An enzymatic component of the insect blood-brain barrier: Implications of DAB (3,3′-diaminobenzidine) oxidation

The oxidation of DAB (3,3′-diaminobenzidine) was examined in intact brains of the European corn borer, Ostrinia nubilalis. The deposition of oxidized DAB was examined by electron microscopy and found to be localized in glial cells. A semi-quantitative comparison of this reaction was made in vitro between horseradish peroxides, catalase, and insect brain. Inhibition characteristics indicate that the brain reaction is not the result of an endogenous peroxidase or catalase. The use of specific inhibitors (i.e. sesamex, tranylcypromine) indicate that this reaction could be attributed to an amine oxidase (monoamine: O₂ oxido-reductase (deaminating), E.C. 1.4.3.4) of microsomal origin.     

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland

Summary A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.     

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.     

Cytochemical Detection of Cell Wall Bound Peroxidase in Rust Infected Broad Bean Leaves*

Cell wall bound peroxidase activity, cytochemically detected by 3,3-diaminobenzidine (DAB), appears unevenly distributed in areolae of broad bean (cv. Aguadulce) leaves infected by the rust fungus Uromyces viciae-fahae (Pers.) Schroet. DAB positivity is not significantly traceable at the periphery of the areola and close to the site of uredinium formation; heavy DAB deposits occur on walls of haustoria containing mesophyll cells. Induction of localized peroxidase activity appears strictly related to the haustorium mother cell differentiation, i.e. to the incipient parasitic phase; it does not, however, counteract the intracellular growth of the haustorium.     

Tyrosinase and superoxide dismutase activities of peroxidase in the vacuoles of beet roots

In the absence of H₂O₂, isoforms of vacuolar phenol-dependent peroxidase (PO) in beet (Beta vulgaris) roots oxidized phenolic compounds like tyrosinases. Tyrosinase activity of PO manifested a clearly expressed pH-dependence with the optimum at pH of 8.0–9.0; peroxidase activity was the highest at pH 5.0–7.0. The inhibitory analysis confirmed a specificity of observed reactions. Along with tyrosinase activity, PO manifested SOD-like activity, which was also expressed in the absence of H₂O₂ and depended on some factors, for example, on the way of analyzed sample preparation. This activity appeared at a long-term dialysis and low temperature. SOD-like PO activity was observed in the presence of such substrates as 3,3′-diaminobenzidine and IAA. The results obtained allow a conclusion that vacuolar PO, as well as PO of other localization and origin is a polyfunctional enzyme, which, under definite conditions, can catalyze reactions of oxidase type.     

Ultrastructural Localization of Hydrogen Peroxide Production in Ligninolytic Phanerochaete chrysosporium Cells

Previous studies have shown that the hydroxyl radical derived from hydrogen peroxide (H₂O₂) is involved in lignin degradation by Phanerochaete chrysosporium. In the present study, the ultrastructural sites of H₂O₂ production in ligninolytic cells of P. chrysosporium were demonstrated by cytochemically staining cells with 3,3′-diaminobenzidine (DAB). Hydrogen peroxide production, as evidenced by the presence of oxidized DAB deposits, appeared to be localized in the periplasmic space of cells from ligninolytic cultures grown for 14 days in nitrogen-limited medium. When identical cells were treated with DAB in the presence of aminotriazole, periplasmic deposits of oxidized DAB were not observed, suggesting that the deposits resulted from the H₂O₂-dependent peroxidatic oxidation of DAB by catalase. Cells from cultures grown for 3 or 6 days in nitrogen-limited medium or for 14 days in nitrogen-sufficient medium had little ligninolytic activity and low specific activity for H₂O₂ production and did not contain periplasmic oxidized DAB deposits. The results suggest that in cultures grown in nitrogen-limited medium, there is a positive correlation between the occurrence of oxidized DAB deposits, the specific activity for H₂O₂ production in cell extracts, and ligninolytic activity.     

Cytochemical localization of peroxidase activity in the submandibular salivary gland of the hamster

Summary Peroxidase activity has been localized to duct cells of the submandibular salivary gland of the hamster using a 3,3-diaminobenzidine (DAB)-H₂O₂ medium. In cryostat sections of glutaraldehyde-fixed tissue the enzyme activity is found in the proximal part of the duct system of the gland. In Epon sections studied in the light microscope or thin sections studied in the electron microscope the peroxidase activity is observed in cytoplasmic granules in cells of the convoluted tubules of the ducts. No activity is seen in the acini or in cells of the intralobular striated ducts. The submandibular gland of the rat was negative with respect to peroxidase reaction. The findings are discussed with special reference to the possible correlation between peroxidase activity and iodine metabolism in salivary glands.     

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.     

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.     

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.     

