Cytochemical demonstration of catalasepositive particles (peroxisomes?) in fibroblasts

Summary The connective tissue of the mucosa of the respiratory tract, of the gastric mucosa and of the mucosa of the tongue was investigated in mice. The tissue was fixed in glutaraldehyde and incubated in an alkaline DAB-medium to demonstrate the peroxidatic activity of catalase. In fibroblasts and fibrocytes, as well as in lymphoid cells, membrane bounded particles from 0.10 to 0.25 m in diameter were found, whose matrices were intensely stained by the histochemical reaction. The reaction is inhibited by the addition of 2×10^–2 M 3-amino-1,2,4-triazole. In connective tissue cells of specimens, which were not reacted to demonstrate catalase activity, these organelles show a granular matrix of moderate electron density. They lack a crystalline core. The possibility that these catalase-positive particles (CPs) represent peroxisomes is discussed.This investigation was kindly supported by a grant of Hochschuljubiläumsstiftung der Stadt Wien.     

Localization of β-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues

Peroxisomes from spinach leaves, mungbean hypocotyls, and potato tubers catalyze a palmitoyl-CoA-dependent, KCN-insensitive O₂ uptake. In the course of this reaction O₂ is reduced to H₂O₂ in a 1:1 stoichiometry and palmitoyl-CoA oxidized, in a 1:1 stoichiometry, to a product serving as substrate for enoyl-CoA hydratase. These findings demonstrate the existence of a peroxisomal acyl-CoA oxidase in these tissues. Enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) are also associated with the peroxisomes from mung-bean hypocotyls and potato tubers (as well as with spinach leaf peroxisomes as recently reported; Gerhardt 1981, FEBS Lett. 126, 71). The low activities of these enzymes in mitochondrial fractions seem to be due to contaminating peroxisomes since the ratio of β-oxidation enzyme activities to catalase activity did not significantly differ between peroxisomal and mitochondrial fractions isolated on sucrose density gradients. The proof of localization of β-oxidation enzymes in peroxisomes without glyoxysomal function leads to the concept that fatty-acid oxidation is a consistent basic function of the peroxisome in cells of higher plants.     

Ultrastructural localization of L-α-hydroxy acid oxidase in rat liver peroxisomes

Summary The localization of L--hydroxy acid oxidase activity in rat liver peroxisomes was studied using slight modifications of the Shnitka and Talibi (1971) method. Best results were obtained with formaldehyde fixation and incubation with glycolate as substrate. Following incubation the copper ferrocyanide reaction product was amplified with 3,3-diaminobenzidine according to Hanker et al. (1972a, b). Dense reaction product was visible in hepatocyte peroxisomes by light and electron microscopy. Some diffusion of enzyme and/or reaction product into the adjacent cytoplasm occurred around the peroxisomes. Apparent non-specific deposits occurred on the plasmalemma, in the nucleus, and occasionally over mitochondria. Glutaraldehyde fixation severely inhibited enzymatic activity, and the enzyme showed less activity toward L-lactate and DL--hydroxybutyrate.     

How are peroxisomes formed? The role of the endoplasmic reticulum and peroxins

Recent data from studies of peroxisome assembly and the subcellular sorting of peroxisomal matrix and membrane proteins have led to an expansion of the 'growth and division' and 'endoplasmic reticulum-vesiculation' models of peroxisome biogenesis into a more flexible, unified model. Within this context, we discuss the proposed role for the endoplasmic reticulum in the formation of preperoxisomes and the potential for 15 Arabidopsis peroxin homologs to function in the biogenesis of peroxisomes in plant cells.     

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H₂O₂ (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillin N acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillin N) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.     

Peroxynitrite (ONOO?) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress

Background and Aims

Peroxisomes are subcellular compartments involved in multiple cellular metabolic pathways. Peroxynitrite (ONOO⁻) is a nitric oxide-derived molecule which is a nitrating species that causes nitration of proteins. This study used cell biology techniques to explore the potential presence of peroxynitrite in peroxisomes and evaluated its content under stress conditions (excess cadmium).

Methods

Peroxynitrite, nitric oxide and superoxide anion were studied using cell-permeable specific fluorescent probes by confocal laser scanning microscopy in Arabidopsis thaliana transgenic plants expressing cyan fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo.

Key Results

When no stress was applied, peroxynitrite was clearly localized in the peroxisomes of roots and stomatal guard cells. Under cadmium (150 μm) stress, the generation of peroxynitrite, nitric oxide and the superoxide anion (O₂^·–) increased and was localized in peroxisomes and the cytosol, participating in the generation of nitro-oxidative stress.

Conclusions

The results show that peroxisomes are an endogenous source of peroxynitrite, which is over-produced under cadmium stress, suggesting that the metabolism of reactive nitrogen species in peroxisomes could participate in the mechanism of the response to this heavy metal.     

