1,721,068 research outputs found
LIPOTEICHOIC ACID AND FLAGELLIN INDUCE UP-REGULATION OF ART1 INAIRWAY EPITHELIAL CELLS
No full textMono ADP-ribosylation, like phosporylation is a post-translational modification that alters key cellular events. This reaction is catalyzed by ADP-ribosyltransferases (ARTs), a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. The family of known ARTs consists of seven members, except for ART1 and 5, which transfer ADP-ribose unit from NAD to arginine, the amino-acid acceptor of the other ARTs is still unknown. We have shown that apical surface of ciliated and intermediate epithelial cells purified from bronchoalveolar lavage express ART1, 3 and 4. This is consistent with the possibility that these ecto-enzymes may have a role in inflammatory responses in lung. Indeed, A549 cells, which retain features of type II alveolar epithelial cells, represent a good model to study ART functions in epithelia. Using an arginine-specific enzymatic assay, we have identified an ART activity, on the surface of A549 cells. Possible candidates for catalyzing the arginine-specific ADP-ribosylation are ART1 and ART5 that recognize arginine as ADP-ribose acceptor. By reverse transcription-PCR we detected only ART1 mRNA and western blot of A549 membrane proteins with α-ART1 polyclonal antibodies further showed the expression of ART1. Since airway epithelial cells can interact with respiratory pathogens or their toxins, we investigated whether ART1 activity and expression is induced by bacterial components of Gram+ and Gram- bacteria. While lipopolysaccharide, peptidoglycan and Poly-(I:C) had no effect on transferase activity, lipoteichoic acid or flagelllin exerted a 3.7- and a 2.7-fold increase respectively of the transferase activity over the basal levels after 24 h treatment. The same effectors up-regulated ART1 expression, as shown by western blot analysis of cell membrane proteins suggesting as the up-regulation of ART1 is not a general event simply associated with cell activation
Promoter of the Pertussis Toxin Operon and Production of Pertussis Toxin
No full textPertussis toxin (PT), the major virulence factor of Bordetella pertussis, is composed of five different subunits whose genes are organized as an operon. We report the mapping of the promoter region of the PT operon and show that this promoter is only weakly active in Escherichia coli. Bordetella parapertussis and Bordetella bronchiseptica, which do not produce any PT, are shown to have a weaker promoter sequence for this operon and not to produce any detectable PT mRNA. We show that transcription of the PT operon in B. pertussis was constant throughout until the late stationary phase, when transcription significantly decreased. Analysis of the transposon Tn5 mutant BP347 showed that the product of the vir locus was required for transcription of the PT operon. Characterization of the Tn5 mutant BP356 showed that subunit S3 was required for the release of PT into the extracellular medium
Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis
No full textFur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. Four promoters of Neisseria meningitidis predicted to have Fur-binding boxes were selected to study the molecular interactions between Fur and the promoter regions of genes expected to play a central role in survival and pathogenesis. We demonstrate that Fur acts not only as a repressor, but also as an activator of gene expression both in vivo and in vitro. We report that Fur binds to operators located upstream of three promoters that are positively regulated in vivo by Fur and iron, whereas Fur binds to an operator overlapping the classically iron-repressed tbp promoter. Deletion of the upstream operator in the norB promoter abolished activation of transcription in vivo in response to iron and in vitro in response to Fur. The role of such a dual mechanism of Fur regulation during infection is discussed
MONO ADP-RIBOSYLTRANSFERASES EXPRESSED ON THE SURFACE OF PULMONARY EPITHELIAL CELLS ARE REGULATED BY PATHOGEN-ASSOCIATED MOLECULAR PATTERNS
Full text linkMono ADP-ribosylation is a post-translational modification catalyzed by a family of enzymes, related to bacterial toxins, which possess adenosine diphosphate ribosyltransferase activity (ARTs). The expression of ART1, 3 and 4 at the apical surface of airway cells is consistent with the possibility that ARTs may have a role in inflammatory response. Since several studies have evidenced that Toll-like receptors (TLRs) are expressed in lung epithelial cells and given that there are a number of potential TLR agonists in the lung, we evaluated whether airway epithelial cells express transferase activity. We have assessed that A549 cells express ART1 on their surface and shown that lipotheicoic acid (LTA) and flagellin, but not lipopolysaccaride (LPS), peptidoglycan (PG) and poly (I:C), up-regulate ART1. This cell line was used to determine how bacterial components of Gram+ and Gram- bacteria affect ART activity and/or expression. The expression of ART1 relative to the actin obtained by qRT-PCR confirm the up-regulation exerted on ART1 gene by LTA but not by PAM and flagellin. LPS, LTA, PG, flagellin or poly(I:C) did not induce the expression of either ART3 or ART5 mRNA, while a strong stimulatory effect on ART4 mRNA expression was found after stimulation of cells with LPS, LTA and PG but not flagellin and poly(I:C). A pretreatment of cells with mAbs that antagonize TLR4 and TLR2, inhibited ART4 activation indicating TLR4 and TLR2 involved in the mediation of ART4 induction. The incubation with TLR2 antagonist specifically blocks the ART1 up-regulation by LTA indicating that the effect is mediated by this receptor
Expression and Immunological Properties of the 5 Subunits of Pertussis Toxin
No full textPertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough. The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon. We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable. To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31. A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture. The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits. These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin. Mice immunized with the five subunits were not protected from an intracerebral challenge with B. pertussis. Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically. The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxi
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
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