Intracellular appearance of nitrite and nitrate in nitrogen-starved cells of Ankistrodesmus braunii

Summary The occurrence of heterotrophic nitrification in nitrogen-starved cells of Ankistrodesmus braunii was confirmed. The levels of nitrate and nitrite were measured over a period of four weeks. The validity of quantitative determinations in the presence of highly active nitrate and nitrite reductases is discussed. Whereas free hydroxylamine as an intermediate could not be detected, increased hydroxylamine oxidase activity was found in nitrogen-starved cultures. Nitrite reductase and hydroxylamine oxidase can be assigned to particles by sucrose density gradient centrifugation. The possible involvement of microbodies, which were found to be present in Ankistrodesmus, in metabolic processes during nitrogen starvation is discussed.Abbreviations NR nitrate reductase - NiR nitrite reductase - NNEDA N-(1-naphthyl)ethylenediaminedihydrochloride - DCPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid - DAB 3,3-diaminobenzidine - AT 3-amino-1H-1,2,4-triazole - AMP 2-amino-2-methyl-1,3-propanediol     

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.     

Ultrastructural localization of photosynthetic activity in thylakoids during chloroplast development in maize

The localization of photosynthetic activity in developing maize (Zea mays L.) chloroplasts was studied in situ by two electron-microscopic-cytochemical methods. The activity of photosystem I was detected by photooxidation of 3,3-diaminobenzidine (DAB) and the activity of the photosystem II by photoreduction of thiocarbamyl nitrotetrazolium blue (TCNBT). During the transformation of proplastids into chloroplasts, at the base of the leaf blade the DAB reaction appeared before the TCNBT reaction. A positive DAB reaction was observed in the single thylakoids of plastids in cells located only about 0.5 mm above the base. Dark, osmiophilic DAB polymers accumulated in the lumina of the thylakoids. Plastid envelopes and tubules of the prolamellar bodies in immature chloroplasts were DAB-negative. In fully differentiated leaf tissue the DAB reaction was intense in the thylakoids of bundle-sheath chloroplasts, as well as in the stroma thylakoids and the peripheral grana thylakoids of mesophyll chloroplats. The photoreduction of TCNBT started in leaf tissue about 1 mm above the base. Dark granular material of reduced TCNBT appeared mostly in the partitions of grana, i.e. interthylakoidally, but some granules were also attached to the stroma thylakoids. The membranes of plastid envelopes and the tubules of prolamellar bodies showed a negative TCNBT reaction. Young bundle-sheath chloroplasts contained some reduced TCNBT in their grana; these deposits largely disappeared in the course of further differentiation. In mature leaf tissue the photoreduction of TCNBT was conspicuous in the grana of mesophyll chloroplasts, but very weak in the single thylakoids and in the granal rudiments of bundle-sheath chloroplasts.Abbreviations DAB 3,3-diaminobenzidine·4 HCl - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS(I,II) photosystem (I,II) - TCNBT thiocarbamyl nitrotetrazolium blue chloride     

Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophylla/c light-harvesting complex in the diatomPhaeodactylum tricornutum

Summary iserum against two polypeptides of the major fucoxanthin-chlorophylla/c light-harvesting complex of the diatomPhaeodactylum tricornutum and heterologous antiserum against purified photosystem I particles of maize were used to localize these two complexes on the thylakoid membranes ofP. tricornutum. As in many chromophyte algae, the thylakoids are loosely appressed and organized into extended bands of three, giving a ratio of 21 for appressed versus non-appressed membranes. Immunoelectron microscopy demonstrated that the fucoxanthin-chlorophylla/c light-harvesting complex, which is believed to be associated with photosystem II, was equally distributed on the appressed and non-appressed thylakoid membranes. Photosystem I was also found on both types of membranes, but was slightly more concentrated on the two outer non-appressed membranes of each band. Similarly, photosystem I activity, as measured by the photooxidation of 3,3-diaminobenzidine, was higher in the outer thylakoids than in the central thylakoid of each band. We conclude that the thylakoids of diatoms differ from those of green algae and higher plants in their macromolecular organization as well as in their morphological arrangement.Abbreviations BSA bovine serum albumin - DAB 3,3-diaminobenzidine - FCPC fucoxanthin-chlorophylla/c light-harvesting complex - LHC light-harvesting complex - PBS phosphate-buffered saline - PS photosystem     

