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Visualization of Peroxisomes (Microbodies) and Mitochondria with Diaminobenzidine
... Seligman et al. (1968) proposed the use of COX histochemistry for the ultrastructural localisation and visualisation of cytochrome c (COX) in rat liver. Using DAB as an electron acceptor, the visualisation of COX activity can be demonstrated by the electron-dense deposits forming with osmium on the inner mitochondrial membrane and in the intracristae space with EM (Seligman et al., 1968;Beard & Novikoff, 1969;Novikoff & Goldfischer, 1969;Seligman et al., 1970;Reith & Schuler, 1972;Roels, 1974). Since then, many reports of EM studies of COX localisation have appeared with several changes to the original protocol. ...
... It is known that mitochondrial ultrastructural preservation is a common problem in cytochemical studies, since fixation reduces enzymatic function by hindering conformational changes. Furthermore, incubation conditions (Seligman et al., 1967) as well as fixation time, type of fixative (Sabatini et al., 1963) and fixative concentration will also influence the cytochemistry reaction (Sabatini et al., 1963;Seligman et al., 1967;Novikoff & Goldfischer, 1969;Nonaka et al., 1989;Saprunova et al., 2008). Indeed, Hirai and colleagues demonstrated that DAB is inactivated in overfixed tissues and enzyme activity, including COX and peroxidases, is impaired (Hirai, 1971;Roels, 1974;Herzog & Fahimi, 1976). ...
Mitochondrial shape and function are known to be linked, therefore there is a need to combine three-dimensional EM structural analysis with functional analysis. Cytochrome c oxidase labelling is one approach to examine mitochondrial function at the EM level. However, previous efforts to apply this method have had several issues including inconsistent results, disruption to mitochondrial ultrastructure, and a lack of optimisation for volume EM methods. We have used short fixation and microwave processing to address these issues. We show that our method gives consistent cytochrome c oxidase labelling and improves labelling penetration across tissue volume. We also quantify mitochondrial morphology metrics, including in volume EM, to show that ultrastructure is unaltered by the processing. This work represents a technical advance that allows the correlation of mitochondrial function and morphology with greater resolution and volume than has previously been feasible. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Some secretory vesicles of a mucus-secreting cell are stained (arrow) while others are not (arrowhead). ac, absorptive cells; mc, mucus-secreting cells; nu, nuclei; st, stomach; vi, villi For catalase detection, tissue fragments fixed as above were incubated for 1 h at 35 C in medium containing 2 mg/ml 3,3 0diaminobenzidine tetrahydrochloride and 0.06% hydrogen peroxide in 0.2 mol l −1 Tris-HCl buffer pH 8.5 (adapted from Novikoff & Goldfischer, 1969). For both enzymes, post-fixation was carried out for 2 h at room temperature with 1% OsO 4 and 1.5% potassium ferrocyanide in cacodylate buffer, dehydrated and embedded in epoxy resin. ...
The esophageal pouches of Chaetopleura angulata and Acanthochitona fascicularis were investigated using light and transmission electron microscopy. These pouches linked to the posterior region of the esophagus are known as sugar glands as they contain a fluid rich in polysaccharide digesting enzymes. They are the second largest glands in the digestive system of chitons, just after the digestive gland. In both species , the pouches contain a dense array of finger-shaped villi. The villi epithelium includes absorptive cells, basophilic secretory cells, mucus-secreting cells, and basal cells. Some absorptive cells were bordered by a dense cover of long microvilli, whereas other absorptive cells had short and sparse microvilli. Absorptive cells contain several lysosomes, mitochondria, peroxisomes, a few small Golgi stacks, some lipid droplets, and large amounts of glycogen. The basophilic secretory cells are characterized by the presence of many electron-dense vesicles, with a glycoprotein content , a large number of rough endoplasmic reticulum cisternae, and a highly developed Golgi apparatus. Mucus-secreting cells are characterized by large vesicles containing acid polysaccharides and wide Golgi stacks. Basal cells that were found at the base of the epithelium in contact with the basal lamina exhibit histological and ultrastructural features of enteroendocrine cells. We suggest that these glandular pouches are involved in extracellular and intracellular digestion, and accumulate lipid and glycogen reserves. K E Y W O R D S arylsulphatase, digestive system, histochemistry, lysosomes, secretory cells
... The ultrathin sections were double-stained with uranyl acetate and lead citrate and observed with a JEOL 1200 EX electron microscope (JEOL Ltd, Tokyo, Japan). To detect peroxisomes, cytochemical staining for peroxisomal catalase was conducted according to the method of Novikoff and Goldfischer (1969). After washing with PBS, the tissue blocks were incubated in alkaline 3,3′-diaminobenzidine (DAB) at 37 °C for 90 min. ...
