Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.     

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.     

Nafenopin-induced proliferation of peroxisomes in the small intestine of mice.

The effect of nafenopin on the epithelial cells of the small intestine of mice was studied. After 17 days the control and nafenopin-treated groups were sacrificed. The tissues were incubated in alkaline DAB medium. Ultra-thin sections of small intestinal tissue from both groups were examined by electron microscopy. Electron micrographs were prepared and examined stereologically so that any morphologic differences in the epithelial cell peroxisomes and mitochondria between the experimental and control groups could be evaluated quantitatively. In the nafenopin-treated group proliferation of peroxisomes occurred, as indicated by significant increases in volume, and surface and numerical density of these structures compared with controls. No such alterations were found in the mitochondria. Our results show that the response of small intestinal epithelial cells to nafenopin is analogous to that produced in hepatocytes by the same drug. Hepatocyte peroxisomes are supposed to be involved in lipid metabolism and it seems that small intestinal epithelial peroxisomes play a similar role.     

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus

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.     

Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.     

Peroxisomes in airway epithelia and future prospects of these organelles for pulmonary cell biology

Peroxisomes are intimately involved in the metabolism of reactive oxygen species, in the synthesis of ether lipids and of polyunsaturated fatty acids as well as in the β-oxidation of bioactive and toxic lipid derivatives. Therefore, the metabolic pathways of this organelle might play an important role in pulmonary biology by protection of inner pulmonary surface epithelia against oxidative stress, induced by the high oxygen levels in the air and/or by regulation of the lipid homeostasis in pulmonary epithelia and the pulmonary surfactant film. In this article, original results on the distribution of peroxisomal marker proteins, involved in the biogenesis, ROS- and lipid-metabolism of this organelle in the bronchiolar epithelium and the alveolar region of the adult human lung in comparison to newborn and adult murine lungs are presented. In addition, we investigated the expression of the PEX11β-mRNA, encoding a protein involved in peroxisomal division. Our study revealed significant differences in the abundance and distribution of peroxisomal proteins in distinct cell types of the lung and different developmental stages and led to the discovery of species-specific differences in the peroxisomal compartment in pulmonary epithelia between mouse and man. Finally, the structure and general biology of pulmonary airways—with special emphasis on Clara cells—are reviewed and discussed in relation to peroxisomal metabolism and proliferation. Future prospects of peroxisomes and Pex11 proteins for pulmonary cell biology are highlighted. Presented at the 50th anniversary symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Peroxisomes in regenerating human skeletal myofibers

Peroxisomes of the human regenerating skeletal myofibers were studied qualitatively and quantitatively by electron cytochemistry and were compared with those of the mature normal human skeletal muscle fibers. Peroxisomes visualized by electron cytochemistry with 3,3'-diaminobenzidine (DAB) were small round or oval bodies delimited by a single membrane and contained the electron-opaque, coarsely granular matrix. Muscle grafts of the regenerating normal human quadriceps obtained from 4 orthopedic patients were analyzed 2 weeks after transplantation into nude mice; they contained peroxisomes with a mean diameter of 0.25 microns, ranging from 0.12 to 0.67 microns. The group mean density of peroxisomes per 100 microns2 was 2.0 +/- 0.4 (SE), while that of histochemically normal mature human quadriceps femoris myofibers was 0. The cytochemical controls without DAB or with the presence of 3-amino 1,2,4-triazole in the solution containing DAB lacked the electron-opaque reaction, indicating that these reactions were on an enzymatic basis. The results of this study showed clearly that the regenerating normal human skeletal myofibers contained numerous peroxisomes differing from the mature normal human muscle fibers in which the peroxisomes were not observed.     

Molecular Species of Phosphatidylcholine Containing Very Long Chain Fatty Acids in Human Brain: Enrichment in X-Linked Adrenoleukodystrophy Brain and Diseases of Peroxisome Biogenesis Brain

Molecular species of phosphatidylcholine containing unsaturated (i.e., monoenoic and polyenoic) 32- to 40-carbon (very long chain) fatty acids (VLCFA-PC) are present in normal human brain, the fatty acid composition changing significantly with development. There is a marked increase in the concentration and a change in the polyenoic VLCFA composition of these molecular species in brains of patients with inherited defects in peroxisomal biogenesis [Zellweger's syndrome, neonatal adrenoleukodystrophy (ALD), and infantile Refsum's disease]. In contrast, there is a marked increase in monoenoic VLCFA-PC in X-linked ALD whereas molecular species containing polyenoic VLCFA are minor components.     

