquitous single-membrane-bound organelles whose ultrastructure is characterized by a matrix and often a crystalloid core. A unique feature is their capacity to generate and degrade H(2)O(2) via several oxidases and catalase, respectively. Handling of H(2)O(2) within PO is poorly understood and, in contrast to mitochondria, they are not regarded as a default H(2)O(2) source. Using an ultrasensitive luminometric H(2)O(2) assay, we show in real time that H(2)O(2) handling by matrix-localized catalase depends on the localization of H(2)O(2) generation in- and outside the PO. Thus, intact PO are inefficient at degrading external but also internal H(2)O(2) that is generated by the core-localized urate oxidase (UOX). Our findings suggest that, in addition to the PO membrane, the matrix forms a significant diffusion barrier for H(2)O(2). In contrast, matrix-generated H(2)O(2) is efficiently degraded. We further show that the tubular structures in crystalloid cores of UOX are associated with and perpendicularly oriented toward the PO membrane. Studies on metabolically active liver slices demonstrate that UOX directly releases H(2)O(2) into the cytoplasm, with the 5-nm primary tubules in crystalloid cores serving as exhaust conduits. Apparently, PO are inefficient detoxifiers of external H(2)O(2) but rather can become an obligatory source of H(2)O(2)--an important signaling molecule and a potential toxin.

The discovery of the colocalization of catalase with H2O2-generating oxidases in peroxisomes was the first indication of their involvement in the metabolism of oxygen metabolites. In past decades it has been revealed that peroxisomes participate not only in the generation of reactive oxygen species (ROS) with grave consequences for cell fate such as malignant degeneration but also in cell rescue from the damaging effects of such radicals. In this review the role of peroxisomes in a variety of physiological and pathological processes involving ROS mainly in animal cells is presented. At the outset the enzymes generating and scavenging H2O2 and other oxygen metabolites are reviewed. The exposure of cultured cells to UV light and different oxidizing agents induces peroxisome proliferation with formation of tubular peroxisomes and apparent upregulation of PEX genes. Significant reduction of peroxisomal volume density and several of their enzymes is observed in inflammatory processes such as infections, ischemia-reperfusion injury and hepatic allograft rejection. The latter response is related to the suppressive effects of TNFalpha on peroxisomal function and on PPARalpha. Their massive proliferation induced by a variety of xenobiotics and the subsequent tumor formation in rodents is evidently due to an imbalance in the formation and scavenging of ROS, and is mediated by PPARalpha. In PEX5-/- mice with the absence of functional peroxisomes severe abnormalities of mitochondria in different organs are observed which resemble closely those in respiratory chain disorders associated with oxidative stress. Interestingly, no evidence of oxidative damage to proteins or lipids, nor of increased peroxide production has been found in that mouse model. In this respect the role of PPARalpha, which is highly activated in those mice, in prevention of oxidative stress deserves further investigation.

Peroxisomes are ubiquitous subcellular organelles, which are highly dynamic and display large plasticity in response to cellular and environmental conditions. Novel proteins and pathways that mediate and control peroxisome formation, growth, and division continue to be discovered, and the cellular machineries that act together to regulate peroxisome number and size are under active investigation. Here, advances in the field of peroxisomal dynamics and proliferation in mammals and yeast are reviewed. The authors address the signals, conditions, and proteins that affect, regulate, and control the number and size of this essential organelle, especially the components involved in the division of peroxisomes. Special emphasis is on the function of dynamin-related proteins (DRPs), on Fis1, a putative adaptor for DRPs, on the role of the Pex11 family of peroxisomal membrane proteins, and the cytoskeleton.