It was reported that 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide and a possible endocrine disruptor, can disturb spermatogenesis, but the precise mechanism is not understood. Since 2,4-D is a weak peroxisome proliferator in hepatocytes and peroxisome proliferator-activated receptor α (PPARα) is also expressed in Leydig cells, this study aimed to investigate the link between PPARα and 2,4-D-mediated testicular dysfunction. 2,4-D (130 mg/kg/day) was administered to wild-type and Ppara-null mice for 2 weeks, and the alterations in testis and testosterone/cholesterol metabolism in Leydig cells were examined. Treatment with 2,4-D markedly decreased testicular testosterone in wild-type mice, leading to degeneration of spermatocytes and Sertoli cells. The 2,4-D decreased cholesterol levels in Leydig cells of wild-type mice through down-regulating the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 and reductase, involved in de novo cholesterogenesis. However, the mRNAs encoding the important proteins involved in testosterone synthesis were unchanged by 2,4-D except for CYP17A1, indicating that exhausted cholesterol levels in the cells is a main reason for reduced testicular testosterone. Additionally, pregnancy rate and the number of pups between 2,4-D-treated wild-type male mice and untreated female mice were significantly lower compared with those between untreated couples. These phenomena were not observed in 2,4-D-treated Ppara-null males. Collectively, these results suggest a critical role for PPARα in 2,4-D-induced testicular toxicity due to disruption of cholesterol/testosterone homeostasis in Leydig cells. This study yields novel insights into the possible mechanism of testicular dysfunction and male infertility caused by 2,4-D.
... La caractérisation des peroxysomes a été facilitée par la mise au point d'une technique de coloration cytochimique en 1968 par l'équipe de Novikoff et Goldfisher en utilisant la diaminobenzadine (DAB) permettant dans un milieu à pH alcalin de visualiser ces organites sur la base de l'activité peroxydasique de la catalase (Novikoff et Goldfischer, 1969 (Gondcaille et coll., 2005). ...
La déficience en Acyl-CoA oxydase 1 (ACOX1) est une leucodystrophie peroxysomale rare et sévère associée à un déficit dans la beta-oxydation des acides gras à très long chaine. A cause du rôle clé de ce déficit peroxysomal microglial dans la physiopathogenèse de la déficience en ACOX1, nous avons utilisé la lignée microgliale murine BV-2 comme modèle : (i) pour évaluer les propriétés antioxydantes et anti-inflammatoires des extraits de raquettes du cactus Opuntia ficus-indica ; (ii) pour caractériser une nouvelle lignée BV-2 déficiente en ACOX1 générée récemment dans notre laboratoire par édition génique grâce à la méthode CRISPR-Cas9. Dans la première partie des travaux, les cellules BV-2, activées par exposition à quatre sérotypes de lipopolysaccharides (LPS), montre un lien entre la structure du LPS et l’effet sur la -oxydation des acides gras et les enzymes antioxydantes dans le peroxysome : les LPS dérivant d’Escherichia coli diminuent l’activité ACOX1 alors que les LPS de Salmonella minnesota réduisent l’activité catalase. Remarquablement, les différents extraits de cactus stimulent l’activité catalase. Cet effet antioxydant est accompagné par un effet anti-inflammatoire attesté par la réduction de la production LPS-dépendante d’oxyde nitrique dans les BV-2. Nos résultats suggèrent que les extraits de cactus auraient une activité neuroprotectrice sur les cellules microgliales activées à travers l’induction d’activités anti-oxydantes peroxysomales et l’inhibition de la production de NO. Dans la deuxième partie des travaux, la caractérisation de la lignée BV-2 déficiente en ACOX1, portant des mutations alléliques, confirme l’absence de la protéine et de l’activité ACOX1. Bien que ces cellules aient une croissance plus faible, elles ne montrent pas de modifications morphologiques détectables. Par contre, l’activité catalase, due à l’enzyme peroxysomale dégradant l’H2O2, est augmentée. Les études par fractionnement subcellulaire et par ultracentrifugation en gradient Nycodenz révèlent une modification de la densité et de la taille de peroxysomes. De plus, ces cellules microgliales déficientes montrent une profonde modification de l’expression des gènes liés à l’inflammation (IL-1b, IL-4, TNF-alpha) et particulièrement l’expression de la protéine CCL2/MCP-1 impliquée dans la neuro-inflammation. Cette nouvelle lignée microgliale déficiente en ACOX1 révèlent les mêmes dérégulations biochimiques que celles décrites chez les patients déficients en ACOX1 et représente donc un modèle pour l’étude des conséquences du déficit de la -oxydation peroxysomale dans la microglie sur les fonctions peroxysomales, le stress oxydatif, l’inflammation et les fonctions cellulaires.
Cancer cells exhibit a unique phenotype of efficiently altered metabolism that encompasses not only the bioenergetic demands of the cell, but also generation of precursors of biosynthetic molecules, along with the induction of homeostatic pathways that support uncontrolled cell growth and proliferation. Mitochondria, the cellular metabolic hubs, play a key role in cancer pathophysiology. We have only started to understand the complex metabolic interplay between the cancer cells and the microenvironment, especially involving the role of mitochondria and their underlying mechanisms that sustain cancer cell growth. In this chapter, we review the studies uncovering the role of mitochondria from the perspective of a normal cell undergoing changes resulting in uncontrolled growth and tumor formation.
