H. Dariush Fahimi

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

SummaryThe usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.

Mammalian peroxisomes and reactive oxygen species.

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-α on peroxisome function and peroxisome proliferator activated receptor-α. 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.

Reactive oxygen species and peroxisomes: struggling for balance.

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 H2O2‐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.

The peroxisome: still a mysterious organelle

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 β-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.

Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse)

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.

THE SHORT- AND LONG-TERM EFFECTS OF BEZAFIBRATE IN THE RAT*

Bezafibrate is a potent hypolipidemic agent, which causes marked proliferation of peroxisomes in rat liver. At the same dosage, bezafibrate is more effective in male than in female rats. This is probably related to divergent pharmacokinetics, which cause differences in drug level in serum and liver. The volume density of peroxisomes and several of their enzymes such as carnitine acetyl transferase and acyl-COA oxidase increase in a dose-related fashion. The hypolipidemic effect of bezafibrate, however, does not correlate with the used dosage. This implies that peroxisomal proliferation may play only a minor role in the hypolipidemic action of bezafibrate. In animals treated for 26 months with 300, 750, or 1500 ppm bezafibrate, the relative liver weight and serum triglycerides did not differ significantly from controls. Peroxisomal proliferation varied in different cells, being most prominent in single hepatocytes. The liver catalase activity was significantly reduced, but carnitine acetyl transferase was increased. Abnormal peroxisomes and mitochondria with longitudinal cristae were quite frequent. In one focus, catalase activity was severely diminished ahd peroxisomes were markedly reduced. The incidence of liver tumors was the same (1-3%) in treated animals as in controls.

Immunolocalization of four antioxidant enzymes in digestive glands of mollusks and crustaceans and fish liver

Abstract. 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 peroxisome: an update on mysteries

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.

TNF-α downregulates the peroxisome proliferator activated receptor-α and the mRNAs encoding peroxisomal proteins in rat liver

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

Growth and division of peroxisomes.

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.