Heme oxygenase (HO)-1, the inducible isoform of the rate-limiting enzyme of heme degradation, and peroxiredoxin (Prx) I, a thioredoxin-dependent peroxidase, are multifunctional antioxidant stress proteins which are coordinately up-regulated by oxidative stress in cell cultures. HO-1 and Prx I exhibit a different hepatic cellular and subcellular localization. Here, a distinct expression pattern of the two genes was confirmed by in situ hybridization of normal rat liver. Moreover, expression of the HO-1 and Prx I genes was determined in a model of acutely damaged rat liver which was elicited by application of a single dose of carbon tetrachloride (CCl4). The mRNA levels of the HO-1 and Prx I genes were induced in whole livers of CCl4-treated rats with differential kinetics as determined by Northern blot analysis. While HO-1 mRNA was induced up to 48 hr, Prx I exhibited a maximum level of mRNA after 12 hr of treatment with CCl4. CCl4-dependent oxidative stress led to a focal increase of perivenous HO-1 positive liver cells with simultaneous loss of Prx I immunoreactivity. Taken together, the complementary hepatic gene expression pattern of HO-1 and Prx I in response to oxidative stress may suggest a functional interplay of these antioxidant genes.

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor-alpha on peroxisome function and peroxisome proliferator activated receptor-alpha. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5-/- knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.

K-111 has been characterized as a potent peroxisome proliferator-activated receptor (PPAR)alpha activator. Antidiabetic potency and amelioration of disturbed lipid metabolism were demonstrated in rodents, which were accompanied by elevations of peroxisomal enzymes and liver weight. To examine the possible therapeutic application of K-111 we have now assessed its efficacy in non-human primates with high transferability to humans. For this purpose obese, hypertriglyceridaemic, hyperinsulinaemic prediabetic rhesus monkeys were dosed sequentially with 0, 1, 3 and 10mg/kg per day orally over a period of 4 weeks each. In addition, the effect of K-111 on the peroxisome compartment was analyzed in cynomolgus monkeys using liver samples obtained following a 13-week oral toxicity study. In prediabetic monkeys, the reduction of hyperinsulinaemia and improvement of insulin-stimulated glucose uptake rate indicated amelioration of insulin resistance. These effects were nearly maximal at a dose of 3mg/kg per day, while triglycerides and body weight were lowered significantly in a dose-dependent manner. This reduction of body weight contrasts sharply with the adipogenic response observed with thiazolidinediones, another family of insulin-sensitizing agents. In young cynomolgus monkeys at a dosage of 5mg/kg per day and more, K-111 induced an up to three-fold increase in lipid beta-oxidation enzymes with an 1.5- to 2-fold increase in peroxisome volume density. This moderate increase in peroxisomal activity by K-111 in monkeys is consistent with its role as an PPARalpha activator and corresponds to the observations with fibrates in other low responder mammalian species. The increase in beta-oxidation may explain, at least in part, the lipid modulating effect as well as the antidiabetic potency of K-111. This pharmacological profile makes K-111 a highly promising drug candidate for clinical applications in the treatment of type 2 diabetes, dyslipidaemia, obesity and the metabolic syndrome.

During the last decade, peroxisome proliferation has emerged as a novel biomarker of exposure to certain organic chemical pollutants in aquatic organisms. Peroxisome proliferation is mediated by nuclear receptors, peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described in mammals: PPAR alpha, PPAR beta and PPAR gamma. PPARs have also been discovered in several fish species. The aim of the present study was to investigate the expression of PPAR subtypes and their cellular distribution patterns in the liver of gray mullet Mugil cephalus, a fish species widely distributed in estuaries and coastal areas in Europe and used as sentinel of environmental pollution. For this purpose, antibodies were generated against the three subtypes of mouse PPARs and different protocols of antigen retrieval were used. In western blots, main bands were detected of approximately 44 kDa for PPAR alpha, two bands of 44 and 58 kDa for PPAR beta and a single band of 56 kDa for PPAR gamma. Similar results were obtained in mouse liver and may indicate antibody recognition of two forms of the protein in certain cases. PPAR alpha was the subtype most markedly expressed in gray mullet liver, being expressed mainly in melanomacrophages, nuclei of hepatocytes and sinusoidal cells and connective tissue surrounding bile ducts. PPAR beta was expressed in the same cell types but immunolabeling was generally weaker than for PPAR alpha. PPAR gamma showed very weak expression; positivity was mainly found in melanomacrophages and connective tissue surrounding bile ducts. Our results demonstrate that all the three PPAR subtypes are expressed in gray mullet liver but in different intensities. The cellular distribution patterns of PPAR subtypes in gray mullet liver resembled partly those found in mouse liver with PPAR alpha as the main subtype expressed in hepatocytes. The fact that melanomacrophages, cells of the immune system in fish, show strong expression of both PPAR alpha and PPAR beta whereas PPAR gamma expression is almost restricted to this cell type suggest a significant role of PPAR-mediated regulation of cell function in melanomacrophages.