Normal and acatalasemic mice were fed 0.5% clofibrate (CPIB) for 70 days and variations of catalase activity and thermal stability of liver catalase were investigated.
1. The specific catalase activity (catalase activity/g liver weight) in the liver of mice which received CPIB compared to that of controls was calculated. The specific catalase activity was in 2.2 normal mice and 1.2 in acatalasemic mice. A significant increase of activity was recognized in normal mice but not in acatalasemic mice.
2. The specific catalase activity in the blood of mice which received CPIB to that of control was determined. The specific catalase activity was 1.3 in normal mice and 1.1 in acatalasemic mice. A significant increase was not observed in either kind of mice.
3. The heat stability of catalase in the liver of normal or acatalasemic mice which received with CPIB was higher than that of normal or acatalasemic mice which did not received CPIB.
4. Increased levels of ALP and ChE activities and decreased levels of GOT activity and TG concentration were observed in the normal or acatalasemic mice which received CPIB, compared to control mice.
5. Isozyme of ALP were determined by electrophoresis. Increased ALP in the plasma of normal and acatalasemic mice was thought to be derived from liver ALP.
In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.
Nephrotoxicity is a costly health problem (CEC/IPCS, 1989) that arises from exposure to environmental pollutants, industrial chemicals and a broad array of therapeutic agents (see Porter, 1982; Bach and Lock, 1987, 1989; Bach et al., 1988, 1990). These clinical nephropathies have, therefore, been studied extensively in animal models with a view to understanding their molecular basis. All too often in the past, however, lesions of clinical relevance have been documented in terms of gross functional changes (such as elevated serum creatinine, blood urea nitrogen, etc.), and advanced morphological degeneration, i.e. end-stage renal disease, using relatively non-specific histological staining such as Haematoxylin and Eosin. Such information is of little value in understanding renal lesions because mechanistic studies need subtle, early effects to be documented. Many nephrotoxic affect discrete anatomical regions of the kidney, while some of these substances damage only specific renal cell types. This concept of target cell toxicity is important to study the mechanisms of nephrotoxicity, as the basis of such discrete injury suggests that there are biochemical characteristics which predispose these cells to the toxic effects of certain chemicals, while the properties of the unaffected cells protect them or make them less susceptible. Sensitive and highly selective methods are necessary to study the molecular basis of injury to the affected region of the kidney and the time-course of injury and recovery.
Cytochemistry at the ultrastructural level is a broad subject, encompassing considerations of fixation and its effect on enzyme activity, problems with reagent and label penetration of tissues and cells, and problems distinguishing reaction products from other cellular constituents. The aim of this chapter is to introduce some of the different general types of staining procedures frequently employed to demonstrate specific chemical entities associated with cellular surfaces and cytoplasmic contents.
Peroxisomes or microbodies are subcellular particles which are characterized by their content of catalase and H2O2-producing oxidative enzymes. These organelles were first described as catalase and urate oxidase-containing particles in liver which, on differential centrifugation, sedimented between the mitochondrial and microsomal fractions (L or λ-fraction) (1,2). Peroxisomes were subsequently identified as the microbodies of liver and kidney (0.5–1.0 µm diameter) described previously by the electron microscopists (1,3). Further work by Novikoff and coworkers resulted in a catalase-specific cytochemical stain (alkaline diaminobenzidine) which revealed the presence of very small (0.1 µm–0.25 µm diameter) catalase-containing particles which they named microperoxisomes (4,5). While the large-sized peroxisomes are found only in liver and kidney of animals, microperoxisomes occur in all tissues.
Cytochemistry at the ultrastructural level is a broad subject, encompassing considerations of fixation and its effect on enzyme activity, problems with reagent and label penetration of tissues and cells, and problems distinguishing reaction products from other cellular constituents. The aim of this chapter is to introduce some of the different general types of staining procedures frequently employed to demonstrate specific chemical entities associated with cellular surfaces and cytoplasmic contents.
Recent reports on the use of lead precipitating procedures for localizing intracellular phosphatases and criticism of these procedures (see (1) and (2) for the most recent interchange of views) have left undiminished the value of using the following phosphatases as cytochemical “markers” for light and electron microscopy (3, 4): plasma membrane — nucleoside phosphatases (e.g., Mn++ — and Mg++-stimulated “ATPase”, 5’-nucleotidase, Co++-stimulated CMPase, etc.); endoplasmic reticulum (ER) of liver, kidney, endocrine-secreting cells, etc. -- a nucleoside diphosphatase (NDPase) that hydrolyzes IDP, UDP, GDP (also TPP) but not CDP or ADP (5, 6); Golgi apparatus -- NDPase, thiamine pyrophosphatase (TTPase) (see 6)); Golgi-endoplasmic reticulumlysosome (GERL) -- acid phosphatase; and lysosomes -- acid phosphatase. Optimal oxidation at high pH and relatively high H2O2 concentration of diaminobenzidine may be used to “mark” peroxisomes because of their high catalase content (7, 8). Mitochondria oxidize diaminobenzidine optimally at low pH and low H2O2 concentration (7).
