39 research outputs found
Degraded Carrageenan Causing Colitis in Rats Induces TNF Secretion and ICAM-1 Upregulation in Monocytes through NF-κB Activation
Carrageenan (CGN) is a high molecular weight sulphated polysaccharide derived from red seaweeds. In rodents, its degraded forms (dCGN) can induce intestinal inflammation associated with macrophage recruitment and activation. The aim of this study was: 1) to analyze the size-dependent effects of dCGN on colon inflammation in vivo, and 2) to correlate these effects with monocyte/macrophage proliferation, cytokine production and expression of various cell surface antigens including ICAM-1 adhesion molecule. Peripheral blood monocytes (PBM) and THP-1 monocytic cells were cultured in the presence of either 10 or 40 kDa, dCGN. The 40 kDa, but not the 10 kDa dCGN, induced colitis in in vivo. Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype
Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut
Background/Aim: The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-kappa B signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-kappa B modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota. Methods: A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-kappa B binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-kappa B modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition. Results: The two proinflammatory cytokines, TNF-alpha and IL-1 beta, were able to activate the reporter system, translating the activation of the NF-kappa B signaling pathway and NF-kappa B inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-kappa B. Conclusions: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-kappa B regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest
Identification of NF-κB Modulation Capabilities within Human Intestinal Commensal Bacteria
The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations
Caratterizzazione della nitrato reduttasi da foglie di pomodoro ed effetto dello stress salino
Dottorato di ricerca in chimica agraria. 12. ciclo. Coordinatore Giacomo Galvano. Tutore Goffredo PetroneConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal
La xanthine dioxygénase a-kétoglutarate dépendante (une enzyme caractéristique des champignons)
Le sujet de cette thèse est le clonage du gène xanA et le caractérisation de la protéine XanA d'Aspergillus nidulans. XanA est une enzyme qui hydroxyle la xanthine en acide urique. Dans une souche sauvage la purine hydroxylase I (HxA) catalyse l'hydroxylation de l'hypoxanthine en xanthine et de la xanthine en acide urique. L'hypoxanthine peut aussi être hydroxylée par la purine hydroxylase II codée par le gène hxnS. Les purines hydroxylases I et II sont des enzymes associées à un cofacteur à molybdène, alors que XanA ne l'est pas. Nous avons cloné et séquencé le gène xanA. Il code une protéine de 370 acides aminés. XanA présente des similarités avec les protéines de la famille des dioxygénases. Des protéines fortement similaires à XanA n'ont été trouvées que chez les champignons (N. crassa, S. pombe, F. graminearum, P. chrysosporium, C. cinereus, U. maydis, C. albicans). Une mutation dans le gène xanA a été isolée. Cette mutation (xanA1) est une transversion de C à A qui se traduit par le remplacement d'une alanine par une asparagine au niveau du codon 167. Le phénotype de la délétion du gène xanA est identique à celui de xanA1. La surexpression de xanA chez A. nidulans a permis de caractériser l'enzyme. Nous avons démontré qu'il s'agit d'une xanthine dioxygénase dépendante de l'a-kétoglutarate. Le gène xanA (chromosome VIII) et son promoteur sont partiellement dupliqués sur le chromosome II. L'homologue de xanA chez S. pombe (TC3962) ne complémente pas la mutation xanA1 d'A. nidulans, tandis que cette mutation est complementée par l'homologue de xanA chez N. crassa (xan1). L'expression de xanA est soumise au contrôle du facteur GATA AreA et est dépendante du facteur UaY.This work comprises the cloning of the xanA gene and the biochemical characterization of the XanA protein of aspergillus nidulans. This enzyme catalyses the oxidation of xanthine to uric acid. In the wild type strain, purine hydroxylase I (HxA) catalyses both the hydroxylation of hypoxanthine to xanthine and that of xanthine to uric acid. Hypoxanthine is also oxidized to xanthine by a second purine hydroxylase (purine hydroxylase II), which is coded by the hxnS gene. Both purine hydroxylases contain a molybdopterin co-factor while XanA does not. We have cloned and