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A-gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.

In the era of application of molecular biological gene-targeting technology for the generation of knockout mouse models to study human genetic diseases, the availability of highly sensitive and reliable methods for the morphological characterization of the specific phenotypes of these mice is of great importance. In the first part of this report, the role of morphological techniques for studying the biology and pathology of peroxisomes is reviewed, and the techniques established in our laboratories for the localization of peroxisomal proteins and corresponding mRNAs in fetal and newborn mice are presented and discussed in the context of the international literature. In the second part, the literature on the ontogenetic development of the peroxisomal compartment in mice, with special emphasis on liver and intestine is reviewed and compared with our own data reported recently. In addition, some recent data on the pathological alterations in the liver of the PEX5(-/-) mouse with a peroxisomal biogenesis defect are briefly discussed. Finally, the methods developed during these studies for the localization of mitochondrial proteins (respiratory chain complexes and MnSOD) are presented and their advantages and pitfalls discussed. With the help of these techniques, it is now possible to identify and distinguish unequivocally peroxisomes from mitochondria, two classes of cell organelles giving by light microscopy a punctate staining pattern in microscopical immunohistochemical preparations of paraffin-embedded mouse tissues., (Copyright 2003 Wiley-Liss, Inc.)

Recent in vivo observations have revealed that peroxisomes are more dynamic and interactive than previously assumed. The growing recognition of the tubular and reticular morphology of peroxisomes in living cells, their association with microtubules, and the dynamic movements of peroxisomes in vivo and in vitro have inspired the query into the investigation of the cellular machinery that mediates such a complex behaviour. The characterisation of the underlying molecular components of this machinery is providing insight into the mechanisms regulating peroxisomal morphology and intracellular distribution., (Copyright 2003 Wiley-Liss, Inc.)

Peroxisomes are cell organelles that perform multiple functions in the metabolism of lipids and of reactive oxygen species. They are present in most eukaryotic cells. However, they are believed to be absent in spermatozoa and they have never been described in male germ cells. We have used the immortalized germ cell line GC1spg to investigate the expression of peroxisomal proteins in germ cells of mice. The GC1spg cells represent the differentiation state of type B spermatogonia or preleptotene spermatocytes. We could show that peroxisomal membrane proteins like Pmp70 and Pex14p as well as peroxisomal matrix proteins like catalase or acyl CoA oxidase are expressed in GC1spg cells. All these proteins were colocalized in the same structures within the cells. Furthermore, by electron microscopy we have identified subcellular particles with an ultrastructural appearance that is characteristic of peroxisomes. This is the first report demonstrating the peroxisomal compartment in male germ cells of mice., (Copyright 2003 S. Karger AG, Basel)

Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.

The unusual amino acid D-aspartate is present in significant amounts in brain and endocrine glands and is supposed to be involved in neurotransmission and neurosecretion (Wolosker et al. [2000] Neuroscience 100:183-189). D-aspartate oxidase is the only enzyme known to metabolize D-aspartate and could regulate its level in different regions of the brain. We examined the cellular and subcellular distribution of this enzyme and its mRNA in human and rat brain by immunohistochemistry, in situ hybridization, and immunoelectron microscopy. D-aspartate oxidase protein and mRNA are ubiquitous. The protein shows a granular pattern, particularly within neurons and to a significantly lesser extent in astrocytes and oligodendrocytes. No evidence for a synaptic association was observed. Whereas between most positive neurons only gradual differences were observed, in the hypothalamic paraventricular nucleus, neurons with high enzyme content were found next to others with no labeling. cDNA cloning of D-aspartate oxidase corroborates an inherent targeting signal sequence for protein import into peroxisomes. Immunoelectron microscopy showed that the protein is localized in single membrane-bound organelles, apparently peroxisomes., (Copyright 2002 Wiley-Liss, Inc.)