Particular chemicals in vivo specifically damage the glomeruli, the medullary cells or the proximal tubular cells. Elucidation of the mechanism(s) of such unique interactions between the target cell and the toxin has proved most difficult using conventional biochemical methods. Histochemical methods may be helpful, e.g. to define toxin distributions or the location of enzymes that effect requisite activation. Isolated renal cells may be used to study the molecular basis of target cell toxicity, provided that — as we have shown for several such chemicals — the same cell type is damaged in vitro.
Rilopirox (HOE 351) is a hydroxy-pyridone compound with antimycotic properties. Hydroxy-pyridones are generally active against medically important dermatophytes, yeasts and moulds, and they exhibit a significant fungicidal potency even against non-proliferating fungal cells and spores.
Ultrastructural studies in our laboratory have previously shown that alcoholic hyalin and megamitochondria are separate and distinct entities within the hepatic cytoplasm of patients with liver disease associated with alcoholism.1 Alcoholic hyalin presents as aggregates of fibrillar material without a limiting membrane and without special association with any organelle. Enlarged mitochondria or megamitochondria are easily identified, regardless of their size or shape because of their characteristic outer membranes and cristae. Hyalin and megamitochondria are also identifiable in thick Epon sections stained by a modified basic fuchsin-crystal violet stain. We were able, therefore, to make direct correlations of the morphology of any specific structure by studying thick sections with a light microscope and the adjacent thin sections with the electron microscope.
The diaminobenzidine (DAB) technique developed by Graham and Karnovsky (1966) has been widely used in the study of the localization of peroxidatic activity in various types of cell, including macrophages. On the basis of the intercellular distribution of peroxidatic activity, two types of macrophage have been distinguished, viz. resident macrophages and exudate macrophages. There are discrepancies between the findings of different authors as well as differences between the animal species used, as described below.
Methods to localize intracellular sites of enzymes with electron microscopy grew directly from light microscopic histochemistry. Techniques, such as Gomori’s (1952) for acid phosphatase activity, which had as end products heavy metal ions with sufficient mass to scatter electrons, were directly applicable for electron microscopy if noncoagulative fixatives were used. Introduction of catalytic osmiophilic polymer generation (Hanker et al., 1972a) made substrates previously valuable only for light microscopy useful for electron microscopy. In the most widely used strategies for electron microscopic localization of enzymes in microorganisms, enzymes are not viewed directly, but reactions with end products impart electron density to the sites of enzyme activity. There are numerous strategies for the cytochemical localization of enzymes, and this chapter will emphasize three of these: (1) ion capture and precipitation of products; (2) ferricyanide reduction and product amplification; and (3) oxidative polymerization of diaminobenzidine. Thus, this discussion is not exhaustive of all techniques used for microorganisms but is illustrative of rationales used in attempting to identify sites of enzyme activities.
The morphology, function, and kinetics of mononuclear phagocytes are usually studied in peritoneal macrophages obtained from experimentally induced peritoneal exudates. Although it has been convincingly demonstrated that most of the mononuclear phagocytes present in peritoneal exudates are monocytes deriving from the peripheral blood, it cannot be entirely excluded that a probably varying proportion of the mononuclear phagocytes present in the peritoneal exudates may be resident peritoneal macrophages, i.e. cells already present in the peritoneal cavity.
The early research on peroxisomes in plants was centred mainly on three groups, my own at Purdue (later at University of California, Santa Cruz) and those of Tolbert at Michigan State University and Newcomb at Wisconsin. Some of the collaborators from these centres subsequently established their own research laboratories and, as interest in the field expanded, others independently joined the international association. Several authors in the present volume trace their lineage in peroxisomal research either directly or indirectly to these three groups, but progress has been such that the questions they now address are well beyond the vision of their ancestors.
The aim of this brief chapter is to review some of the recent contributions in the general area of cell biology of animal peroxisomes with special emphasis on ultra-structural and cytochemical aspects.
Zoospores, prosporangia, and asexual sporangia were studied with electron microscopy to determine the ultrastructural identification of “chromidia,” granular masses surrounding nuclei that classical mycologists believed to be extruded chromatin used for lipid synthesis. In the zoospore the nucleus was enclosed by an aggregation of ribosomes. In other developmental stages the behavior of microbodies was identical to that described for “chromidia.” A microbody network with interspersed ER surrounded nuclei in young prosporangia. As the prosporangium matured, lipid globules became associated with the microbodies. When the single, large nucleus migrated into the elongate asexual sporangium, microbodies still surrounded the nucleus; but after the nucleus divided and a multinucleate sporangium formed, microbodies were scattered throughout the cytoplasm. When incubated in the diaminobenzidine medium for the cytochemical detection of catalase, reaction product was found in these microbodylike structures, confirming that “chromidia” described in prosporangia and asexual sporangia by classical mycologists are really microbodies. Rather than giving rise to lipid, these microbodies are probably involved in the metabolism of the lipid globules with which they are associated. The “chromidia” in zoospores are not extruded chromatin as suggested earlier, but correspond in their location around the nucleus to an aggregation of ribosomes.
