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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Histochemistry and cell biology 102 (1994), S. 15-27 
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O·− 2, H2O2, and the H2O2-myeloperoxidase (MPO)-halide system. The H2O2-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3′-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H2O2, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H2O2 to yield oxidized DAB, H2O2 was localized to phagolysosomes. The subcellular sites of H2O2 generation were shown using cerium ions which react with H2O2 and precipitate electron opaque cerium perhydroxides (Ce(OH)2OOH and Ce(OH)3OOH). The results suggested that NADPH oxidase is associated with the plasmalemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H2O2 in the phagosome. As O·− 2 is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O·− 2 oxidizes Mn2+ to Mn3+, and Mn3+ oxidizes DAB, a medium containing DAB-Mn2+ was used to localize sites of O·− 2 production in stimulated PMN. The localizations were, as expected, similar to those for H2O2. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.
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  • 2
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Normal skeletal and cardiac striated muscle from adult rats was incubated for the cytochemical detection of acid phosphatase activity with cerium as the capture metal. Results from these experiments show that normal striated muscle has a greater number of acid phosphatase-positive structures, which are presumed to be lysosomes, than has been indicated by several previous cytochemical studies.
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  • 3
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and nonparticulate (phorbol myristate acetate) stimuli. This membraned activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn++, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium.
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  • 4
    ISSN: 1615-6102
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 5
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 23 (1984), S. 2039-2043 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Approximately 15% of mouse oocytes cultured for 16 h in medium containing cytochalasin B (CCB, 5 µg ml 1) underwent pseudocleavage (Fig. 1a and d) This percentage was increased several-fold by the addition of db cyclic AMP (0 2 mM) to the medium5 or by using oocytes which failed to resume ...
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  • 8
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Examination of the toad urinary bladder by freeze-fracture electron-microscopy demonstrates structural specialization of the granular cell's luminal membrane compared to its basal membrane. Although both membranes appear to possess about 1,700 intramembranous particles per μm2, those of the luminal membrane tend to be significantly larger in size. In addition, the fracturing properties of the two membranes are markedly different: the majority of particles are found on fracture face B (outer membrane face), in the case of the luminal membrane, and the majority are found on fracture face A (inner membrane face), in the case of the basal membrane. While the two fracture faces of the basal membrane possess a similar distribution of particle sizes, in the case of the luminal membrane the B face was found to possess particles generally larger than those found on the A face. It was established that the probability of luminal membrane particles adhering to face B instead of face A is closely correlated with the size of the particle. The structural specialization of the granular cell's luminal membrane may have an important relationship to the characteristic permeability properties of this membrane and the capacity of this cell type to respond physiologically to the hormone vasopressin.
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  • 9
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The PMN is exquisitely designed to combat invading micro-organisms. The relationship between structure and function is nowhere more evident than in this cell type. The elaborate biochemical machinery which the PMNs possess for killing ingested micro-organisms works as a highly integrated system, with each step occurring in sequence and at a particular site. In the past decade or so it has become apparent that the Klebanoff system (myeloperoxidase-halide-H2O2) and possibly other active O2 species as well, play an important role in the bactericidal activity of PMNs. Application of cytochemical techniques for oxidative enzymes and for end-products of oxidative reactions has localized the sites within the phagocytosing or stimulated PMN at which these various components of the cidal systems are active and generated. In this fashion, biochemical data have been not only confirmed, but in several instances, the cytochemical approach has led the way in extending our knowledge and thinking regarding PMN metabolism and cidal functions. In our laboratory we have studied the bactericidal machinery of PMNs by cytochemical means. We have established, at the ultrastructural level, that the myeloperoxidase-containing azurophil granules fuse with the phagosome membrane and empty their contents into the phagosome (Baehneret al., 1969). We have shown that H2O2 is generated within the phagosome (Briggset al., 1975b). This established that the myeloperoxidase-H2O2 system could work within the phagosome, since both of these components are present following phagocytosis. We determined that H2O2 could be detected on the cell surface and within the phagosome following phagocytic stimulation of NADH oxidase activity (Briggset al., 1975a). The cell surface localization of H2O2 was an important finding since the phagosome membrane is derived from the plasmalemma. Thus internalization of the plasmalemma, with components capable of generating H2O2, can explain the presence of H2O2 within the phagosome. We have also shown that when PMNs are treated with non-particulate stimuli of the respiratory burst, similar results are found, that is, H2O2 is present on the cell surface and within vesicles, which are presumed to be of surface origin (Badweyet al., 1980). We have shown that D-amino acid oxidase, another enzyme capable of generating H2O2 is cytochemically demonstrable and that it can utilize cell wall components of ingested bacteria as substrates for enzyme activity (Robinsonet al., 1978). The PMNs from CGD patients do not kill certain bacteria. This inability to kill bacteria is related to the low levels of H2O2 produced during phagocytosis. Using the cerium reactioon we determined that PMN from CGD patients produce little cytochemically detectable H2O2 and that what little is present is restricted to the phagosome (Briggset al., 1977). Some PMNs contain other oxidases which are capable of generating H2O2 and O 2 − from O2 consumed during phagocytosis. The guinea-pig PMN (but not human) has an unusual aldehyde oxidase. Cytochemically the aldehyde oxidase activity is restricted to the phagosome (Robinsonet al., 1979). We have also developed a method for localization of sites of O 2 − production following stimulation. In phorbol myristate acetate-stimulated PMNs, reaction product for O 2 − is present within surface-derived vesicles, and in some cases, on the cell surface. Cytochemical detection of enzymes and products of enzymatic activity (H2O2 and O 2 − ) associated with stimulation of the respiratory burst in PMN has thus provided further evidence for the importance of active oxygen species in phagocytosis. Furthermore, the site-specific information obtained from cytochemistry has provided an important link in understanding the structure-function interplay associated with phagocytosis in PMNs. It should be realized, however, that the cytochemical methods we have utilized detect in most instances the end product of an enzymatic reaction (for example, H2O2) and not the site of the enzyme itself. This is important, for instance in the case of H2O2, because this entity appears to begenerated on the surface of the plasmalemma or on the luminal surface of the phagosomal membrane. However, the enzyme responsible may well be situated on the cytoplasmic side of these membranes, and the generation of the H2O2 may involve an electron shuttle across the membrane. Such a mechanism may involve cytochrome and quinone compounds as carriers (Segal & Jones, 1979; Millardet al., 1979). Experiments are now being designed to localize the sites of the enzymesper se by immunocytochemistry. This approach should help resolve these important questions.
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  • 10
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: By repeated selection for longer survival in an isotonic solution of glycerol, a stable subline of Novikoff rat hepatoma cells has been isolated. The cells exhibit markedly increased resistances to osmotic lysis in isotonic solutions of glycerl. They are twice as large and have twice as many chromosomes as cells of the parental line. It is suggested that the osmotic stress procedure can be extended for the selection of numerous kinds of mutants and can be used as a method of analysis of membrane properties.
    Additional Material: 7 Ill.
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