Zellweger syndrome (cerebro-hepato-renal syndrome) is the most severe form of the peroxisomal biogenesis disorders leading to early death of the affected children. To study the pathogenetic mechanisms causing organ dysfunctions in Zellweger syndrome, we have recently developed a knockout-mouse model by disrupting the PEX5 gene, encoding the targeting receptor for most peroxisomal matrix proteins (M Baes, P Gressens, E Baumgart, P Carmeliet, M Casteels, M Fransen, P Evrard, D Fahimi, PE Declercq, D Collen, PP van Veldhoven, GP Mannaerts: A mouse model for Zellweger syndrome. Nat Genet 1997, 17:49-57). In this study, we present evidence that the absence of functional peroxisomes, causing a general defect in peroxisomal metabolism, leads to proliferation of pleomorphic mitochondria with severe alterations of the mitochondrial ultrastructure, changes in the expression and activities of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial compartment in various organs and specific cell types (eg, liver, proximal tubules of the kidney, adrenal cortex, heart, skeletal and smooth muscle cells, neutrophils). The changes of mitochondrial respiratory chain enzymes are accompanied by a marked increase of mitochondrial manganese-superoxide dismutase, as revealed by in situ hybridization and immunocytochemistry, suggesting increased production of reactive oxygen species in altered mitochondria. This increased oxidative stress induced probably by defective peroxisomal antioxidant mechanisms combined with accumulation of lipid intermediates of peroxisomal beta-oxidation system could contribute significantly to the pathogenesis of multiple organ dysfunctions in Zellweger syndrome.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)

We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusion-fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen retrieval combined with biotin-avidin-HRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal beta-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of peroxisomes. (J Histochem Cytochem 49:155-164, 2001)

The aim of this work was to determine the immunolocalization of the antioxidant enzymes catalase, Cu,Zn-superoxide dismutase (SOD), Mn-SOD, and glutathione peroxidase (GPX) in the bivalve mollusks Mytilus galloprovincialis and Crassostrea sp., the crab Carcinus maenas, and the teleostean fish Mugil cephalus. By immunoblotting, crossreactivity between antibodies and the corresponding proteins in the digestive gland/hepatopancreas of invertebrates and the fish liver was demonstrated. Immunohistochemical studies showed that the stomach epithelium was strongly immunostained for catalase in mollusks. In crabs, ducts showed stronger immunostaining than tubules and in mullet hepatocytes the reaction appeared in discrete granules corresponding to peroxisomes. With regard to Cu,Zn-SOD, the apex of the tubule cells in mussels and crabs was distinctly immunostained, whereas in oysters the reaction was more marked in ducts and in mullet liver a uniform diffuse cytoplasmic staining was found. Mn-SOD was strongly positive in mollusk and crab ducts and in mullet periportal hepatocytes. Finally, GPX was not detected in mussels while in oysters a slight reaction was noted in all cell types. In crabs, connective tissue cells and the apex of duct cells were immunostained, but in mullet liver only erythrocytes appeared reactive. Immunoelectron microscopy revealed that catalase was localized in peroxisomes with a dense labeling in fish and less intense labeling in invertebrates. Cu,Zn-SOD was mainly a cytosolic protein although additional positive subcellular sites (peroxisomes, nuclei) were also observed, while Mn-SOD was restricted to mitochondria. GPX was localized in the cytosol, nucleus, and lysosomes, occurring also in peroxisomes of the fish liver. The results presented here provide a basis for future application of the immunodetection techniques to study the possible differential induction of antioxidant enzymes in aquatic organisms subjected to oxidative stress as a result of exposure to environmental pollutants.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3'-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.

