1,720,956 research outputs found
Redox‐active cysteines in TGACG‐BINDING FACTOR 1 (TGA1) do not play a role in salicylic acid or pathogen‐induced expression of TGA1‐regulated target genes in Arabidopsis thaliana
No full textSummary Salicylic acid (SA) is an important signaling molecule of the plant immune system. In Arabidopsis thaliana, SA biosynthesis is indirectly modulated by the closely related transcription factors TGACG‐BINDING FACTOR 1 and 4 (TGA1 and TGA4, respectively). They activate expression of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1, the gene product of which regulates the key SA biosynthesis gene ISOCHORISMATE SYNTHASE 1. Since TGA1 interacts with the SA receptor NONEXPRESSOR OF PATHOGENESIS‐RELATED GENES 1 (NPR1) in a redox‐dependent manner and since the redox state of TGA1 is altered in SA‐treated plants, TGA1 was assumed to play a role in the NPR1‐dependent signaling cascade. Here, we identified 193 out of 2090 SA‐induced genes that require TGA1/TGA4 for maximal expression after SA treatment. One robustly TGA1/TGA4‐dependent gene encodes for the SA hydroxylase DOWNY MILDEW RESISTANT 6‐LIKE OXYGENASE 1, suggesting an additional regulatory role of TGA1/TGA4 in SA catabolism. Expression of TGA1/TGA4‐dependent genes in mock/SA‐treated or Pseudomonas‐infected plants was rescued in the tga1 tga4 double mutant after introduction of a mutant genomic TGA1 fragment encoding a TGA1 protein without any cysteines. Thus, the functional significance of the observed redox modification of TGA1 in SA‐treated tissues remains enigmatic.Dorothea‐Schlözer‐Fellowship (Göttingen University)Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/50110000165
Identification of the Components Involved in Cyclic Di-AMP Signaling in Mycoplasma pneumoniae
No full textBacteria often use cyclic dinucleotides as second messengers for signal transduction. While the classical molecule c-di-GMP is involved in lifestyle selection, the functions of the more recently discovered signaling nucleotide cyclic di-AMP are less defined. For many Gram-positive bacteria, c-di-AMP is essential for growth suggesting its involvement in a key cellular function. We have analyzed c-di-AMP signaling in the genome-reduced pathogenic bacterium Mycoplasma pneumoniae. Our results demonstrate that these bacteria produce c-di-AMP, and we could identify the diadenylate cyclase CdaM (MPN244). This enzyme is the founding member of a novel family of diadenylate cyclases. Of two potential c-di-AMP degrading phosphodiesterases, only PdeM (MPN549) is active in c-di-AMP degradation, whereas NrnA (MPN140) was reported to degrade short oligoribonucleotides. As observed in other bacteria, both the c-di-AMP synthesizing and the degrading enzymes are essential for M. pneumoniae suggesting control of a major homeostatic process. To obtain more insights into the nature of this process, we have identified a c-di-AMP-binding protein from M. pneumoniae, KtrC. KtrC is the cytoplasmic regulatory subunit of the low affinity potassium transporter KtrCD. It is established that binding of c-di-AMP inhibits the KtrCD activity resulting in a limitation of potassium uptake. Our results suggest that the control of potassium homeostasis is the essential function of c-di-AMP in M. pneumoniae
Molecular basis for the enzymatic inactivity of class III glutaredoxin ROXY9 on standard glutathionylated substrates
Full text linkAbstract Class I glutaredoxins (GRXs) are nearly ubiquitous proteins that catalyse the glutathione (GSH)-dependent reduction of mainly glutathionylated substrates. In land plants, a third class of GRXs has evolved (class III). Class III GRXs regulate the activity of TGA transcription factors through yet unexplored mechanisms. Here we show that Arabidopsis thaliana class III GRX ROXY9 is inactive as an oxidoreductase on widely used model substrates. Glutathionylation of the active site cysteine, a prerequisite for enzymatic activity, occurs only under highly oxidizing conditions established by the GSH/glutathione disulfide (GSSG) redox couple, while class I GRXs are readily glutathionylated even at very negative GSH/GSSG redox potentials. Thus, structural alterations in the GSH binding site leading to an altered GSH binding mode likely explain the enzymatic inactivity of ROXY9. This might have evolved to avoid overlapping functions with class I GRXs and raises questions of whether ROXY9 regulates TGA substrates through redox regulation
