1,721,074 research outputs found
Prereplicative increase of nuclear matrix-bound DNA polymerase-alpha and primase activities in HeLa S3 cells following dilution of long-term cultures.
No full textWe investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85\% of the cells were in the G1 phase of the cycle, whereas 8\% were in the S phase. After dilution with fresh medium, 18-22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38\% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10\% of nuclear activity. As judged by [3H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activities was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60\% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures
Redistribution of DNA topoisomerase II β after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy
No full textUsing confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase IIβ (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37°or 42°C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0°C in a buffer containing spermine/spermidine/KC1 or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0°C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37°or 42°C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KC1, the incubation buffer also contained 5 mM Mg++ and the temperature was 37°C. If the stabilization was performed at 42°C, Mg++ failed to maintain the original distribution of DNA topoisomerase IIβ, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37°C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37°C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0°C incubated nuclei; they showed a certain degree of shrinkage in 0°C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37°or 42°C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37°C, but not at 42°C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus
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
Nuclear diacylglycerol produced by phosphoinositide-specific phospholipase C is responsible for nuclear translocation of protein kinase C- α
No full textIt is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition
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
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