The subcellular compartmentalization of urate oxidase (UOX) in the digestive glands of mussels, Mytilus galloprovincialis Lmk, was studied by means of immunoblotting and immunocytochemistry, using an antibody raised in rabbit against rat liver UOX. Western blot analysis of subcellular fractions revealed an immunoreactive polypeptide with a molecular weight similar to the corresponding mammalian hepatic protein. This crossreactive polypeptide of 32 kDa was particle-bound yet not peroxisome-associated. In paraffin sections the antiserum specifically labeled the plasma membrane of the digestive gland epithelial cells and discrete regions within the perinuclear and apical portions of the digestive tubules and duct cells. By electron microscopy gold particles representing antigenic sites were found on the microvilli and the lateral plasma membrane as well as the membranes of the secretory/ endocytic compartments, that is, the Golgi complex, secretory and some endocytic vesicle membranes. Since the peroxisomal UOX-antibody exhibits a comparable immunoreactivity towards a urate-transporter channel protein in rat kidney proximal tubules and has been used for its molecular cloning (Leal-Pinto et al., 1997, J. Biol. Chem. 272, 617-625), we suggest that the membrane protein identified in mussel digestive glands could represent a homologous urate-transporter protein.

Peroxisomes of the digestive glands of mussels, Mytilus galloprovincialis Lmk, were investigated by immunoblotting and immunohistochemistry using rabbit antibodies against several mammalian hepatic peroxisomal proteins. Western blot analysis of main subcellular fractions revealed immunoreactive polypeptides with molecular weights comparable to those of the corresponding mammalian hepatic proteins. They could be localized to the peroxisomal matrix in the case of catalase, multifunctional enzyme (PH), and palmitoyl-CoA oxidase (AOX), and to the peroxisomal membrane in respect to PMP 70. The purification of peroxisomes by metrizamide density gradient centrifugation revealed the existence of two subpopulations with densities of 1.16 and 1.20 g cm(-3) exhibiting different protein compositions. In paraffin sections, positive immunolabeling for catalase was distributed along the apical cytoplasm of the epithelia of digestive ducts and stomach and throughout the cytoplasm of digestive tubule cells. The peroxisomal beta-oxidation enzymes, AOX and PH, also appeared predominantly in the ducts and the stomach epithelia with a weaker immunolabeling in the tubules. At the electron microscopic level a clear labeling with gold particles was observed in the peroxisomal matrix with the anti-guinea pig catalase antibody. In addition to peroxisomes, the anti-PH antibody also labeled the mitochondria. The similarity in the protein composition of molluscan and mammalian peroxisomes as revealed by the present study indicates that those proteins have been well conserved in evolution suggesting that functionally peroxisomes in molluscs could also be involved in the metabolism of lipids and in detoxification of xenobiotics. Thus, the antibodies tested could provide useful tools for detection of peroxisomal induction in molluscan biomonitoring programs for the assessment of aquatic environmental pollution.

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3'-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of "preperoxisomes" is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins. (J Histochem Cytochem 47:1219-1232, 1999)

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (rho = 1.22-1.24 g/cm(3)) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.

Peroxisomes and the activities of their enzymes have been reported to be significantly reduced in various types of tumors including the colon carcinoma. Therefore, the present study was designed to investigate the gene expression of several peroxisomal proteins in human colon carcinoma and additionally those of the peroxisome proliferator activated receptor alpha (PPARalpha) and PEX5, a receptor protein involved in the import of most peroxisomal matrix proteins. Samples from adenocarcinomas and adjacent normal colon were analyzed by immunohistochemistry and western blotting. The mRNA content was assessed by a novel sensitive dot blot RNase protection assay and northern blotting. By immunohistochemistry, peroxisomes were distinctly visualized in normal colonocytes but were not detected in colon carcinoma cells. The protein levels of catalase (CAT), acyl-CoA oxidase as well as the 22 and 70 kDa peroxisomal membrane proteins (PMP22 and PMP70) were all significantly decreased in carcinomas. The corresponding mRNAs for CAT and PMP70, however, were unchanged. In contrast, the mRNA of PEX5 was significantly increased. The expression of PPARalpha was not altered in tumors, neither at protein nor mRNA levels. These observations show that the reduction of peroxisomes and their proteins in colon carcinoma is not due to a generalized reduction of transcription of their genes. It seems more likely that this phenomenon is regulated at a post-transcriptional or translational level. Alternatively, and more likely, an impairment of the biogenesis of the organelle could account for the paucity of peroxisomes in colon carcinoma.