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     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues

Summary Cytochrome oxidase activity was demonstrated in unfixed root segments from Lupinus albus at the ultrastructural level using the osmiophilic reagent 3,3-diaminobenzidine (DAB). Precipitate, the formation of which was completely inhibited by 0.01 M KCN, and observed almost entirely on mitochondrial cristae, is considered to be produced by cytochrome oxidase activity. Heterogeneity of mitochondria as to the intensity of the reaction in the same cell could not be established with certainity. However, mitochondria of the root tip cells and cells belonging to the plerome consistently did not show histochemically demonstrable cytochrome oxidase activity.     

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.     

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     

Reflectance enzyme histochemistry (REH): visualization of cerium-based and DAB primary reaction products of phosphatases and oxidases in cryostat sections by confocal laser scanning microscopy

In the present study the reflectance mode of confocal laser scanning microscopy was adapted to detect and to assess semiquantitatively cerium-based primary reaction products of oxidases [Ce(IV) perhydroxide] and phosphatases [Ce(III) hydroxyphosphate converted into Ce(IV) perhydroxyphosphate] as well as of the 3,3-diaminobenzidine (DAB)-based primary reaction product of cytochromec oxidase in cryostat sections. Confocal laser scanning microscopy offers a unique way of making visible histochemical reaction products which are weakly absorbant but sufficiently reflective. It was easily possible to record simultaneously the reflectance signals at the wavelength of the exciting laser (preferentially 488 nm) and the autofluorescence signals (>580 nm in our set-up) of glutaraldehyde-fixed tissue. The results of an imbibition study of cerium-containing model precipitates indicate that the cerium, generally, should be oxidized prior to observation because the index of refraction of Ce(IV) compounds is considerably higher than that of the corresponding Ce(III) compounds. An attempt at comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is presented. The proposed technique may open new possibilities in enzyme- and immunohistochemistry.     

Demonstration of sensory cutaneous nerve fibers in guinea-pig lip using endogenous oxidase histochemistry

Summary We developed a new histochemical method based on endogenous oxidase activity for the detection of cutaneous nerves in guinea-pig lips. After the application of a reaction mixture containing 0.14 mM 3,3-diaminobenzidine(DAB) and 15 mM nickel ammonium sulfate in Tris-HCl buffer (pH 7.6), many blue-black sensory fibers were observed, i.e., dermal nerve fiber bundles, perifollicular plexuses, Merkel-cell endings, and Meissner-like nerve endings. The autonomic fibers of the skin were not stained. Electron microscopy revealed that the reaction product was mainly localized on mitochondrial cristae. The addition of 0.01 M sodium azide completely inhibited the reaction. This technique is simple and preferentially stains sensory nerves.     

Isolation of mutants of the cyanobacterium,Anabaena variabilis,impaired in photoautotrophy

Mutants ofAnabaena variabilis Kütz. that have a decreased ability to grow photoautotrophically have been isolated by a modification of the techniques used to isolate auxotrophic mutants of that filamentous cyanobacterium, and have been stably propagated. Three mutants have a reduced content of phycocyanin and, as determined by in situ assays of partial reaction sequences of photosynthesis, an impairment in photosystem II. Three other strains, all of which appear to have a normal complement of carotenoids when grown heterotrophically, are sensitive to light.Abbreviations Used TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid, sodium salt - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, sodium salt - MV methylviologen - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DAB 3,3-diaminobenzidine - P-BQ p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fecy K-ferricyanide - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Specialization of the inner cortex for ureide production in soybean root nodules

Summary The possibility that cells in the inner cortex of determinate root nodules participate in ureide production from recently fixed N₂, as do the uninfected (interstitial) cells of the infected central region, has been investigated in soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. Like the interstitial cells, cells of the three innermost cortical layers produce enlarged peroxisomes and a meshwork of tubular ER during differentiation. These changes are most pronounced in the innermost cortical layer, are successively less so in the 2nd and 3rd layers, and are usually undetectable in more distant layers. Peroxisomes in the inner three layers are stained in the DAB (3,3-diaminobenzidine) test for uricase (EC 1.7.3.3) activity, indicative of the potential for ureide formation, but peroxisomes in more distant cortical cells are not stained. A nodulespecific uricase also is demonstrable in the inner three cortical layers by immunogold labeling enhanced with silver for visualization in the light microscope. The observations suggest that with respect to ureide production the cells of the inner layers of the cortex are functionally similar to the interstitial cells of the infected region despite the apparent distinctiveness of the two regions anatomically.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