Cytochemical localization by ferricyanide reduction of α-hydroxy acid oxidase activity in peroxisomes of rat kidney

Summary Conditions are described for the use of ferricyanide as an electron acceptor for the cytochemical demonstration by light and electron microscopy of mammalian L--hydroxy acid oxidase activity in peroxisomes of rat kidney. Enzyme activity survives brief fixation in cold formaldehyde or in Karnovsky's fixative. Cytochemical localization of -hydroxy acid oxidase activity in cryostat sections, or in finely chopped tissue blocks, is based on a simulaneous coupling reaction, in which ferrocyanide (produced by the enzymatic reduction of ferricyanide) is captured by copper to yield an insoluble, amorphous, electron-opaque deposit of cupric ferrocyanide (Hatchett's Brown). Under cytochemical conditions, the enzyme is most active in the presence of D,L--hydroxy butyric acid. The staining reaction requires the presence of substrate, and is abolished by heat treatment of sections. The use of rubeanic acid (dithiooxamide) is recommended for the visualization of the copper-containing reaction product by light microscopy. The cytochemical localization obtained is specific for peroxisomes located in cells of the proximal tubule of the rat nephron. By light microscopy, renal peroxisomes can be distinguished from lysosomes and mitochondria on the basis of their size, shape, number, and intracellular distribution. At an ultrastructural level, amorphous, electronopaque cupric ferrocyanide reaction product is precisely localized to the nucleoid and peripheral portion of the matrix of the peroxisome in lightly stained areas, and throughout the organelle, where staining is more intense. Staining results with the ferricyanide method for L--hydroxy acid oxidase, reported herein, are compared with those obtainable with the tetrazolium technic developed by Alien and Beard for the same enzyme, and with the 3,3-diamino-benzidine (DAB) method for catalase.This study was supported by grants MT-1273 and MT-1341 from the Medical Research Council of Canada.     

Immunocytochemical localization of L-α-hydroxyacid oxidase in dense bar of dumb-bell-shaped peroxisomes of monkey kidney

Localization of the B of L-hydroxyacid oxidase (HOX-B) in monkey kidney peroxisomes was investigated by immunoelectron microscopic techniques. Kidneys of Japanese monkeys,Macaca fuscata, were fixed with 4% paraformaldehyde+0.25% glutaraldehyde and embedded in LR White resin. Thin sections were stained for HOX-B and catalase by the immunogold technique. HOX-B was localized in the marginal plates of normal peroxisomes and the dense bar of dumb-bellshaped peroxisomes. Catalase was detected in the matrix of normal peroxisomes and in the terminal dilatations of dumb-bell-shaped peroxisomes. There were no gold particles indicating presence of catalase associated with the marginal plates or with the dense bars. Immunoblot analysis of monkey kidney homogenate showed that HOX-B has a molecular mass of 42 kDa that was slightly larger than that of rat kidney HOX-B (39 kDa). The results show that the dense bar of dumb-bell-shaped peroxisomes in monkey kidney is composed of at least HOX-B and is a variation of the marginal plates.     

Mitochondria and peroxisomes: Are the ‘Big Brother’ and the ‘Little Sister’ closer than assumed?

Mitochondria and peroxisomes are essential subcellular organelles in mammals. Despite obvious differences, both organelles display certain morphological and functional similarities. Recent studies have elucidated that these highly dynamic and plastic organelles share components of their division machinery. Mitochondria and peroxisomes are metabolically linked organelles, which are cooperating and cross-talking. This review addresses the dynamics and division of mitochondria and peroxisomes as well as their functional similarities to provide insight as to why these organelles share the fission machinery in evolutionary aspects.     

On the role of peroxisomes in the metabolism of lipids — evidence from studies on mammalian tissues in vivo

Summary Recent investigations into the role of peroxisomes in mammalian lipid metabolism have employed double isotope methodologies to examine the influence of peroxisomal agents on lipid turnover in the liver and extra hepatic tissues of the living animal.The action of these agents, all of which caused extensive changes in the flux of lipid metabolism in the treated animals, may best be viewed in relation to their effects on the common pathway of fatty acid oxidation in peroxisomes.Clofibrate, for example, acts through induction of peroxisomal oxidases and catalase; glycolate and ethanol through activation of this pathway; and aminotriazole and allylisopropylacetamide through inhibition of the catalase step in the sequence.The data from these studies provide support for the concept of an important contributory and regulatory role of peroxisomes in relation to the overall balance of lipid metabolism, and emphasize that these organelles play a significant role in the oxidation of common fatty acids, as well as a potential for the elimination of fatty acids that are poorly oxidized by mitochondria.Additionally, the data raise intriguing questions on the extension of peroxisomal influence to include phospholipid metabolism and the substantial degree of inter-tissue communication which is involved in the balance of lipid metabolism in the whole animal.     

Immunological detection of the mitochondrial F1-ATPase α subunit in the matrix of rat liver peroxisomes: A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the α-subunit of the mitochondrial F₁-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No β-subunit of the mitochondrial F₁-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature α-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the α-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Révision du genre Perrindema Lacroix 1997 (Insecta : Coleoptera : Scarabaeoidea : Melolonthidae : Pachydeminae)

La synonymie entre Perrindema Lacroix 1997, et Zanitanus Lacroix 2001, est proposée. Le genre Perrindema est révisé. Une nouvelle combinaison est établie pour Cephaloncheres lindiensis Moser 1919. Deux nouvelles espèces sont décrites du Mozambique : Perrindema quiterajoensis n. sp. et P. pembaensis n. sp. Un historique du genre est donné ainsi qu’une clé de détermination. La position systématique de ce genre au sein des Pachydemini Burmeister 1855, est discutée. La conservation de l’utilisation du nom Pachydeminae au détriment de Tanyproctinae Erichson 1847, est argumentée.