The electron microscopical studies presented here show that characteristic morphological alterations in mitochondria are a very early hallmark of the hepatocellular apoptotic program. Before chromatin condensation occurs, the outer mitochondrial membrane is focally disrupted and the inner membrane protrudes through this gap forming a hernia. The demonstration of cytochrome oxidase in mitochondria revealed a very strong activity in pre-apoptotic and apoptotic cells as well as in apoptotic bodies.

We have studied the alterations of peroxisomes in the human hepatoblastoma cell line HepG2, induced to differentiate by long-term cultivation (20 days without passaging) using morphological and biochemical techniques as well as mRNA analysis. Ultrastructural studies revealed alterations in shape and size of peroxisomes, with significant increases in mean diameter and formation of small clusters exhibiting heterogeneous staining for catalase after 20 days in culture. These alterations of peroxisomes correspond to the changes described during the maturation process from prenatal to adult human hepatocytes. As revealed by Northern and Western blotting there was marked elevation of the mRNA (190%) and protein (180%) of the peroxisomal branched-chain acyl-CoA oxidase. This protein is the key regulatory enzyme for the side chain oxidation of cholesterol for bile acid synthesis, a pathway associated with mature hepatocytes. Concomitantly a marked increase of bile canaliculi was noted by light and electron microscopy. This differentiation process was confirmed also by the increase of albumin synthesis (mRNA: 160%; protein: 190%) which is generally used as a differentiation marker of hepatocytes in culture. Interestingly, the mRNA for peroxisome proliferator-activated receptor alpha (PPAR alpha) increased drastically by almost 390% and its corresponding protein by 150%, suggesting its involvement in maturation of the peroxisomal compartment in differentiating HepG2 cells. In contrast to the wellknown increases during the drug-induced peroxisome proliferation of cytochrome P450 4A, multifunctional enzyme 1, palmitoyl-CoA oxidase and the 70-kDa peroxisomal membrane protein, those proteins were either not altered or only slightly elevated during the differentiation process, suggesting that peroxisome proliferation and maturation are two distinct and differentially regulated processes.

Background: During allograft rejection, cytokines and lipid mediators contribute to cell injury and organ failure. Peroxisomes play a crucial role in lipid metabolism, including the degradation of lipid mediators by peroxisomal beta-oxidation. Therefore, we investigated the alterations of hepatic peroxisomes after allogeneic rat liver transplantation., Methods: MHC-incompatible Dark Agouti (RT1a) donor rats and Lewis (RT1(1)) recipient rats were used for allogeneic transplantation. For immunosuppression, a group of these animals received cyclosporine (CsA) intraperitoneally (1 mg/kg body weight per day). Lewis rats were used for isogeneic transplant combination. Ten days after transplantation, livers were investigated using morphometrical methods for determination of peroxisomal diameter and volume density. The activities of peroxisomal catalase (CAT) and acyl-coenzyme A oxidase (AOX) were determined, and the corresponding proteins were evaluated by quantitative immunocytochemistry and immunoblotting. The expressions of mRNAs encoding CAT and AOX were investigated by Northern blotting., Results: The volume density and diameter of peroxisomes were significantly decreased in allogeneic transplanted livers but were unchanged in CsA-treated animals. Both the activities of CAT and AOX and their protein levels were significantly reduced in liver allografts. Moreover, the corresponding mRNA levels of CAT and AOX were decreased significantly in liver allografts, whereas CsA treatment led to an increase of those mRNAs. Isogeneic transplanted livers showed only a slight reduction of the corresponding enzyme values., Conclusions: Peroxisomes are severely affected both morphologically and functionally after allogeneic liver transplantation. These results suggest that impairment of peroxisomal lipid beta-oxidation could contribute to the pathogenesis of the rejection process by decreased catabolism of lipid mediators involved in the regulation of the inflammatory response. CsA, in addition to its immunosuppressive effects, may contribute to allograft survival by maintenance of those important peroxisomal functions.

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.

Immune free-flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen-specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH approximately 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG-coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post-mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.