The cells of the supracaudal gland of guinea pig have abundant cytoplasm, round and ovoid mitochondria, abundant free ribosomes, a few rough cisternae, abundant vesicular reticulum, complex agglomerates of tubular reticulum, a few residual bodies and a few multivesicular bodies. Golgi complex is small. Electron lucid lipid droplets are numerous. Cell membranes interdigitate. Desmosomes and tonofibrils are frequent. Single membrane limited particles 200-300 mμ in diameter show close spatial relations to cisternae and lipid droplets (Fig. 1). Glutaraldehyde fixed tissue reacted for demonstration of catalase identifies these particles as microbodies at light (Fig. 3) and EM (Fig. 5) level. Aminotriazole abolishes reaction.
The uropygial or preen gland of hen is composed of similar cells. Some lipid droplets contain lamellar or reticular material (Fig. 2). Rough reticulum is moderately abundant and lumina contain pale droplets or liposomes. Particles with dense matrix contain pale granules with lamellar substructure. Microbodies (Fig. 2) 200-800 mμ in diameter are numerous. They are associated with ER, lipid or cisternae containing dense fibrils (Fig. 7). DAB reaction is positive at light (Fig. 4) and EM (Fig. 6) level. It is abolished by aminotriazole. Specific activities of catalase in homogenates of both glands are about 50% of those found in livers.
The effects of chloramphenicol (CAP) on mitochondrial respiratory activity in the wild strain (ST) and in a cytoplasmic CAP-resistant mutant (STR 1 ) of Tetrahymena pyriformis were studied by determining oxygen consumption, by spectrophotometry, and by cytochemistry. In the absence of CAP both strains had the same respiration capacity, and the low-temperature spectra of their isolated mitochondria were similar. Furthermore, the mitochondria of both strains showed a positive reaction with diaminobenzidine, denoting a similar cytochrome oxidase activity. However, when cells were grown in CAP for 24 or 48 h, the peaks of cytochrome oxidase and cytochrome b were almost absent in the wild type. In this type the oxygen consumption was greatly decreased, and the mitochondria were no longer stained by diaminobenzidine. In the mutant, the peaks of cytochrome oxidase and cytochrome b were decreased only; respiration was less affected than in the wild type, and cytochrome oxidase activity was still disclosed by the diaminobenzidine reaction. These results show that CAP inhibits the synthesis of two cytochromes ( b and oxidase) which are partially translated into the mitochrondria of T. pyriformis. In the mutant, CAP reduces only the mitochondrial translation, resulting in reduced mitochondrial activity and reduced growth rate of the cell. These results are compared with the nucleo-mitochondrial regulation mechanisms discussed in our previous works.
We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.
Successful electron microscope cytochemistry invariably requires a consideration of the initial methods for tissue processing, particularly the fixation procedure. Preservation of enzyme activities usually requires a mild fixation, for instance, with formaldehyde or low concentrations of glutaraldehyde, because strong fixatives, such as osmium tetroxide, or high concentrations of glutaraldehyde usually inhibit enzyme activity. However, weak fixatives may lead to less optimal structural preservation, and a suitable balance has to be reached between adequate fixation and ultrastructural preservation. Therefore, the preparatory procedures may differ for different enzymes or for other substances. The choice of fixative and fixation procedure is crucial to the outcome of enzyme cytochemistry because enzymes vary considerably with respect to their sensitivity to different fixatives. Potassium-dependent p-nitrophenylphosphatase, which is a partial enzyme activity of Na,K-ATPase, can only be preserved with formaldehyde fixatives of low strength but is inhibited by glutaraldehyde fixation. In contrast, acid phosphatase is much more resistant and is active even after fixation in 3% glutaraldehyde for 2 hours.
The Leydig cells of the rat testis which are involved in testosterone production contain an abundance of smooth endoplasmic reticulum and mitochondria (Figs. 2,6). These cells also possess many peroxisomes, lysosomes and multivesicular bodies (MVB's). On the cell surface, the plasma membrane contains numerous short microvilli, small invaginations and large plasmalemmal folds which appear to engulf extracellular fluid. There are also many large dilated vacuoles adjacent to the cell surface. The purpose of the present study is to determine if these cells show endocytic activity and to differentiate by various cytochemical means lysosomal elements from peroxisomes.
To identify lysosomes, tissue chopper sections of 2% glutaraldehyde-fixed testes (containing 2.5% dextran) were incubated in media containing thiamine monophosphate as a substrate (Lalli, 1983) to demonstrate the presence of acid phosphatase or in media containing P-nitrocatechol sulfate for the demonstration of arylsulfatase (Hopsu-Havu et al., 1967).
