Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues

Specific cytochemical reactions have been instrumental in the illucidation of compounds within tissues, whether these compounds are hormones, enzymes, or molecules, such as certain nerve transmitter agents. Many cytochemical reaction products depend upon some complex, which is an electron dense deposit. Several types of cytochemical procedures can be used to visualize agents related to synaptic transmission at the junctional complex. One method which has been used with considerable success has been the cytochemical localization of biogenic amines (BAs), i.e., norepinephrine (NE) and dopamine (DA). For the past few years, a chrome complex formed with certain BAs and glutaraldehyde has been utilized to localize BAs at the electron microscopic level and the specificity of the reaction has been verified biochemically.

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Biochemical Properties and Cytochemical Localization of Ouabain-insensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in Rat Atrial Myocytes

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704500204 ◽

1997 ◽

Vol 45 (2) ◽

pp. 177-187 ◽

Cited By ~ 6

Author(s):

Vadim S. Zinchuk ◽

Toshihiro Kobayashi ◽

Eva Garcia del Saz ◽

Harumichi Seguchi

Keyword(s):

Ventricular Myocytes ◽

Reaction Medium ◽

Biochemical Properties ◽

Atrial Myocytes ◽

Electron Microscopic ◽

Cytochemical Localization ◽

X Ray ◽

T Tubules ◽

P Type ◽

Triton X

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3, Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

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A CYTOCHEMICAL LOCALIZATION OF REDUCTIVE SITES IN A GRAM-NEGATIVE BACTERIUM

The Journal of Cell Biology ◽

10.1083/jcb.20.3.377 ◽

1964 ◽

Vol 20 (3) ◽

pp. 377-387 ◽

Cited By ~ 38

Author(s):

Woutera van Iterson ◽

W. Leene

Keyword(s):

Plasma Membrane ◽

Electron Scattering ◽

Gram Negative Bacteria ◽

Proteus Vulgaris ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Gram Negative ◽

Living State ◽

Granular Matrix ◽

Reducing Activity

In order to obtain information on the exact location of the respiratory enzyme chain in Gram-negative bacteria, an electron microscopic study was made of the sites of reducing activity of cells that had, in the living state, incorporated tellurite. In the test object Proteus vulgaris, the reduced tellurite was found to be deposited in bodies contiguous with the plasma membrane but different in structure from those described in the Gram-positive Bacillus subtilis (2). In fact, the bodies proved to consist of a conglomerate of elements which contained the strongly electron-scattering reduced tellurite and a delicately granular "matrix." A limiting membrane was not observed around these complexes. In serial sections details of the complexes are illustrated. Reduced tellurite was not deposited in the plasma membrane to any important degree. Since no other sites of deposition of the reduced product were revealed, it is assumed that the complexes represent the mitochondrial equivalents in the investigated organism. In addition, the bodies might function as the basal granules of the flagella.

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D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.9.1974901 ◽

1990 ◽

Vol 38 (9) ◽

pp. 1377-1381 ◽

Cited By ~ 12

Author(s):

M E Beard

Keyword(s):

Amino Acid ◽

Oxidase Activity ◽

Electron Microscopic Level ◽

Acid Oxidase ◽

Electron Microscopic ◽

Amino Acid Oxidase ◽

Cytochemical Localization ◽

Peroxisomal Enzyme ◽

Cytochemical Study ◽

Biochemical Studies

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.

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Electron microscopic cytochemical localization of Ca-ATPase in the rod outer segments of the toadBufo marinus

The Anatomical Record ◽

10.1002/ar.1092210312 ◽

1988 ◽

Vol 221 (3) ◽

pp. 761-768 ◽

Cited By ~ 6

Author(s):

Walter L. Davis ◽

H. K. Hagler ◽

Gene R. Farmer ◽

James H. Martin ◽

George Bridges

Keyword(s):

Rod Outer Segments ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Outer Segments

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Electron microscopic cytochemical localization of?-hydroxyacid oxidase in rat kidney cortex

Histochemistry ◽

10.1007/bf00982671 ◽

1986 ◽

Vol 85 (5) ◽

pp. 411-418 ◽

Cited By ~ 18

Author(s):

S. Angerm�ller ◽

C. Leupold ◽

K. Zaar ◽

H. D. Fahimi

Keyword(s):

Rat Kidney ◽

Kidney Cortex ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Rat Kidney Cortex

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THE CYTOCHEMICAL LOCALIZATION OF GLYCOLYTIC AND OXIDATIVE ENZYMES WITHIN MITOCHONDRIA OF SPERMATOZOA OF SOME PULMONATE GASTROPODS

Journal of Histochemistry & Cytochemistry ◽

10.1177/18.11.783 ◽

1970 ◽

Vol 18 (11) ◽

pp. 783-793 ◽

Cited By ~ 13

Author(s):

WINSTON A. ANDERSON ◽

PAUL PERSONNE

Keyword(s):

Reductase Activity ◽

Periodic Acid ◽

Oxidative Enzymes ◽

Tetrazolium Salt ◽

Phenazine Methosulfate ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Cytochemical Study ◽

