Cardiac Lineage Protein-1 (CLP-1) Regulates Cardiac Remodeling via Transcriptional Modulation of Diverse Hypertrophic and Fibrotic Responses and Angiotensin II-transforming Growth Factor β (TGF-β1) Signaling Axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Host nutritional status as a contributory factor to the remodeling of schistosomal hepatic fibrosis

Weaning Swiss mice were percutaneously infected with 30 cercariae of Schistosoma mansoni and submitted to a shifting either from a deficient to a balanced diet or vice-versa, for 24 weeks. The nutritional status was weekly evaluated by measurements of growth curves and food intake. Hepatic fibrosis and periovular granulomas were studied by histological, morphometric and biochemical methods. All mice fed on a deficient diet failed to develop periportal "pipestem" fibrosis after chronic infection. An unexpected finding was the absence of pipestem fibrosis in mice on normal diet, probably related to the sample size. The lower values for nutritional parameters were mainly due to the deficient diet, rather than to infection. Liver/body weight ratio was higher in "early undernutrition" group, after shifting to the balanced diet. Volume density and numerical density of egg granulomas reached lowest values in undernourished animals. The amount of collagen was reduced in undernourished mice, attaining higher concentrations in well-fed controls and in "late undernutrition" (balanced diet shifted to a deficient one), where collagen deposition appeared increased in granulomas. That finding suggested interference with collagen degradation and resorption in "late" undernourished animals. Thus, host nutritional status plays a role in connective tissue changes of hepatic schistosomiasis in mice.