An ultrastructural and cytochemical investigation of the colonial green algaPediastrum tetras during zoospore formation

Summary Ultrastructural changes during zoospore formation and aggregation into motile, aggregating zoospores were examined in the colonial green algaPediastrum tetras. Developing zoospores are characterized by irregularly shaped nuclei, presence of peripheral networks of rough endoplasmic reticulum, chloroplasts with tightly apposed thylakoids and dictyosome cisternae which are compressed and reduced in size. A single membrane bound organelle with a fine granular matrix of moderate electron density of diameter ranging from 0.2 m to 0.6 m and associated with chloroplasts, mitochondria and endoplasmic reticulum was found only in adult cells. Although this organelle has the morphology of a microbody, it did not stain with 3,3-diaminobenzidine (DAB) at pH 9.6 or pH 7.6, whereas mitochondrial membranes stained. No DAB staining was observed along the cell wall or the plasma membrane of zoospores, or associated with endoplasmic reticulum, plastid membranes or dictyosomes.     

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.     

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.     

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.     

Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions

The development of peroxisomes has been studied in cells of the yeast Hansenula polymorpha during growth on methanol in batch and chemostat cultures. During bud formation, new peroxisomes were generated by the separation of small peroxisomes from mature organelles in the mother cells. The number of peroxisomes migrating to the buds was dependent upon environmental conditions. Aging of cells was accompanied by an increase in size of the peroxisomes and a subsequent increase in their numbers per cell. Their ultimate shape and substructure as well as their number per cell was dependent upon the physiological state of the culture. The change in number and volume density of peroxisomes was related to the level of alcohol oxidase in the cells. Development of peroxisomes in cells of batch cultures was accompanied by an increase in size of the crystalline inclusions in the organelles; they had become completely crystalline when the cells were in the stationary phase. Peroxisomes in cells from methanol-limited chemostat cultures were completely crystalline, irrespective of growth rate. Results of biochemical and cytochemical experiments suggested that alcohol oxidase is a major component of the crystalline inclusions in the peroxisomes of methanol-grown Hansenula polymorpha. Possible mechanisms involved in the ultrastructural changes in peroxisomes during their development have been discussed.Abbreviations DAB 3,3-diaminobenzidine - OD optical density (663 nm)     

Phycoerythrin is absent from the pyrenoid of Porphyridium cruentum: photosynthetic implications

The thylakoid lamellae which traverse the pyrenoid of the unicellular red alga Porphyridium cruentum (Agardh) Nägeli appear to lack phycobilisomes. We have confirmed by immuno-electron microscopy that phycoerythrin (PE), an important structural component of the phycobilisomes of red algae, is absent from the pyrenoid. To characterize pyrenoid thylakoids further, electron-microscopic cytochemical methods were employed to detect photosystem activity. Photosystem (PS) I activity was demonstrated in both stromal and pyrenoid thylakoids by the photooxidation of 3,3-diaminobenzidine. In contrast, the localization of photoreduced distyryl nitroblue tetrazolium demonstrated that PSII activity was restricted to stromal thylakoids. The observed partitioning of PE and PSII activity within the plastid may be related to another observation, that being the localization of nearly all ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) within the pyrenoid of this alga. It is possible that the pyrenoid of P. cruentum functions as a specific metabolic compartment where CO₂ fixation is enhanced by the absence of photosynthetic O₂ evolution.Abbreviations DAB 3,3-diaminobenzidine-4HCl - DS-NBT distyryl nitroblue tetrazolium chloride - EF exoplasmic face - LSU large subunit of RuBisCO - PE phycoerythrin - PS photosystem - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and Robert MacColl (New York State Department of Health, Albany) for providing us with the antibodies used in this study. We also thank Dr. C.E. Smith for use of the Zeiss MOP-3 digital analyser and Dr. Geneviève Bricheux for kindly providing Lowicryl-embedded samples of P. cruentum. Aatrex^® was kindly donated by Ciba-Geigy. This research was supported by the Natural Sciences and Engineering Research Council of Canada (grant No. A-2921).