Marine mussels are used as bioindicators of water pollution in marine and estuarine environments in the so-called "Mussel Watch" programs because of their capacity to accumulate numerous organic xenobiotics including aromatic hydrocarbons. In this study, we have analyzed the effects of two xenobiotics [benzo(a)pyrene and the water accommodated fraction of a lubricant oil] and two typical (rodent) peroxisome proliferators (clofibrate and dioctyl phthalate) on structure and function of peroxisomes in digestive glands of mussels Mytilus galloprovincialis, either following water exposure (for 1, 7, and 21 days) or after direct injection through the adductor muscle (for 1 and 7 days). The activities of catalase (CAT), acyl-CoA oxidase (AOX), and D-amino acid oxidase were determined in whole homogenates of digestive glands. In addition, stereological methods were applied on sections stained histochemically for demonstration of catalase activity in order to quantify the morphological changes of peroxisomes. The peroxisomal acyl-CoA oxidase and D-amino acid oxidase were increased in mussels injected for 7 days with benzo(a)pyrene, phthalate, and clofibrate and a similar trend was noted for benzo(a)pyrene and lubricant oil in water exposure experiments (21 days). The catalase activity was reduced or unchanged depending on the mode of exposure of animals. By stereology, significant increases of numerical and volume densities of peroxisomes were found in animals injected for 7 days with lubricant oil or clofibrate. These observations indicate that peroxisomal oxidases in mussels are induced at moderate rates in response to different xenobiotics and that their determination could provide a (sensitive) marker for detection of effects of some toxic pollutants, particularly the lubricant oils which in addition induce significant structural alterations of mussel peroxisomes.

We showed recently the plasticity of the peroxisomal compartment in the human hepatoblastoma cell line HepG2 as evidenced by the presence of elongated tubular peroxisomes measuring up to 5 microm next to much smaller spherical or rod-shaped ones (0.1-0.3 microm). Since the occurrence of tubular peroxisomes in a given cell in culture is synchronized, with neighboring cells containing either small spherical or elongated tubular peroxisomes, cell counting of immunofluorescence preparations stained for catalase was used for the quantitative assessment of the dynamics of the peroxisomal compartment and the factors regulating this process. Initial studies revealed that the formation of tubular peroxisomes is primarily influenced by the cell density as well as by lipid- and protein-factors in fetal calf serum, being independent of an intact microtubular network. Biochemical studies showed that the occurrence of tubular peroxisomes correlated with the expression of the mRNA for 70 kDa peroxisomal membrane protein (PMP70), but not with that of matrix proteins. By cultivation of cells in serum- and protein-free media specific factors were identified which influenced the formation of tubular peroxisomes. Among several growth factors tested, nerve growth factor (NGF) was the most potent one inducing tubular peroxisomes and its effect was blocked by K252b, a specific inhibitor of neurotrophin receptor pathway, suggesting the involvement of signal transduction in this process. Furthermore, from several polyunsaturated fatty acids (PUFA) which all induced tubular peroxisomes, the arachidonic acid (AA) was the most potent one. Our observations suggest that tubular peroxisomes are transient structures in the process of rapid expansion of the peroxisomal compartment which are induced either by specific growth factors or by polyunsaturated fatty acids both of which are involved in intracellular signaling.

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.

Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta-oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of cytochrome P450 IVA and the peroxisome proliferation in both sexes support the notion of a functional relationship. In view of the almost exclusive periportal localization of DHEA-induced tumors in female rats in contrast to the pericentral localization of the peroxisomal proliferation shown by this study, it seems likely that other factors in addition to peroxisome proliferation may contribute to the hepatocarcinogenic effect of DHEA.

We have studied the effects of TNF-alpha on the mRNAs coding for the peroxisome proliferator activated receptor alpha (PPAR-alpha), and for catalase (Cat), acyl-CoA oxidase (AOX), multifunctional enzyme (PH), and beta-actin in rat liver. Total RNA was isolated from livers of male SD-rats 16 h after administration of a single dose of 25 microg TNF-alpha and mRNAs were analyzed by a novel dot blot RNase protection assay. The mRNAs for PPAR-alpha and for Cat, AOX and PH were significantly reduced by TNF-treatment. In addition, the level of PPAR-alpha protein was also decreased after TNF. In contrast, the mRNA for beta-actin was markedly increased implying that the effect of TNF on PPAR-alpha and the peroxisomal mRNAs is highly selective. This effect may have important implications in perturbation of the lipid metabolism induced by TNF-alpha.