Metal compound intensification of the electron-density of diaminobenzidine

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.     

Cytotoxicity of 1,4-diamino-2-butanone,a putrescine analogue,to RKO cells: mechanism and redox imbalance

Abstract

α-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. In vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive α-oxoaldehyde and H₂O₂. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05–10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC₅₀ ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 μM) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.     

Plasmodium falciparum: discovery of peroxidase active organelles

Staining with 3,3' diaminobenzidine tetrahydrochloride (DAB) is a common method used for the detection of peroxidases. Using this histochemical staining method in conjunction with transmission electron microscopy, we observed oxidation of DAB that was localized to a discrete set of organelles displaying morphological similarity to small (75-90 nm diameter) versions of higher eukaryotic microbodies or peroxisomes. These single membrane bounded organelles were characterized by an asymmetrical matrix capable of oxidizing DAB to an electron dense inclusion. Oxidation of DAB was further found to be dependent upon hydrogen peroxide (H2O2) as a substrate. Given a lack of peroxisomal import proteins and enzymes, it is unlikely that these represent conventional peroxisomes. Rather, they likely represent specialized organelles containing endogenous peroxidase or pseudo-peroxidase activity.     

Effects of low molecular weight fungal compounds on inflammatory gene transcription and expression in mouse alveolar macrophages

1,2-Diacetylbenzene (DAB) is a neurotoxic minor metabolite of 1,2-diethylbenzene or naphthalene reaction product with OH radical. DAB causes central and peripheral neuropathies that lead to motor neuronal deficits. However, the potent effects and molecular mechanisms of DAB on neural progenitor cells and hippocampus are unknown. In the current study, we report the DAB damage at lower doses (less than 50 μM) to neural progenitor cell (NPC) invitro and hippocampal neurogenesis invivo. DAB significantly suppressed NPC proliferation with increased reactive oxygen species (ROS) production in a dose-dependent manner. The suppression of NPC proliferation was effectively blunted by the action of an antioxidant, N-acetyl cysteine. Six-week-old male C57BL/6 mice were treated with 1 or 5 mg/kg DAB for 2 weeks. DAB significantly suppressed NPC proliferation in the dentate gyrus of the hippocampus, indicating impaired hippocampal neurogenesis. Increased ROS production and the formation of oxidative stress-associated dinitrophenyl adducts were detected in the hippocampal homogenates of DAB-treated mice. DAB activated Mac-1-positive immune cells which are involved in inflammatory process in the hippocampus. Taken together, these results confirm that oxidative stress by DAB might be cause of adverse effects in NPC proliferation and hippocampal neurogenesis.     

Ovoperoxidase activity in ionophore treated mouse eggs. I. Electron microscopic localization

A mammalian ovoperoxiadase activity has been detected in ionophore activated mouse eggs. The peroxidase activity was demonstrated at the electron microscopic level using the 3,3′-diaminobenzidine (DAB) histochemical method. A positive DAB reaction was detected in a portion of the intact cortical granules of untreated or DMSO treated control eggs. In the ionophore activated eggs, the DAB reaction product was routinely detected by electron microscopy, predominantly on the cell surface, that is on the zona pellucida, in the perivitelline space, and in association with the cortical granule exudates. Furthermore, the peroxidase inhibitors phenylhydrazine and sodium sulfite prevented DAB staining in ionophore activated oocytes. These results indicate the presence of an ovoperoxidase, possibly of cortical granule origin, on the surface of activated mammalian eggs, detectable by histochemical means.     

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.     

Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve

p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.     

Staining of Xylem Parenchyma Mitochondria with Photo-Oxidized 3,3'-Diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldebyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.     

Stereological analysis of peroxisomes and mitochondria in intestinal epithelium of patients with peroxisomal deficiency disorders: Zellweger's syndrome and neonatal-onset adrenoleukodystrophy

Peroxisomes, participants in lipid metabolism, have been shown to be altered in liver in two metabolic diseases in which long-chain fatty acids accumulate in tissues: Zellweger's syndrome and neonatal adrenoleukodystrophy (ALD). The intestine also plays a role in lipid metabolism, and we have had the opportunity to compare peroxisomes in normal intestinal epithelium with those from patients with Zellweger's syndrome and neonatal ALD at the electron microscopic level by using the combined techniques of cytochemistry and stereological analysis. Peroxisomes were numerous in intestinal epithelium of the normal individuals. They were ellipsoidal in shape with average diameters of 0.37 by 0.56 micron and filled with coarsely granular, DAB+ content. Peroxisomes in the intestinal epithelium of the ALD patient were similar in appearance and number but smaller in size (0.28 by 0.44 micron). Peroxisomes of normal appearance were absent from the intestinal epithelium of patients with Zellweger's syndrome; DAB+ content, however, was observed in rare, membrane-bound structures of much smaller size (0.12 by 0.19 micron). In liver of patients with Zellweger's syndrome, peroxisomes are lacking; in neonatal ALD they are abnormal in appearance and greatly reduced in number. The presence of rare minute peroxisomes in the intestinal epithelium in Zellweger's syndrome and of small peroxisomes in this epithelium in neonatal ALD indicate that peroxisomes in the intestinal epithelium are affected in these diseases, but to a lesser extent than in the liver. In the ALD intestinal epithelium, DAB+ material was also seen in long, sinuous, tubular or cisternal elements intermingled and occasionally in continuity with peroxisomes. It is suggested that these represent the early stages of peroxisome formation, the peroxisomal reticulum as originally envisioned by Lazarow, while the rare structures seen in Zellweger's represent rudiments of such a reticulum. Lamellar inclusions and clear spaces occurred in the cytoplasm adjacent to these structures indicating either that material accumulated there had been extracted during fixation or that these regions are more susceptible to autolysis. Mitochondria are also involved in lipid metabolism and have been reported to be abnormal in Zellweger's tissue. No qualitative differences were observed in the mitochondria of the intestinal epithelia examined in this study. Although quantitation revealed a greater mean volume, number, and surface density of mitochondria in the intestinal epithelia of neonatal ALD, it was not a statistically significant difference in all cases.     