Mitochondrial Derivative ◽

The Matrix

In this electron microscopic cytochemical study, the periodic acid-thiosemicarbazide-silver proteinate procedure was used to demonstrate glycogen stores within the mitochondrial derivative of sperm of pulmonate gastropods. In the presence of phenazine methosulfate and tetrazolium salt, enzymatic activity for glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is shown in the matrix and in the compartment containing glycogen, but in the absence of phenazine methosulfate, tetrazolium reductase activity in the matrix is emphasized. Activity for NADH2-tetrazolium reductase and succinate dehydrogenase is also demonstrated in the matrix. Using 3,3'-diaminobenzidine tetra-HCl, cytochrome c oxidase activity is shown in the paracrystalline mitochondrial structure. The interrelation between glycolytic and oxidative pathways in this highly compartmentalized mitochondrion is considered.

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Cytochemical localization of peroxidase activity in wound vessel members of Coleus

Canadian Journal of Botany ◽

10.1139/b72-118 ◽

1972 ◽

Vol 50 (5) ◽

pp. 977-983 ◽

Cited By ~ 54

Author(s):

Peter K. Hepler ◽

Rita M. Rice ◽

William A. Terranova

Keyword(s):

Peroxidase Activity ◽

Cell Walls ◽

Secondary Wall ◽

Primary Wall ◽

Electron Microscopic ◽

Cytochemical Localization ◽

Secondary Thickening ◽

Vessel Elements ◽

Microscopic Investigations

Peroxidase activity has been localized in the cell walls and cytoplasm of wound vessel elements of Coleus which had been fixed in glutaraldehyde, incubated in diaminobenzidine (DAB) and H2O2, and postfixed in OSO4. Electron microscopic investigations revealed prominent staining in the reticulate secondary wall and in the primary wall where the secondary thickenings attach. The stain in the secondary wall is finely textured and heavier towards its periphery than towards its core. The staining of the primary wall, however, is coarsely granular. In the cytoplasm of differentiating vessel elements electron-opaque deposits are observed in the plasmalemma, especially where it overlies the secondary thickening, and in the dictyosomes and their associated vesicles. Staining also occurs on the internal membranes of developing chloroplasts where it is most likely the result of photooxidation of DAB.Staining, except in chloroplasts, appears to be due specifically to peroxidase, since either removal of H2O2 or preincubation with KCN markedly reduces staining, whereas preincubation with aminotriazole, an inhibitor of catalase, does not. The similarity of localization of peroxidase and lignin in the walls of Coleus wound vessel elements supports the postulate that the enzyme participates in lignification.

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CYTOCHEMICAL LOCALIZATION OF NUCLEIC ACIDS BY AN ACRIFLAVINE-PHOSPHOTUNGSTATE COMPLEX FOR FLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY

Journal of Histochemistry & Cytochemistry ◽

10.1177/18.9.609 ◽

1970 ◽

Vol 18 (9) ◽

pp. 609-627 ◽

Cited By ~ 22

Author(s):

VICTORIA CHAN-CURTIS ◽

W. DUANE BELT ◽

CHARLES T. LADOULIS

Keyword(s):

Electron Microscopy ◽

Fluorescence Microscopy ◽

Nucleic Acids ◽

Phosphotungstic Acid ◽

Phosphomolybdic Acid ◽

Testicular Tissue ◽

Aqueous System ◽

Single Step ◽

Electron Microscopic ◽

Cytochemical Localization

An acriflavine-phosphotungstate complex (A-PTA) was synthesized from a reaction between acriflavine and phosphotungstic acid, with the former in excess. The purified product is fluorescent and electron-dense, and was studied for its use as a cytochemical reagent for fluorescence microscopy and electron microscopy. A selectivity for nucleic acids and sulfated cerebroside esters that excludes sulfated glycoaminoglycans is proposed. The reaction for fluorescence microscopy is rapid, sensitive and reproducible. The electron-dense A-PTA complex localized nucleic acids for electron microscopy. Known sites of DNA and RNA in cells of pancreatic, cerebellar and testicular tissue have been demonstrated with the reaction. The reaction is administered as a single step, interposed in the electron microscopic preparative procedure, and can be used in either an alcoholic or an aqueous system. The alcoholic A-PTA reaction produced adequate contrast on short duration (2 hr); the aqueous block reaction was found most satisfactory when prolonged to 48 hr. Uranyl acetate, phosphomolybdic acid and phosphotungstic acid can be used as supplementary stains in addition to the A-PTA reaction. Under these conditions, DNP and RNP sites were demonstrated. The A-PTA complex is a prototype of the coupled dye-metal histochemical reagents, and its selectivity for nucleic acids represents a new approach in cytochemistry. Its reactions can be investigated by fluorescence microscopy and electron microscopy, and in solution. Other dye-metal reagents can probably be formulated on a similar design as detection systems for lipids, polysaccharide or specific groups in proteins.

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