Lipid metabolism in peroxisomes in relation to human disease

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1, fatty acid β-oxidation 2, fatty acid α-oxidation 3, synthesis of cholesterol and other isoprenoids 4, ether-phospholipid synthesis and 5, biosynthesis of polyunsaturated fatty acids. In Chapter 2–6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attentin is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for trransport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

The subcellular localization of phytanic acid oxidase in rat liver

Peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia) show a series of enzymatic defects related to peroxisomal dysfunctions. Accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) has been found in several of these patients, caused by a defect in the alpha-oxidation mechanism of this acid. The fact that the alpha-oxidation of phytanic acid is defective in the peroxisomal disorders as well as in classical Refsum's disease makes it likely that this oxidation normally takes place in the peroxisomes. A series of experiments preformed to localize the phytanic acid oxidase in subcellular fractions of rat liver show, however, that the alpha-oxidation of phytanic acid is a mitochondrial process. Free phytanic acid is the substrate, and the only cofactors necessary are ATP and Mg2+.     

Immunohistochemical localization of peroxisomal enzymes during rat embryonic development.

Peroxisomes are cytoplasmic organelles involved in a variety of metabolic pathways. Thus far, the morphological and biochemical features of peroxisomes have been extensively characterized in adult tissues. However, the existence of congenital peroxisomal disorders, primarily affecting tissue differentiation, emphasizes the importance of these organelles in the early stages of organogenesis. We investigated the occurrence and tissue distribution of three peroxisomal enzymes in rat embryos at various developmental stages. By means of a highly sensitive biotinyl-tyramide protocol, catalase, acyl-CoA oxidase, and ketoacyl-CoA thiolase were detected in embryonic tissues where peroxisomes had not thus far been recognized, i.e., adrenal and pancreatic parenchyma, choroid plexus, neuroblasts of cranial and spinal ganglia and myenteric plexus, and chondroblasts of certain skeletal structures. In other tissues, i.e., gut epithelium and neuroblasts of some CNS areas, they were identified earlier than previously. In select CNS areas, ultrastructural catalase cytochemistry allowed identification of actively proliferating organelles at early developmental stages in several cell types. Our data show that in most organs maturation of peroxisomes parallels the acquirement of specific functions, mainly related to lipid metabolism, thus supporting an involvement of the organelles in tissue differentiation.     

Germ cells of male mice express genes for peroxisomal metabolic pathways implicated in the regulation of spermatogenesis and the protection against oxidative stress

Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.     

Peroxisomes,lipid metabolism and lipotoxicity

Peroxisomes play an essential role in cellular lipid metabolism as exemplified by the existence of a number of genetic diseases in humans caused by the impaired function of one of the peroxisomal enzymes involved in lipid metabolism. Key pathways in which peroxisomes are involved include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis, and (3.) fatty acid alpha-oxidation. In this paper we will describe these different pathways in some detail and will provide an overview of peroxisomal disorders of metabolism and in addition discuss the toxicity of the intermediates of peroxisomal metabolism as they accumulate in the different peroxisomal deficiencies.     

Central role of peroxisomes in isoprenoid biosynthesis

Peroxisomes contain enzymes catalyzing a number of indispensable metabolic functions mainly related to lipid metabolism. The importance of peroxisomes in man is stressed by the existence of genetic disorders in which the biogenesis of the organelle is defective, leading to complex developmental and metabolic phenotypes. The purpose of this review is to emphasize some of the recent findings related to the localization of cholesterol biosynthetic enzymes in peroxisomes and to discuss the impairment of cholesterol biosynthesis in peroxisomal deficiency diseases.     

Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

MicroRNAs in alcoholic liver disease: Recent advances and future applications

Alcoholic liver disease (ALD) is characterized by hepatocyte damage, inflammatory cell activation, and increased intestinal permeability leading to the clinical manifestations of alcoholic hepatitis. Selected members of the family of microRNAs (miRNAs) are affected by alcohol, resulting in an abnormal miRNA profile in the liver and circulation in ALD. Increasing evidence suggests that miRNAs that regulate inflammation, lipid metabolism and promote cancer are affected by excessive alcohol administration in mouse models of ALD. This communication highlights recent findings in miRNA expression and functions as they relate to the pathogenesis of ALD. The cell-specific distribution of miRNAs, as well as the significance of circulating extracellular miRNAs, is discussed as potential biomarkers. Finally, the prospects of miRNA-based therapies are evaluated in ALD.     

Peroxisomes in plant reproduction and seed-related development

Peroxisomes are small multi-functional organelles essential for plant development and growth. Plant peroxisomes play various physiological roles, including phytohormone biosynthesis, lipid catabolism, reactive oxygen species metabolism and many others. Mutant analysis demonstrated key roles for peroxisomes in plant reproduction, seed development and germination and post-germinative seedling establishment; however,the underlying mechanisms remain to be fully elucidated. This review summarizes findings that reveal the importance and complexity of the role of peroxisomes in the pertinent processes. The b-oxidation pathway plays a central role, whereas other peroxisomal pathways are also involved. Understanding the biochemical and molecular mechanisms of these peroxisomal functions will be instrumental to the improvement of crop plants.