Identification of small RNAs in Pseudomonas species

Summary: Bacterial small RNAs (sRNAs) are transcripts most of which have regulatory functions. Sequence and secondary structure elements enable numerous sRNAs to interact with mRNAs or with regulatory proteins resulting in diverse regulatory effects on virulence, iron storage, organization of cell envelope proteins or stress response. sRNAs having high affinity for RsmA-like RNA-binding proteins are important for posttranscriptional regulation in various Gram-negative bacteria. In Pseudomonas spp., the GacS/GacA two component system positively controls the production of such sRNAs. They titrate RsmA-like proteins and thus overcome translational repression due to these proteins. As a consequence, secondary metabolites can be produced that are implicated in the biocontrol capacity of P. fluorescens or in the virulence of P. aeruginosa. A genome-wide search carried out in P. aeruginosa PAO1 and in closely related Pseudomonas spp. resulted in the identification of 15 genes coding for sRNAs. Eight of these are novel, the remaining seven have previously been observed. Among them, the 1698 sRNA gene was expressed under GacA control, whereas the transcription of 1887 sRNA gene was transcribed under the control of the anaerobic regulator Anr in an oxygen-limited environment. Overexpression of 1698 sRNA in P. fluorescens strain CHAO did not affect the expression of the GacA-regulated hcnA gene (first gene of the operon coding for HCN synthase), indicating that 1698 sRNA is probably not part of the secondary metabolite regulation pathway. The expression of 1698 sRNA was positively regulated by RpoS in both P. aeruginosa PAO 1 and P. ,fluorescens CHAO and appeared to be modulated temporarily by oxidative stress conditions. However, the effect of 1698 sRNA on oxidative stress survival has not yet been established. Hfq protein interacted with 1698 sRNA in vitro and improved 1698 sRNA expression in vivo in P. aeruginosa. In P. fluorescens, GacA and Hfq were both required for expression of rpoS and GacA showed a positively control on the hfq expression; therefore, at least in this organism, GacA control of 1698 sRNA expression may act indirectly via Hfq and RpoS. Different methods were employed to find abase-pairing target for 1698 sRNA. In a proteomic analysis carried out in P. aeruginosa, positive regulation by 1698 sRNA was observed for Soda, the iron-associated superoxide dismutase, an enzyme involved in oxidative stress resistance. A sequence complementary with 1698 sRNA was predicted to be located in the 5' leader of soda mRNA. However, base-pairing between soda mRNA and 1698 sRNA remains to be proven. In conclusion, this work has revealed eight novel sRNAs and novel functions of two sRNAs in Pseudomonas spp. Résumé Les petits ARNs non-codants (sRNAs) produits par les bactéries sont des transcrits ayant pour la plupart des activités régulatrices importantes. Leurs séquences nucléotidiques ainsi que leurs structures secondaires permettent aux sRNAs d'interagir soit avec des RNA messagers (mRNAs), de sorte à modifier l'expression des protéines pour lesquelles ils codent, soit avec des protéines régulatrices liant des rnRNAs, ce qui a pour effet de modifier l'expression de ces mRNAs. Des sRNAs sont impliqués dans diverses voies de régulation, telles que celles qui régissent la virulence, le stockage du fer, l'organisation des protéines de l'enveloppe bactérienne ou la réponse au stress. Chez les Pseudomonas spp., le système à deux composantes GacS/GacA contrôle la production de métabolites secondaires. Ceux-ci sont engagés dans l'établissement du biocontrôle, chez P. fluorescens, ou. de la virulence, chez P. aeruginosa. La régulation génique dirigée par le système GacS/GacA fait intervenir les sRNAs du type RsmZ, capables de contrecarrer l'action au niveau traductionnel exercée par les protéines régulatrices du type RsmA. Une recherche au niveau du génome a été menée chez P. aeruginosa PAO1 de même que chez des espèces qui lui sont étroitement apparentées, débouchant sur la mise en évidence de 15 gènes codant pour des sRNAs. Parmi ceux-ci, huit ont été découverts pour la première fois et sept confirment des travaux publiés. L'expression du gène du sRNAs 1698 s'avère être régulée par GacA, vraisemblablement de manière indirecte. La transcription du gène du sRNA 1887 montre une dépendance envers Anr, régulateur de l'anaérobiose, et envers une carence en oxygène. La surexpression du sRNA 1698 chez P. fluorescens CHAO n'affecte pas l'expression de hcnA, un gène du régulon GacA, laissant supposer que le sRNA n'intervient pas dans la régulation des métabolites secondaires. Chez P. aeruginosa PAOI et chez P. fluorescens CHAO, RpoS, le facteur sigma du stress, est nécessaire à l'expression du sRNA 1698, et la concentration de ce dernier est modulée par des conditions de stress oxydatif. Toutefois, un effet du sRNA 1698 quant à la survie suite au stress oxydatif n'a pas été établi. Par ailleurs, l'interaction entre le sRNA 1698 et Hfq, la protéine chaperone de RNAs, in vitro ainsi qu'un rôle positif de Hfq pour l'expression du sRNA 1698 in vivo ont été démontrés chez P. aeruginosa. L'induction de l'expression par GacA de rpoS et de hfq a été confirmée chez P. fluorescens CHAO, suggérant que la régulation par GacA du sRNA 1698 pourrait se faire par l'intermédiaire de RpoS et Hfq. Diverses méthodes ont été employées pour identifier un transcrit qui puisse être apparié par le sRNA 1698. Une analyse de protéome chez P. aeruginosa montre que l'expression de Soda, la superoxyde dismutase associée au fer, est positivement régulée par le sRNA 1698. Soda est une enzyme impliquée dans la résistance au stress oxydatif. Une séquence de complémentarité avec le sRNA 1698 a bien été prédite sur le leader 5' du mRNA de soda. Cependant, l'appariement entre le sRNA et son transcrit cible est encore à prouver. En conclusion, ce travail a dévoilé huit nouveaux sRNAs et de nouvelles fonctions pour deux sRNAs chez les Pseudomonas.

Macrophage migration inhibitory factor: a regulator of innate immunity.

For more than a quarter of a century, macrophage migration inhibitory factor (MIF) has been a mysterious cytokine. In recent years, MIF has assumed an important role as a pivotal regulator of innate immunity. MIF is an integral component of the host antimicrobial alarm system and stress response that promotes the pro-inflammatory functions of immune cells. A rapidly increasing amount of literature indicates that MIF is implicated in the pathogenesis of sepsis, and inflammatory and autoimmune diseases, suggesting that MIF-directed therapies might offer new treatment opportunities for human diseases in the future.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Detection of cytotoxic necrotizing factor types 1 and 2 among fecal Escherichia coli isolates from brazilian children with and without diarrhea

The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.