Peroxidase activity has been identified in the monocyte of several species, but could not be demonstrated in the rabbit monocyte except in the rough endoplasmic reticulum (RER) and digestive vacuoles of "resident" macrophages. Aminotriazole-sensitive catalase activity has been demonstrated in the RER of guinea pig monocytes, but has not been demonstrated in their granules or in the rabbit monocyte. Recently, we attempted to localize catalase activity in the monocyte and to cytochemically differentiate between catalase and myeloperoxidase (MPO) reactivity.
Peroxisomes in normal human hepatocytes are catalase-containing rounded or oval organelles with an average diameter of 0.63 μm (l). They are usually surrounded by single tripartite membranes. Their presence can be easily demonstrated by the use of 3,3' diaminobenzidine (DAB) stain in the presence of H 2 0 2 (2). This paper represents an ultrastructural study of peroxisomes in tne liver biopsy of an alcoholic patient with preoperative diagnosis of fatty infiltration.
Biopsy tissues were fixed in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% OsO 4 and embedded in epoxy resin. Thin sections were stained with lead citrate and uranyl acetate and examined with a Zeiss EM 9 S-2.
Light microscopic study of the liver biopsy reveals an intact hepatic architecture. The hepatocytes appear slightly edematous and possess many eosinophilic granules.
Electron microscopy (Fig l) reveals that there is a marked increase in the number of randomly distributed peroxisomes in alcoholic human hepatocytes.
A number of compounds of osmium VIII including osmiamates and coordination complexes of OsO4 with various heterocyclic nitrogen compounds have been synthesized, characterized and evaluated as substitutes for OSO4 in cytochemistry. The compounds synthesized include ammonium osmiamate (NH 4 OsO 3 N), potassium osmiamate (KOSO 3 N), and addition complexes of OSO4 with hexamethylenetetramine, pyridine, quinoline, piperazine, and sym. collidine.
The ammonium osmiamate was relatively unstable. The potassium osmiamate, although stable, was insoluble. Pyridine formed a stable yellow complex with OsO4 which was insoluble in water and sublimed readily at room temperature. All of the other nitrogen heterocycles formed unstable molecular addition complexes which decomposed in a short period of time with the exception of hexamethylenetetramine. The latter formed a stable complex, which had a negligible vapor pressure of OSO4, and did not sublime like many of the other octavalent osmium compounds. Spectroscopic studies, potentiometric titration,elemental analysis, and atomic absorption studies with an osmium lamp indicated the structure shown in Fig. 1 (top).
Biochemical and differential centrifugation studies have demonstrated that the oxidative enzymes of Acanthamoeba sp . are localized in mitochondria and peroxisomes (microbodies). Although hartmanellid amoebae have been the subject of several electron microscopic studies, peroxisomes have not been described from these organisms or other protozoa. Cytochemical tests employing diaminobenzidine-tetra HCl (DAB) and hydrogen peroxide were used for the ultrastructural localization of peroxidases of trophozoites of Hartmanella sp. (A-l, Culbertson), a pathogenic strain grown in axenic cultures of trypticase soy broth.
The fat body of insects has always been compared functionally to the liver of vertebrates. Both synthesize and store glycogen and lipid and are concerned with the formation of blood proteins. The comparison becomes even more apt with the discovery of microbodies and the localization of urate oxidase and catalase in insect fat body.
The microbodies are oval to spherical bodies about 1μ across with a depression and dense core on one side. The core is made of coiled tubules together with dense material close to the depressed membrane. The tubules may appear loose or densely packed but always intertwined like liquid crystals, never straight as in solid crystals (Fig. 1). When fat body is reacted with diaminobenzidine free base and H 2 O 2 at pH 9.0 to determine the distribution of catalase, electron microscopy shows the enzyme in the matrix of the microbodies (Fig. 2). The reaction is abolished by 3-amino-1, 2, 4-triazole, a competitive inhibitor of catalase. The fat body is the only tissue which consistantly reacts positively for urate oxidase. The reaction product is sharply localized in granules of about the same size and distribution as the microbodies. The reaction is inhibited by 2, 6, 8-trichloropurine, a competitive inhibitor of urate oxidase.
Myeloid cells are known to contain myeloperoxidase (MPO) and catalase. This study has used MPO and catalase replete and deficient myeloid cell lines to clarify the localization of these components using 3,3’-diaminobenzidine (DAB) ultrastructural cytochemistry. Conditions of DAB incubation can be modified to preferentially stain catalase (alkaline at pH 9.7) or MPO (neutral at pH 7.0-7.6), but crossreactivity persists, preventing the discrimination between catalase and peroxidase. Biochemical assays demonstrated both MPO and catalase in HL60 cells; similar amounts of catalase but no MPO activity in the A7 cell line; increased amounts of catalase but no MPO activity in the HP50 and HP100 cell lines; and neither MPO nor catalase in the KG1 cell line. Neutral DAB stained MPO (pH 7.4; [DAB] 5 or 20 mg/10 mL 0.05 M Tris; 30 min or 120 min; 24° or 37°C; 0.01% H 2 O 2 ) in HL60 (Fig. 1), but not in A7. Alkaline DAB intensely stained catalase (pH 9.7; 20 mg/10 mL; 120 min; 37°C; 0.01% or 0.03%) in A7.
