H2O2 destruction by ascorbate-dependent systems from chloroplasts.

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3'-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated. System A is membrane-bound, reacts with 3,3'-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide. The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide. Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H2O2 which is produced in these organelles by photosynthetic O2 reduction.     

Electron histochemistry and ultrastructural localization of carbohydrate-containing substances in the sheath of Volvox.

The location and characteristics of carbohydrate-containing structures within the intact sheath of Volvox were studied by 3,3'-diaminobenzidine tetrahydrochloride-osmium, colloidal iron, colloidal thorium, ruthenium red and periodic acid-silver methenamine staining. The sheath consists of external and internal fibrillar layers separated by a tripartite structure. The external layer reacts positively with 3,3'-diaminobenzidine tetrahydrochloride, colloidal iron, colloidal thorium and ruthenium red, indicating that it contains acid mucosaccharides. Staining in the external layer is abolished by Ba(OH)2 treatment. The tripartite structure and internal fibrillar layer contain periodic acid reactive groups which do not occur in the external layer. Under certain conditions, reactions between the cationic dyes and the internal material were also observed. It is postulated that the internal matrix of the sheath contains glycoproteins or a mixture of acid mucosaccharides and glycoproteins. Possible functions of the sheath material are discussed.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Ultrastructure, peroxisomes and lipid peroxidation in reperfused myocardium

Reperfusion injury was studied in dog myocardium using a transmission electron microscope and 3,3'-diaminobenzidine (DAB) to locate areas of peroxidatic activity. Dark electron dense DAB reaction product was observed in peroxisomes and damaged mitochondria. These results suggest attack by reactive oxygen species on mitochondrial membranes, which might result in the formation of lipid peroxides and prostaglandin-like compounds. It is suggested that the release of lipid peroxide or prostaglandins from the injured cells may contribute to reactive hyperaemia, ventricular fibrillation and angina.     

A simple enhancement method for the silver-gold-intensified diaminobenzidine reaction in the light microscopic immunoperoxidase technique.

We describe a simple and sensitive method for enhancement of the silver-gold-intensified 3,3'-diaminobenzidine (DAB) reaction demonstrating peroxidase activity. After completing silver-gold intensification of the preparations immunostained by the avidin-biotin-peroxidase method with DAB as the chromogen, the preparations were immersed in a solution containing uranyl nitrate. This new method appeared to increase the sensitivity by at least one order of magnitude as compared with silver-gold intensification alone.     

Three-Way Differentiation of Chinese Hamster Ovary Chromosomes by Immunoperoxidase Technique Using a Monoclonal Anti-Bromodeoxyuridine Antibody

A new permanent staining procedure for sister chromatid differentiation (SCD) in cultured Chinese hamster ovary cells has been established by combining the three-way differentiation in third mitosis (M3) chromosomes and the immunoperoxidase reaction developed with 3,3'-diaminobenzidine using a monoclonal anti-bromodeoxyuridine (BrdU) antibody. This procedure allows SCD at very low BrdU concentrations, and the evaluation of the sister chromatid exchange frequencies on a per cell-cycle basis.     

Cytochemical Evidence for the Occurrence in Plants of a Novel Microbody that Contains Peroxidase

A cytochemical study with 3,3'-diaminobenzidine revealed thepresence of peroxidase in the microbodies which were recentlyfound in the xylem parenchyma cells of wintering poplar (Sagisakaand Asada 1981). The peroxidative reaction was not inhibitedby 3-amino-l,2,4-triazole, but was completely inhibited by KCN.There was no detectable staining in the absence of added H₂O₂. (Received March 6, 1986; Accepted August 27, 1986)     

Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody.

Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.     

Ultrastructure of methanol-utilizing yeast cells: appearance of microbodies in relation to high catalase activity.

Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.     

Optimisation of the enzyme-based determination of hydrogen peroxide using the quartz crystal microbalance

The benzidines, 3,3'-diaminobenzidine (DAB), 3,3'-dimethoxybenzidine (DMOB) and 3,3',5,5'-tetramethylbenzidine (TMB) were enzymatically oxidised to detect hydrogen peroxide, using the quartz crystal. The oxidised product mainly remains in suspension, resulting in a limited quartz sensor signal. We have used two non-ionic surfactants, Tween 80 and Triton X-100 to interact with the oxidised amphiphilic products to increase their solubility and surface activity, and their ability to adsorb to the crystal surface. Tween 80 exhibits optimised response effects for DAB, DMOB and TMB at 0.012, 0.005, and 0.002% (v/v), respectively, whereas Triton X-100 is optimum at 0.1, 0.2, and 0.006% (v/v), respectively. As a result, we have improved the quartz crystal sensor sensitivity to peroxide. The use of Triton X-100 gave an improved response time.     