Functional analysis of the Arabidopsis thaliana glutaredoxin ROXY9
Full text linkGlutaredoxins are nearly ubiquitious small enzymes which protect the thiol groups of cellular proteins from oxidative stress, contribute to iron-sulfur cluster biogenesis or regulate protein activities via redox modification. Three different classes of glutatreoxins are distinguished by their active site motif: CPYC-type glutaredoxins catalyze the reduction and oxidation of thiol groups and mediate defense against oxidative stress; CGFS-type glutaredoxins are weak catalysts but associate with iron-sulfur clusters, possibly transferring them to target proteins. Whereas CPYC- and CGFS-type glutaredoxins are found in all types of organisms, CC-type glutaredoxins, which are called ROXYs in Arabidopsis thaliana, are restricted to land plants. In contrast to their relatively well characterized relatives, CC-type glutaredoxins are biochemically poorly understood. Information about these glutaredoxins is only available from in vivo studies. Mutant analysis and studies with plants ectopically expressing ROXYs have shown that they modulate TGACG binding (TGA) factor activity. As an example, the CC-type glutaredoxin ROXY9 was shown to repress TGA1-mediated hyponastic growth when overexpressed. Since TGA1 was shown to be redox-regulated in plants treated with the defense hormone salicylic acid, it was speculated that ROXY9 represses TGA1 via catalytic activity. Therefore, this study was set up to analyze whether recombinant ROXY9 exhibits oxidoreductase and/or iron-sulfur cluster binding activity. Up to now, difficulties in the purification of CC-type glutaredoxins has been the bottleneck for their biochemical characterization. Similarly, ROXY9 could not be purified in a soluble, non-aggregated state when using Escherichia coli as an expression host. Strikingly, expression in insect cells resulted in large amounts of monomeric and soluble ROXY9 fused to a strep-MBP-tag. However, the protein turned out to oxidize quickly under aerobic conditions. Reduction of the oxidized protein by glutathione might be inefficient, which could explain the inactivity of ROXY9 towards the typical glutaredoxin substrates bis(2-hydroxyethyl)disulphide (HEDS), insulin, and the redox-sensitive green fluorescent protein (roGFP). Still, a weak reductase activity of ROXY9 towards glutathionylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was observed, suggesting that ROXY9 in principle can reduce target proteins. In contrast to its weak reductase activity, ROXY9 turned out to glutathionylate roGFP efficiently with the help of glutathione disulfide, suggesting that ROXY9 might repress TGA1 in vivo via glutathionylation. However, because all these observations might have been influenced by the oxidation of the protein, the catalytic activity of ROXY9 has to be reproduced with fully reduced protein. Aside from catalysis, ROXY9 appeared to bind an iron-sulfur cluster under anaerobic conditions. This raises the alternative hypothesis that it could repress TGA1 via recruitment of an iron-sulfur cluster-dependent repression complex. In vivo analysis of the repression capacity of overexpressed mutated ROXY9 versions suggested that ROXY9 requires the second cysteine and the tyrosine of its extended active site motif CCLCY for its activity. Future experiments under anaerobic conditions will have to clarify whether ROXY9 requires these amino acids for catalysis or for iron-sulfur cluster binding. In an initial experiment to address the repression mechanism of TGA1 by ROXY9 in vivo, a TGA1 version with mutated cysteine residues was constructed and expressed in the tga1 tga4 mutant. This mutant protein is resistant to oxidation and was as active in vivo as wildtype TGA1 regarding hyponastic growth and flowering. However, the experimental setup might not allow the detection of a weak contribution of the TGA1 redox state to hyponastic growth and flowering; thus, this experiment does currently not allow to conclude whether TGA1 is redox-controlled. To test this hypothesis in the future, ROXY9 will have to be overexpressed in tga1 tga4 mutants complemented with wildtype TGA1 and the TGA1 cysteine mutant to find out, whether ROXY9 can still repress the TGA1 cysteine mutant
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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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