Diabetes mellitus (DM) is a heterogeneous set of multifactorial pathogenesis syndrome where the common nexus is metabolic disorder, mainly chronic hyperglycemia and alterations in lipid and protein metabolism. The effects of DM, include long-term damage, dysfunction, and failure of various organs. It especially affects eyes, kidneys, muscle, nerves, heart, and blood vessels. The primary goal of diabetes treatment is the prevention of macrovascular complications (e.g., myocardial infarction, heart failure, and ischemic stroke) as well as the microvascular complications (e.g., retinopathy, neuropathy, and nephropathy). Abnormalities in mitochondrial function are common in the pathophysiology of diabetes that include modifications in the redox state and oxidative and nitrosative stress, as well dysregulation of mitochondrial complex activities. Oxidative stress is a factor that contributes to the development of complications in diabetes; however, its effects can be counteracted using exogenous antioxidants that are found in some plants, which is why people turn to traditional medicines in the search for therapeutic treatment. Identification of major compounds in extracts of medicinal plants can contribute to ameliorate hyperglycemia and oxidative stress due to exacerbated mitochondrial dysfunction in diabetes. The growing need to find alternatives for the treatment of diabetes justifies the study of medicinal plants used in traditional medicine. In this study, we aimed to review information related to possible treatments with bioactive compounds from medicinal plants on diabetes that affect several organs, including liver, heart, brain, muscle, and kidney with exacerbated oxidative stress originated mainly in mitochondria.
Reliable visual identification of peroxisomes is important in developmental, clinical, and investigational research. The current technique employed in most laboratories uses a specific electron dense label for the demonstration of peroxisomes by transmission electron microscopy by applying 3,3'- Diaminobenzidine Tetrahydrochloride (DAB) directly to freshly fixed tissue samples to react with endogenous peroxisomal catalase. After routine processing, ultrastructural examination of tissue sections is conducted either with light staining or without post-staining of grids. While peroxisomes are easily identified using this method, remaining tissue architecture is difficult to visualize due to the opacity of the tissue. Additionally, if grids are post-stained with heavy metal solutions, they must be modified to allow for enough staining to visualize cellular components without compromising the quality of the peroxisome label. We will describe a technique whereby DAB-reacted tissues are stained with a postfixative solution including potassium ferricyanide that imparts density to cell membranes and cellular components thereby enhancing identification and interpretion of data.
Cultivation in liquid Kessler’s medium containing 0.2 % methanol stimulated the growth of Chlamydomonas reinhardtii autotrophic batch culture. To elucidate the mechanism, we examined the effects of methanol on the enzymatic activity of catalase, catalase gene (CAT1) expression, and ultrastructure of C. reinhardtii cells. CAT1 relative expression was detected by real time RT-PCR. The cellular ultrastructure was investigated using transmission electron microscopy (TEM). Catalase-mediated oxygen evolution from H2O2 was assayed with a Clark-type electrode. The localization of catalase activity in C. reinhardtii cells grown in the presence of methanol was studied by a method based on cytochemical staining with 3,3'-diaminobenzidine-tetrahydrochloride (DAB). The cytochemical data obtained in this study confirmed catalase localization in mitochondria of C. reinhardtii cells. It was shown biochemically that mitochondrial catalase activity of the alga can be enhanced by methanol. CAT1 gene expression after 4 h of growth with methanol was 16-fold higher than in the control samples. Methanol addition induced quantitative changes in ultrastructure of C. reinhardtii cells: the volume of C. reinhardtii cells and fractions of cell area occupied by chloroplasts decreased, the areas of vacuoles, mitochondria, and plastoglobules increased. The results suggest that mitochondrial catalase takes part in the oxidation of methanol.
The ultrastructure of dormant basidiospores of Psilocybe cubensis is described. The spore wall is characterized by three distinct layers and a germ pore. A pore cap is described for the first time in a species of Psilocybe. The protoplast is surrounded by a well defined plasma membrane with many distinct invaginations. Internally, large numbers of nonmembrane-bound lipids occur at both ends of the spore. Two nuclei are typically present and the nuclear envelope has many nuclear pores. Mitochondria with only a few, but well delineated, plate-like cristae are present. There is scant endoplasmic reticulum. Ribosomes occur regularly attached to the ER and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. Variously sized vacuoles were demonstrated cytochemically to contain acid phosphatase. Microbody-like organelles are described but cytochemical tests to determine if these organelles are functionally similar to those of higher plants were unsuccessful. Cell-free extracts of basidiospores exhibit catalase and isocitrate lyase activity but no malate synthase activity.
The ultrastructure of microbodies in zoospores, hyphae and developing sporangia was studied in Monoblepharella sp. Catalase activity in these organelles was demonstrated with the diaminobenzidine cytochemical technique. In zoospores elongate microbodies lay adjacent to lipid globules, ribosomal aggregations, and rumposomes. Hyphal microbodies varied in morphology and were either oval, elongate, reticulate or ring shaped. Microbodies in developing sporangia were elongate, branched and closely appressed to lipid globules. This study supports the validity of the microbody-lipid-globule complex as a possible phylogenetic marker in Chytridiomyoetes.