Cytochemical localization of peroxisomes in Tetrahymena pyriformis.

Pronounced staining of Tetrahymena pyriformis peroxisomes was demonstrated when glutaraldehyde-fixed cells were incubated in an alkaline medium containing 3,3'-diaminobenzidine and hydrogen peroxide. A variable amount of electron-opague deposit was observed when cells were incubated in diaminobenzidine and H2O2 for 1 hr while an intense deposit followed incubation for 4 hr in the same medium. The staining was abolished completely when 3-amino-1,2,4-triazole, KCN or NaN3 was added to the incubation medium. Based on these cytochemical observations and the morphologic identification by size, shape and other ultrastructural details, it is suggested that this study presents evidence for a conclusive morphologic identification of Tetrahymena peroxisomes.     

Identification of an endosome-specific antigen.

We have used successive density gradient centrifugation with vesicles prepared from a human hepatoma Hep G2 post nuclear supernatant to obtain a highly enriched preparation of early endosomes. A monoclonal antibody (8E4) raised against this early endosome preparation recognizes a single polypeptide highly enriched in light vesicle membranes. The antigen has a molecular weight of 195 kDa by SDS-PAGE in the presence or absence of a reducing agent. Western blot analysis shows that the 8E4 antigen is detectable only in light vesicle membranes and not among heavy membranes, whole cytosol, or nuclear pellet proteins. The 8E4 antigen appears to be an integral membrane protein as it is precipitated by Triton X-114. The distribution of the 8E4 antigen in a Nycodenz density gradient fractionation of light vesicle membranes is identical to the distribution of 125I-ASOR-labeled early endosomes but distinct from the distribution of the plasma membrane enzyme, alkaline phosphodiesterase. In addition, incubation of cells with a horseradish peroxidase-transferrin conjugate followed by 3,3'-diaminobenzidine cytochemistry specifically quenches 8E4 antigen detection by protein dot blot analysis. These data strongly suggest that the 8E4 antigen is an integral membrane protein primarily located in endocytic vesicles.     

Cationic heme undecapeptide as stain for detecting glycosaminoglycans on cellulose acetate strips

A rapid and sensitive method has been developed for detecting glycosaminoglycans on cellulose acetate strips. The method is based on the electrostatic interactions between glycosaminoglycans and cationic heme undecapeptide. The complexes are identified by peroxidatic reaction using 3,3'-diaminobenzidine as H2 donor. The detection limit is 2.5-10 ng for sulfated glycosaminoglycans and 60 ng for hyaluronic acid. The sensitivity of this method is higher than of those using Alcian blue or toluidine blue (500 ng).     

Peroxidatic activity in heart effluent: a biochemical parameter for the assessment of experimental ischemia-reperfusion injury

Peroxidatic activity in heart effluent was defined as a new biochemical parameter for the experimental study of myocardial ischemia. The peroxidatic reaction was determined by dot blot analysis with 3,3'-diaminobenzidine as hydrogen donor. After ischemia, the level of peroxidatic activity in heart effluent was 2-3 times higher than before. The effects in experimental modulation of ischemia, such as nicorandil or aprikalim protection, and the reversibility of protection by glibenclamide, could accurately be noted using the level of peroxidatic activity in heart effluent as a biochemical parameter. The results were in good agreement with those obtained for other enzymes used as biochemical parameters in experimental heart ischemia-reperfusion studies.     

Oxidation catalyzed by H+ ions improves the silver intensification of 3,3'-diaminobenzidine staining by strongly suppressing tissue argyrophilia

We found that a 15-min treatment of tissue sections with 2 M/l H2O2 dissolved in 6 M/l sulphuric acid strongly suppresses the argyrophilia of tissue elements, thus facilitating selective silver intensification in histochemical procedures. When applied inserted between the 3,3'-diaminobenzidine reaction and physical development, this treatment allows the selective and sensitive visualization of very low levels of peroxidase activity.     

Cytochemical localization of peroxidase activity in the mitochondria of Hymenolepis diminuta.

A cytochemical study of mitochondria of Hymenolepis diminuta indicates the presence of a mitochondrial peroxidase. Utilizing a 3,3'-diaminobenzidine (DAB) medium at pH 9.7, the reaction product is localized in the intracristal space, and between the inner and outer membranes of the mitochondria. No inhibitory effects are exerted on the peroxidase reaction by cyanide, azide, or aminotriazole. In addition, the mitochondria appear to have an enzyme which is cytochemically similar to vertebrate cytochrome c-oxidase. The possible physiological significance of the peroxidase is discussed.     

Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.