1,721,034 research outputs found
Investigating the role of AMPK in inflammation
No full textIn addition to the well-characterized role of AMPK in the regulation of nutrient metabolism, it is increasingly clear that AMPK activation has multiple actions on inflammatory signalling. Here we describe methods to identify effects of AMPK activity on pro-inflammatory signalling, specifically (1) the nuclear localization of the key inflammatory mediators nuclear factor-κB (NFκB) and phosphorylated c-Jun N-terminal kinase (JNK), (2) preparation of conditioned medium to analyze the secretion of cytokines/chemokines, and (3) the pro-inflammatory adhesion of leukocytes to cultured cells
Identifying the heterotrimeric complex stoichiometry of AMPK in skeletal muscle by immunoprecipitation
No full textThe 5'-AMP-activated protein kinase is a complicated enzyme consisting of three different subunits, each of which is expressed as two or three isoforms. This gives the possibility of 12 different heterotrimeric complexes, which could have diverse functions within the cell. To map out which of these complexes are present and to what extent in skeletal muscle, we have used the immunoprecipitation technique and analyzed both the precipitates and the remaining supernatants for coprecipitation of complex partners. We have fine-tuned this method to give the best results on lysates from the skeletal muscle, liver, and heart muscle from mouse to man.</p
Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK
No full textAMP-activated protein kinase (AMPK) is an energy sensor that is activated by increases in the cellular AMP/ATP and ADP/ATP ratios by three mechanisms: (1) allosteric activation, (2) promotion of phosphorylation at Thr172 on the α subunit by upstream kinases, and (3) inhibition of dephosphorylation of Thr172 by protein phosphatases. All of these effects are triggered by the binding of AMP or ADP at one or more of three sites on the γ subunit, where they displace ATP. AMPK is also activated by ligands that bind in the ADaM site, which is located between the α and β subunits. In this chapter we describe cell-free assays that can be used to study these varied activation mechanisms.</p
Kinase activity determination of specific AMPK complexes/heterotrimers in the skeletal muscle
No full textMeasuring the kinase activity of the 5'-AMP-activated protein kinase (AMPK) is an essential part of understanding the regulation of this metabolic master switch. The AMPK heterotrimer can exist in 12 different constellations with potentially diverse activation patterns. It is therefore important to be able to measure heterotrimer-specific activity to discriminate between these patterns. In this chapter we describe how to measure the AMPK activity of specific heterotrimeric complexes by consecutive immunoprecipitations and how the assay can be performed in a medium throughput fashion using 96-well plates.</p
Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation
No full textAMP-activated protein kinase (AMPK) is extremely sensitive to cellular stress, so that nonphysiological activation of the kinase can readily occur during harvesting of cells or tissues. In this chapter we describe methods to harvest cells and tissues, and for kinase assays, that preserve the physiological activation status of AMPK as far as possible. Note that similar care with methods of cell or tissue harvesting is required when AMPK function is monitored by Western blotting, rather than by kinase assays. We also describe methods to determine whether compounds that activate AMPK in intact cells do so indirectly by interfering with cellular ATP synthesis or directly by binding to AMPK and, if the latter, whether this occurs by binding at the AMP-binding sites on the γ subunit or at the ADaM site located between the α and β subunits.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Is TAK1 a Direct Upstream Kinase of AMPK?
No full textAlongside Liver kinase B1 (LKB1) and Ca2+/Calmodulin-dependent protein kinase kinase 2 (CaMKK2), Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1) has been suggested as a direct upstream kinase of AMP-activated protein kinase (AMPK). Several subsequent studies have reported on the TAK1-AMPK relationship, but the interpretation of the respective data has led to conflicting views. Therefore, to date the acceptance of TAK1 as a genuine AMPK kinase is lagging behind. This review provides with argumentation, whether or not TAK1 functions as a direct upstream kinase of AMPK. Several specific open questions that may have precluded the consensus are discussed based on available data. In brief, TAK1 can function as direct AMPK upstream kinase in specific contexts and in response to a subset of TAK1 activating stimuli. Further research is needed to define the intricate signals that are conditional for TAK1 to phosphorylate and activate AMPK at T172
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe 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
- …