下丘脑室旁核内NPY神经元与CCK神经元相互关系的免疫电镜双标研究

应用包埋前免疫电镜双标技术对大鼠下丘脑室旁核的神经肽Y(NPY)和胆囊收缩素(CCK)神经元的相互关系进行了研究。用Norgren法进行免疫电镜双标染色。结果在电镜下观察到:在室旁核内侧部,NPY样免疫反应产物呈电子密度高的颗粒状或絮状,弥漫分布于胞浆;CCK样免疫反应产物则呈电子密度高的针状或块状,散在分布于胞浆,偶见于核内。有时,在一个神经末梢内既有浓重的颗粒状DAB反应产物,又有典型的针状TMB反应产物。在室旁核内,NPY和CCK神经元胞体互相混杂、交错存在,两者均为中等大细胞。在超微结构水平,NPY和CCK神经元的树突和轴突可由非NPY、非CCK神经末梢接受传入突触联系;CCK神经元的树突还可接受其他CCK神经末梢的传入性自调节突触;CCK神经元胞体可接受NPY神经末梢的传入性突触,后者的突触前成分内可能有CCK与NPY共存。     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

Histamine content of peritoneal and tissue mast cells of growing rats

Summary p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm, Lumbricus terrestris L

The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H2O2 was omitted. Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Oxidation of diaminobenzidine in the heterocysts ofAnabaena cylindrica

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the honeycomb region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H₂O₂, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O₂-labile nitrogenase.     

DISTRIBUTION OF PEROXISOMES (MICROBODIES) IN THE NEPHRON OF THE RAT : A Cytochemical Study

The distribution of peroxisomes (microbodies) in the rat nephron was studied cytochemically, using glutaraldehyde- or formaldehyde-fixed tissue, by means of α-hydroxy acid oxidase activity in light microscopy or oxidation of 3,3'-diaminobenzidine (DAB) at pH 9 in both light and electron microscopy.The two cytochemical methods show peroxisomes to be nearly sperical particles found only in cells of the proximal convoluted tubule. Lysosomes were identified in the same or parallel sections, with β-glycerophosphate or 5'-cytidylic acid as substrate. They are found in all cells of the nephron. These cytochemical methods visualize the two organelles for light microscopy; they also permit unequivocal differentiation of all kidney peroxisomes from lysosomes in electron micrographs. Peroxisomes are larger and more reactive in the cells of the pars descendens (P₃ segment) of the proximal convolution, located in the outer medulla and medullary rays, than in the cells of the pars convoluta (P₁ and P₂ segments), situated in the cortex. In contrast, lysosomes are much smaller in the P₃ segment and larger and more reactive in the P₁ and P₂ segments. In all cells of the proximal convolution, peroxisomes tend to be concentrated nearer the base of the cells than do lysosomes. Mitochondria in P₃ cells also show low levels of DAB oxidation at pH 6, in contrast to those in P₁ and P₂ cells. The possibility is discussed that P₃ cells possess an extramitochondrial means of oxidation in which peroxisome oxidases play an important role.     

Changes in microsomal enzyme activities during DAB carcinogenesis

Microsomal fractions were isolated from livers of rats that been fed p-dimethylaminoazobcnzene (DAB) for 2 months sygnificant decreases were noted in the specific activities of four of the membrane-bound enzymes, Sudies on membrane composition and the interactions between membranc components were carried out in an attempt to explain the mechanisms responsable for these decreases. Changes in the lipid components of the membrane were brought about as a result of DAB feeding. No changes were detected in the protein components of the membrane. 1-Anilinonaphthalene-8-Sulfonic acid (ANS) fluorescence enhancement was decreased by 25 % in membranes delived from DAB-fed animals. The response of the membranebound enzymes to pretreatment with NH₄OH and Triton X-100 was examined. The glucose-6-phosphatase of membranes derived from carcinogen-fed animals was more activated by Triton than the enzyme from control. It is concluded that altered membrane conformation is a result of DAB carcinogensus, and that such alterations may be responsible for loss of enzyme activity.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm,Lumbricus terrestris L.

Summary The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H₂O₂ was omitted.Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals

There is a great deal of evidence to support a pathogenic role of oxidative stress in Alzheimer's disease (AD), but the sources of reactive oxygen species have not been directly demonstrated. In this study, using a novel in situ detection system, we show that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. Pretreatment with deferoxamine or diethylenetriaminepentaacetic acid abolishes the ability of the lesions to catalyze the H2O2-dependent oxidation of 3,3'-diaminobenzidine (DAB), strongly suggesting the involvement of associated transition metal ions. Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion-dependent catalytic redox reactivity. Although DAB oxidation can also detect peroxidase activity, this was inactivated by H2O2 pretreatment before use of DAB, as shown by a specific peroxidase detection method. Model studies confirmed the ability of certain copper and iron coordination complexes to catalyze the H2O2-dependent oxidation of DAB. Also, the microtubule-associated protein tau, as an in vitro model for proteins relevant to AD pathology, was found capable of adventitious binding of copper and iron in a redox-competent manner. Our findings suggest that neurofibrillary tangles and senile plaques contain redox-active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment.     

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.