Intracellular location of cytochrome oxidase active in vivo in the cyanophytes,Synechocystis sp. PCC6714 andAnacystis nidulans Tx20 and R2, grown under various conditions

Summary Intracellular location of cytochrome oxidase (cytoxidase) active in vivo was studied cytochemically with four strains of cyanophytes, using 3,3-diaminobenzidine (DAB) oxidation. DAB was oxidized in the dark bySynechocystis sp. PCC6714 and two strains ofAnacystis nidulans (Tx20 and R2) grown under photoautotrophic, heterotrophic and salt-stressed conditions, respectively. Electron microscopic observations showed that DAB-oxidation in the dark occurred in the thylakoids, but was insignificant on or around the cytoplasmic membrane. However, deposition of DAB-oxidation product around the cytoplasmic membrane was observed with cells of the thylakoid-less cyanophyteGlaeobacter violaceus ATCC29082. All DAB oxidations observed with the four strains were inhibited completely by cyanide, the inhibitor of cyt-oxidase, but not by 3-amino-1,2,4-triazole, the inhibitor of peroxidase. The results show that (1) DAB was oxidized by the cyt-oxidase functioning in the respiratory system, and that (2) cyt-oxidase in thylakoids was active in vivo.Abbreviations AT 3-amino-1,2,4-triazole - Cyt oxidase cytochrome oxidase - ETS electron transport system - DAB 3,3-diaminobenzidine     

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.     

The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays—especially enzyme-linked immunosorbent assays (ELISAs)—are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3′,5′-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r = 0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.     

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     

Evidence for haemoglobin uptake by oöcytes of Chironomus thummi

Developing oöcytes of a haemoglobin-containing fly, Chironomus thummi, have been investigated using the 3,3′-diaminobenzidine method for their endogeneous peroxidase activity. In the previtellogenic oöcytes the reaction product, which is thought due to the peroxidatic activity of the haemoglobins, is not observed within the oöcytes. Vitellogenic oöcytes appear active in the uptake and incorporation of the externally derived peroxidese-active material into the yolk. The reaction product which is first visualized in the extracellular spaces within the follicle, then the pinosomes and multivesicular bodies of the oöcyte, is later seen in the mature yolk granules. These observations are discussed in terms of their relation to the accumulation of haemoglobin as a part of the yolk.     

Some cytochemical correlations between oxidase activity (cytochrome and peroxidase) and chemical structure of bis(phenylenediamines)

Summary A series of 17 bis(phenylenediamine) derivatives have been prepared and compared with 3,3-diaminobenzidine (DAB) with regard to their ability to demonstrate cytochrome oxidase activity, peroxisome activity, horseradish peroxidase activity, erthrocytic peroxidase activity in cytochemical preparations, and bovine catalase activity in in vitro experiments. The results are tabulated, some illustrative photomicrographs are included and interesting correlations are discussed.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U. S. Public Health Service.Acknowledgement for technical assistance is due Charles Hatton and William Brown.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Effect of high-level oxygen exposure on the peroxidase activity and the neuromelanin-like pigment content of the nerve net in the earthworm, Lumbricus terrestris

Histochemical examination of 1-μm tissue sections from the dorsal nerve plexus of the earthworm, Lumbricus terrestris, reveals multiple brown intraneuronal granules. These granules contain material morphologically and histochemically consistent with neuromelanin. When viewed with transmission electron microscopy, these were seen as single membrane-enclosed biphasic granules with diameters of 370–730 nm. Exposure of L. terrestris to high-level environmental oxygen resulted in an increase in the number of neuromelanin-like pigment granules within the neurons of the circular muscle layer. As measured by ortho-phenylenediamine hydrochloride, the endogenous peroxidase activity of extracts from worms incubated in high-level environmental oxygen was 51% more than controls. The endogenous peroxidase activity was localized in situ with 3,3-diaminobenzidine (DAB) and was found to increase in and around the neuromelanin-like pigment-containing neurons within the circular muscle layer. These studies suggest that the nerve net of L. terrestris may serve as a model to study the role of neuromelanin production in oxidative stress and its relationship to endogenous peroxidases.     

Actin localization and function in higher plants

Summary Two different cytochemical methods were used to study the localization of uricase (EC 1.7.3.3) and catalase (EC 1.11.1.6) in developing root nodules of soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. One of the methods employs DAB (3,3-diaminobenzidine) and detects uricase activity indirectly by coupling it to endogenous catalase activity. The other method utilizes cerium chloride to detect uricase activity directly. These methods were modified to obtain not only a strong staining reaction but also improved ultrastructural preservation. With the indirect DAB method, intense staining indicative of both uricase and catalase activity was obtained in the enlarged peroxisomes of older uninfected cells. Similar staining was observed in enlarging peroxisomes of younger uninfected cells, and in the material of associated sacs whose bounding membranes appear to arise as distensions of the ER. The observations are discussed in relation to the controversial role of the ER in peroxisome biogenesis. Although the small peroxisome-like organelles of infected cells did not give a clearly positive reaction in the indirect DAB method, they reacted positively in the cerium chloride method, and are considered to be peroxisomes.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.