Because microbodies in many filamentous fungi fail to stain with the 3,3′-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalasc. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.
Balantioides coli is a ciliated protozoon that inhabits the intestine of pigs, non-human primates and humans. Light microscopy studies have described over 50 species of the genus Balantioides but their validity is in doubt. Due to the limited information about this genus, this study is aimed to identify morphological characteristics of Balantioides coli isolated using fluorescence microscopy and both scanning (SEM) and transmission electron microscopy (TEM). Trophozoites isolated from the feces of pig and macaque were washed and subjected to centrifugation. These cells were fixed with paraformaldehyde for immunofluorescence. Other aliquots of these trophozoites were fixed with glutaraldehyde, post fixed with osmium tetroxide and processed for SEM and TEM. Immunofluorescence studies revealed microtubules with a longitudinal distribution to the main axis of the parasite and in the constitution of cilia. SEM demonstrated a high concentration of cilia covering the oral apparatus and a poor presence of such structures in cytopyge. TEM revealed in the plasma membrane, several associated structures were observed to delineate the cellular cortex and mucocysts. The cytoskeleton of the oral region was observed in detail and had an organization pattern consisting of microtubules, which formed files and nematodesmal networks. Organelles such as hydrogenosomes like and peroxisomes were observed close to the cortex. Macronuclei were observed, but structures that were consistent with micronuclei were not identified. Ultrastructural morphological analysis of isolates confirms its similarity to Balantioides coli . In this study were identified structures that had not yet been described, such as hydrogenosomes like and cytoskeletal structures.
La révélation in situ de l'activité de la catalase par cytochimie ultrastructurale a permis de suivre l'évolution des peroxysomes hépatiques au cours de la métamorphose naturelle ou induite par la triiodothyronine (T3) chez les amphibiens anoures. Les activités enzymatiques de la catalase et de diverses oxydases peroxysomales ont été analysées par dosages spectrophotometriques pendant le développement post-embryonnaire d'alytes obstetricans et de xenopus laevis. Des différences s'établissent dans le comportement des peroxysomes hépatiques entre les animaux dont la métamorphose s'accompagne d'un changement d'habitat (alyte) et ceux qui restent aquatiques (xenope). Le traitement des larves d'alytes obstetricans a permis de montrer que les peroxysomes étaient sensibles à l'action de la T3. Les effets du clofibrate sur les peroxysomes hépatiques ont été recherchés chez les amphibiens anoures. Cette substance est sans effet apparent sur les peroxysomes des larves d'alytes obstetricans. Elle provoque une prolifération des peroxysomes hépatiques ainsi qu'une induction de leurs enzymes en particulier celles de la -oxydation chez rana esculenta adulte (espèce terrestre) et n'induit que les activités enzymatiques peroxysomales dans le foie de xenopus laevis adulte (espèce aquatique). L'expression de la catalase et de la d-aminoacide oxydase post-embryonnaire a été étudiée chez ces différentes espèces d'amphibiens par électrophorèse monodimensionnelle et western blotting à partir des protéines extraites de fractions hépatiques enrichies en peroxysomes.
Neutrophil myeioperoxidase standard 3,3'-Diaminobenzidine Tetrahydrochioride (DAB) procedure:
When staining neutrophils, a cell suspension would be preferred, but a finely minced buffy coat can be used, if you thick section and locate the stained cells before thin sectioning. Staining should be carried out as soon as possible after fixation, The myeioperoxidase (MPO) activity falls off such that we process samples within 2-3 weeks of fixation.
Note that fixation is for 1 hour. Enzyme activity may be destroyed if tixed for longer periods.
The main objectives of this article are to survey current advances about selected aspects of biochemistry, molecular biology and physiological role of catalases in higher plants. The reader is also referred to other recent reviews dealing with specific aspects of catalases (Willekens et al, 1995; Scandalios et al, 1997; Zámocký and Koller, 1999; Nicholls et al, 2001). Catalase is a very effective enzyme with a high turnover number but a weak affinity for its substrate hydrogen peroxide. Catalase works as a single enzyme in plants at high hydrogen peroxide concentrations or in co-operation with other antioxidative enzymes to prevent the generation of reactive oxygen species. Therefore, the biological function of catalases becomes more and more complex. Catalases work in a bifunctional mode (see later). It is also necessary to distinguish between monofunctional catalases (typical catalases) and catalase-peroxidases like ascorbate peroxidases in plants which participate in a network system of hydrogen peroxide metabolizing enzymes. The substrate hydrogen peroxide is proposed to act as a signal molecule in cellular signal transduction and also influences processes like plant pathogen response. In this chapter, we focus on catalase acting in non-stressed plant cells. Catalases of algae will not be included into this review and data resulting from studies on catalases of heterotrophic organisms are only integrated when needed in context.
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