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Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca2+ with fura-2.
No full textThe fluorescent reporter dyes quin2 and fura-2 are used by many investigators to quantitate the cytosolic free-calcium concentration [Ca2+]i in different types of cells. For the dyes to measure accurately [Ca2+]i, they must be selectively and uniformly distributed within the cytoplasmic matrix of the cells. However, in a number of cell types fura-2 does not remain within the cytoplasmic matrix; it accumulates within intracellular compartments, is secreted from the cells entirely, or both. This chapter reviews the evidence for fura-2 sequestration and secretion by cells and describes the use of organic-anion transport inhibitors to ameliorate problems caused by these processes. Mouse macrophages possess organic-anion transporters that remove fluorescent dyes, including fura-2, from the cytoplasmic matrix of these cells. The dyes are sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. The transporters that promote dye sequestration and secretion are inhibi..
Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels
No full textAbstract
Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i
Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions
No full textAbstract
Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types
Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms
No full textWe have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only
Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers
No full textFura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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