sequenced the xanA gene. xanA encodes a protein 370 amino acids long. the sequence of XanA has confirmed that it's not a molybdenum-containing enzyme; we found some similarities with proteins of the dioxygenase family. Proteins with high similarity to XanA were found only in fungi in N. crassa, S. pombe, F. graminearum, p: chrysosporium, C. cinereus, U. maydis, C.albicans. One mutation (xanA1) has been isolated. The xanA1 allele is a C to A transversion resulting in an alanine to asparagine change in codon 167. The phenotype of the xanA1 mutation is identical to that of xanA deletion. Overexpression of the xanAgene in A. nidulans has permitted a preliminary characterization of the enzyme. We have shown it is an a-ketoglutarate dependent xanthine dioxygenase. The xanA gene (chromosome VIII), including its promoter is partially duplicated in chromosome II. The S. pombe homologue of xanA, TC3962, is not able to complement the mutation xanA1. As all other enzymes of the purine utilization pathway xanA expression is under the control of the GATA factor AreA and the pathway specific transcription factor UaY.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
Complementary and dose-dependent action of AtCCS52A isoforms in endoreduplication and plant size control.
International audienceThe dimension of organs depends on the number and the size of their component cells. Formation of polyploid cells by endoreduplication cycles is predominantly associated with increases in the cell size and implicated in organ growth. In plants, the CCS52A proteins play a major role in the switch from mitotic to endoreduplication cycles controlling thus the number of mitotic cells and the endoreduplication events in the differentiating cells. * Arabidopsis has two CCS52A isoforms; AtCCS52A1 and AtCCS52A2. Here we focused on their roles in endoreduplication and cell size control during plant development. We demonstrate their complementary and dose-dependent actions that are dependent on their expression patterns. Moreover, the impact of CCS52A overexpression on organ size in transgenic plants was dependent on the expression level; while enhanced expression of the CCS52A genes positively correlated with the ploidy levels, organ sizes were negatively affected by strong overexpression whereas milder overexpression resulted in a significant increase in the organ sizes. * Taken together, these finding support both complementary and dose-dependent actions for the Arabidopsis CCS52A isoforms in plant development and demonstrate that elevated ectopic CCS52A expression positively correlates with organ size, opening a route to higher biomass production
Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells
The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells
ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways
Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolic products of carbohydrate fermentation by the microbiota and constitute the main source of energy for host colonocytes. SCFAs are also important for gastrointestinal health, immunity, and host metabolism. Intestinally produced angiopoietin-like protein 4 (ANGPTL4) is a secreted protein with metabolism-altering properties and may offer a route by which microbiota can regulate host metabolism. Peroxisome proliferator-activated receptor (PPAR)-gamma has previously been shown to be involved in microbiota-induced expression of intestinal ANGPTL4, but the role of bacterial metabolites in this process has remained elusive. Here, we show that the SCFA butyrate regulates intestinal ANGPTL4 expression in a PPAR-gamma-independent manner. Although PPAR-gamma is not required for butyrate-driven intestinal ANGPTL4 expression, costimulating with PPAR-gamma ligands and SCFAs leads to additive increases in ANGPTL4 levels. We suggest that PPAR-gamma and butyrate rely on two separate regulatory sites, a PPAR-responsive element downstream the transcription start site and a butyrate-responsive element(s) within the promoter region, 0.5 kb upstream of the transcription start site. Furthermore, butyrate gavage and colonization with Clostridium tyrobutyricum, a SCFA producer, can independently induce expression of intestinal ANGPTL4 in germ-free mice. Thus, oral administration of SCFA or use of SCFA-producing bacteria may be additional routes to maintain intestinal ANGPTL4 levels for preventive nutrition or therapeutic purposes
A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria
Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds