1,721,044 research outputs found
Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli
No full textThe efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed
FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence- structure-function relationships.
No full textFlgM proteins, also known as Anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. Recently, a three-dimensional structure of the Aquifex aeolicus sigma28/FlgM complex (PDB code: 1rp3) was determined by X-ray crystallography at 2.3 A resolution. Furthermore, experimental data on bacterial FlgM, including site-directed mutagenesis and structural characterization by NMR are also available. However, an interpretation of the sequence-structure-function relationships combining X-ray and NMR data with the evolutionary information extracted from the increasing number of FlgM-related sequences annotated in databases is not available. In the present study, we combined database sequence searches and sequence-analysis tools to update the multiple sequence alignment of a previously characterized cluster of orthologs (COG2747) and the PFAM classification of protein domains (PF04316) for the FlgM family. A phylogenetic analysis of 77 protein sequences revealed the presence of at least three major sequence clades within the FlgM family. Besides, we predicted functional residues using a SequenceSpace method. We also generated homology models for Bacillus subtilis and Salmonella typhimurium FlgM proteins, for which sequence-structure-function relationship data are available, and used the docking program ClusPro to hypothesize about the dimer association between FlgM proteins. In conclusion, the analysis presented in this work will be useful in designing new experiments to understand better protein-protein interactions between FglM, sigma factors, and putative molecules from the flagellar export apparatus
Structural characterization of l-aspartate oxidase and identification of an interdomain loop by limited proteolysis.
No full textl-Aspartate oxidase is the first enzyme in the de novo biosynthesis of pyridinic coenzymes in facultative aerobic organisms. The enzyme is FAD dependent and it shares common features with both the oxidase and the fumarate reductase classes of flavoproteins. In this report we focused our attention on the supersecondary structure of the molecule by means of limited proteolysis studies. Moreover the polymerization state of the protein at different pH and the interactions with NAD and its analogues are described. The results suggest that l-aspartate oxidase is a monomer at pH values lower than 4.5 and a dimer at pH values higher than 6.5. The protein is organized in two major domains connected by a flexible loop located in the 120-140 region. The data obtained by limited proteolysis of the holo and the apo form in the presence and in the absence of substrates (fumarate and menadione), inhibitors (succinate) and NAD allows the proposition that both domains are involved in the binding of the flavin coenzyme. Moreover the data reported in this manuscript suggest that NAD inhibits l-aspartate oxidase activity by competing with the flavin for the binding to the enzyme
Overproduction and characterization of the Bacillus subtilis anti-sigma factor FlgM.
No full textFlgM is an anti-sigma factor of the flagellar-specific
sigma (s) subunit of RNA polymerase in Bacillus subtilis,
and it is responsible of the coupling of late flagellar
gene expression to the completion of the hook-basal
body structure. We have overproduced the protein in
soluble form and characterized it. FlgM forms dimers as
shown by gel exclusion chromatography and native
polyacrylamide gel electrophoresis and interacts in
vitro with the cognate sD factor. The FlgMzsD complex is
a stable heterodimer as demonstrated by gel exclusion
chromatography, chemical cross-linking, native polyacrylamide
gel electrophoresis, and isoelectric focusing.
sD belongs to the group of sigma factors able to bind to
the promoter sequence even in the absence of core RNA
polymerase. The FlgMzsD complex gave a shift in a DNA
mobility shift assay with a probe containing a sD-dependent
promoter sequence. Limited proteolysis studies
indicate the presence of two structural motifs, corresponding
to the N- and C-terminal regions, respectively
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
Association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation.
No full textThe sialylation pattern of serum α1-acid glycoprotein (AGP) in non-symptomatic cats infected by feline coronavirus (FCoV) and its possible relationship with the amount of FCoVs shed in faeces were investigated. Blood from three specific pathogen-free cats (group A) and from 10 non-symptomatic FCoV-positive cats from catteries with low (group B, three cats) or high (group C, seven cats) levels of faecal shedding were collected monthly. AGP was purified from serum and Western blotting followed by lectin-staining of α(2,3)-linked and α(2,6)-linked sialic acid. Faecal shedding was quantified in group C by quantitative polymerase chain reaction. Variations of AGP sialylation were recorded only in cats from group C, on which viral shedding peaked before the occurrence of feline infectious peritonitis (FIP) in the cattery, and decreased 1 month later, when serum AGP had an increase of α(2,3)-linked sialic acid. These results suggest that hypersialylation of AGP may be involved in host–virus interactions
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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