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Commentary to: Mutations of the PI3KCA gene in ovarian and breast cancer
No full textObjective: to conduct a mutational analysis of the PIK3CA gene
in ovarian and breast tumors and correlate the
molecular results with histological types
The erythroid differentiation of K562 cells depends on the nuclear translocation of Akt
No full textPrevious findings have demonstrated that Erythropoietin (EPO) treatment during erythroid differentiation of human erythroleukaemia cells activates PI3K (1,2) and that an active PI3K translocates into the nucleus (3). When PI3K activity is inhibited, cellular differentiation is blocked, showing the requirement of this kinase during cell differentiation (3). It is known that in the PI3K signalling pathway, the Ser/Thr kinase Akt is a downstream enzyme involved in many cellular processes including differentiation (4).
Therefore we wanted to analyze the role of nuclear Akt during the erythroid differentiation process in K562 erythroleukaemia cells, following EPO treatment.
Materials and methods: K562 cells coltures, EPO treatment, preparation of whole cell homogenates, isolation of nuclei, immunoblotting, Akt kinase assay and immunocytochemistry were accomplished as previously reported (3).
Results: In whole cells homogenates after 10 min of EPO exposure we observed the highest level of Ser473 phosphorylated Akt. This kinase increases rapidly and transiently in response to EPO treatment its intranuclear amount, with a delay when compared with the cell homogenates. Infact we observed that into the nucleus Ser473 phosphorylated Akt was observable at highest levels after 15 min.
In situ analysis by means of immunocytochemistry showed the nuclear translocation of Akt and of its phosphorylated form on Ser473 that peaked at 15 min and returned to control conditions after 30 min.
Enzyme translocation and erythroid differentiation were blocked by the specific Akt pharmacological inhibitor acting on its PH activation domain, 1L-6-Hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (from Alexis).
Almost all Akt kinase observed into the nucleus was phosphorylated on Serine 473 of the hydrophobic regulative activation domain, as demonstrated by using double round of immunoprecipitation (IP) experiments in which the first round of IP was performed with anti phospho- Akt (Ser473) antibody and the second round with total anti Akt antibody.
Using kinase assay we observed a peak of Akt activity after 10 min of EPO treatment in whole cell homogenates and an intranuclear peak of activity after 15 min of EPO exposure, that were both blocked by pre-treatment of cells by Akt inhibitor.
Conclusions: EPO induces the nuclear translocation of Akt in a time dependent and ordered manner. Furthermore, when cells were treated with an Akt inhibitor, the nuclear translocation of the enzyme was blocked as well as the differentiation process. These findings strongly suggest the requirement of active Akt translocation into the nucleus as an important step in the PI3K/Akt signaling pathway to enter into EPO-mediated erythroid differentiation
Nucleolin: a nuclear Akt substrate
No full textNucleolin is one of the most representative proteins in nucleoli of actively dividing cells, potentially involved in a large number of nuclear processes like transcription, chromatin organization, rRNA maturation and ribosome assembly (1-2). We observed the presence in nucleolin of the Akt phosphorylation motif, which is highly conserved in several species, raising the hypothesis that nucleolin could be a specific substrate of Akt. However, modulation of nucleolin functions by the PI3K/Akt signaling pathway has not been still investigated. With this study we aimed to demonstrate the functional interaction between Akt kinase and nucleolin. Since Akt translocates into the nucleus after stimulation of several cell lines (3), our goal was to explore Akt and nucleolin binding and Akt enzymatic activity toward this potential nuclear substrate after treatment in different cell models.
Materials and methods: we used HL60, Jurkat and PC12 cell in coltures, stimulated with different growth factors. Preparation of whole cell homogenates, immunoprecipitation, immunoblotting and Akt kinase assay were accomplished as previously reported (4-6).
Results: nucleolin was recognized in immunoblotting after immunoprecipitation with anti Akt antibody; conversely, Akt was detected in immunoblotting after nucleolin immunoprecipitation. Interestingly, bands displayed various intensity in different cell models, being more intense in cell lines with the PI3K/Akt signaling pathway iper-activated (7-8), such as HL60-AR (apoptosis resistant) and Jurkat cells. In vitro Akt kinase assay on nucleolin, performed using the immunoprecipitated proteins, showed the functional interaction between the two molecules, since nucleolin was recognized by the antibody against the phosphorylated Akt sequence (PAS). Moreover, second round of immunoprecipitation using antibody against nucleolin after a first round using anti-PAS antibody, showed a faint amount of unphosphorylated nucleolin, thus showing elevated levels of phospho-nucleolin in Jurkat cells. Nucleolin phosphorylation was modulated by cell treatment, as demonstrated in HL60 human acute promyelocytic leukaemia cells stimulated with IGF-I and in PC12 murine pheochromocytoma cell line treated with NGF. Nevertheless, HL60-AR and Jurkat cell lines, which have the PI3K/Akt signaling pathway iper-activated, showed high basal level of nucleolin phosphorylation.
Conclusions: We showed that nucleolin is a direct target for Akt kinase activity. Nucleolin and Akt differently interact in several cell lines, as shown by their higher binding in models where the PI3K/Akt signaling pathway is activated. Our findings strongly document the Akt and nucleolin functional interaction that seems to have a role in relaying signals from cell surface to the nucleus in several cell lines
AKT1 (v-akt murine thymoma viral oncogene homolog 1). Atlas Genet Cytogenet Oncol Haematol. May2009. http://AtlasGeneticsOncology.org/Genes/AKT1ID355ch14q32.html (This paper should be referenced as such)
No full textThe Gly331Ser mutation in factor VII in Europe and the Middle East
No full textThe Gly331Ser mutation in factor VII (FVII) was found in
the homozygous condition in several unrelated FVII deficient
subjects from Southern Italy and from Germany, one of Turkish
origin. Genotyping for several polymorphisms revealed
that the mutation, occurring at a CpG site, is associated with
two haplotypes, suggesting multiple occurrence
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
Residual factor VII activity and different hemorrhagic phenotypes in CRM+ factor VII deficiencies (Gly331Ser and Gly283Ser)
No full textTwo cross-reacting material-positive (CRM(+)) factor VII (FVII) mutations, associated with similar reductions in coagulant activity (2.5%) but with mild to asymptomatic (Gly331Ser, c184 [in chymotrypsin numbering]) or severe (Gly283Ser, c140) hemorrhagic phenotypes, were investigated. The affected glycines belong to structurally conserved regions in the c184 through c193 and c140s activation domain loops, respectively. The natural mutants 331Ser-FVII and 283Ser-FVII were expressed, and in addition 331Ala-FVII and 283Ala-FVII were expressed because 3 functional serine-proteases bear alanine at these positions. The 331Ser-FVII, present in several asymptomatic subjects, showed detectable factor Xa generation activity in patient plasma (0.7% +/- 0.2%) and in reconstituted system with the recombinant molecules (2.7% +/- 1.1%). The reduced activity of recombinant 283Ala-FVII (7.2% +/- 2.2%) indicates that the full function of FVII requires glycine at this position, and the undetectable activity of 283Ser-FVII suggests that the oxydrile group of Ser283 participates in causing severe CRM(+) deficiency. Furthermore, in a plasma system with limiting thromboplastin concentration, 283Ser-FVII inhibited wild-type FVIIa activity in a dose-dependent manner
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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