A role for extracellular amastigotes in the immunopathology of Chagas disease

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

IPH response to Department for Social Development consultation on future regulation of gambling in Northern Ireland

The Department for Social Development (DSD) recently undertook a review of Northern Ireland's gambling law sought views to help strike a balance between developing gambling as a leisure pursuit and minimising its potential negative consequences.  Following the consultation period, DSD aims to produce a balanced package of reforms which will strengthen the regulatory regime while easing some of the current restrictions on industry development.

Bcl9/Bcl9l are critical for Wnt-mediated regulation of stem cell traits in colon epithelium and adenocarcinomas.

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

Medicinal products: regulation of biosynthesis in space and time

We live in a "Demon-Haunted World". Human health care requires the ever increasing resistance of pathogens to be confronted by a correspondingly fast rate of discovery of novel antibiotics. One of the possible strategies towards this objective involves the rational localization of bioactive phytochemicals. The conceptual basis of the method consists in the surprisingly little known gearings of natural products with morphology, ecology and evolution of their plant source, i. e. an introspection into the general mechanisms of nature.

Brain-derived neurotrophic factor enhances the expression of the monocarboxylate transporter 2 through translational activation in mouse cultured cortical neurons.

MCT2 is the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate. It is suggested that MCT2 is upregulated to meet enhanced energy demands after modifications in synaptic transmission. Brain-derived neurotrophic factor (BDNF), a promoter of synaptic plasticity, significantly increased MCT2 protein expression in cultured cortical neurons (as shown by immunocytochemistry and western blot) through a translational regulation at the synaptic level. Brain-derived neurotrophic factor can cause translational activation through different signaling pathways. Western blot analyses showed that p44/p42 mitogen-activated protein kinase (MAPK), Akt, and S6 were strongly phosphorylated on BDNF treatment. To determine by which signal transduction pathway(s) BDNF mediates its upregulation of MCT2 protein expression, the effect of specific inhibitors for p38 MAPK, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), p44/p42 MAPK (ERK), and Janus kinase 2 (JAK2) was evaluated. It could be observed that the BDNF-induced increase in MCT2 protein expression was almost completely blocked by all inhibitors, except for JAK2. These data indicate that BDNF induces an increase in neuronal MCT2 protein expression by a mechanism involving a concomitant stimulation of PI3K/Akt/mTOR/S6, p38 MAPK, and p44/p42 MAPK. Moreover, our observations suggest that changes in MCT2 expression could participate in the process of synaptic plasticity induced by BDNF.

Determinació de la regió del genoma o gens implicats en la síntesi del polièster extracel•lular present en la variant llisa de Mycobacterium vaccae mitjançant transformació

Estudi realitzat a partir d’una estada a la Universidad de Zaragoza, Espanya, entre novembre del 2007 i abril del 2008. Mycobacterium vaccae és un micobacteri ambiental de creixement ràpid molt estudiat pel seu interès com a possible ús immunoterapéutic en el tractament de la tuberculosis i altres malalties. M.vaccae a l’igual que altres micobacteris presenta dues morfologies colonials: llisa i rugosa. M.vaccae ATCC15483T té originàriament una morfologia llisa. Quant aquest es cultiva en medi sòlid a 30ºC apareixen espontàniament variants rugoses estables que no reverteixen a llises. El motiu pel qual aquest procés té lloc no es coneix, encara que s’ha descrit en Mycobacterium smegmatis i en Mycobacterium avium que els lípids de la paret cel•lular es troben involucrats en aquest canvi de morfologia colonial. L’anàlisi dels contingut en lípids i glicolípids de la paret cel•lular de les dos variants morfològiques de M.vaccae, ens ha indicat que les soques llises presenten un compost extracel•lular que no es troba en les rugoses i que mitjançant l’anàlisi estructural d’aquest compost ha sigut identificat com un polièster extracel•lular de cadena llarga. El present estudi s’ha centrat en determinar els gens implicats en la síntesis d’aquest compost. Per a realitzar aquest anàlisi genètic s’ha construit una llibreria de mutants per transposició de la soca llisa de M. vaccae mitjançant un plàsmid ts/sac i un transposó. S’han obtingut colònies de morfologia rugosa on el plàsmid s’ha insertat en la zona del genoma que codifica per aquest compost extracel•lular. Aquests nous mutants s’han analitzat mitjançant tècniques moleculars (PCR, Southern y seqüenciació). A mès, s’ha construit una llibreria genòmica amb DNA de la soca llisa en plàsmids replicatius de micobacteris derivats de pAL5000 i s’ha transformat la soca rugosa seleccionant per a un fenotip llis estudiant els gens que complementen.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Extracellular matrix and platelet function in patients with musculocontractural Ehlers-Danlos syndrome caused by mutations in the CHST14 gene.

We report on a consanguineous, Afghani family with two sisters affected with characteristic facial features, multiple contractures, progressive joint and skin laxity, hemorrhagic diathesis following minor trauma and multisystem fragility-related manifestations suggestive of a diagnosis of musculocontractural Ehlers-Danlos syndrome (EDS). This novel form of connective tissue disorder was recently reported in patients of Japanese, Turkish, and Indian descent who were formerly classified as having EDS type VIB and has now been recognized to be a part of spectrum including patients previously classified as having adducted thumb-clubfoot syndrome. We identified a previously unreported mutation in the CHST14 gene, which codes for the enzyme dermatan 4-O-sulfotransferase. We discuss the prenatal presentation, detailed clinical manifestations, and neurological findings in two sisters with this newly described musculocontractural EDS-CHST14 type. We demonstrate that fibroblasts from one of our patients produce more chondroitin sulfate than normal and show lower than normal deposition of collagens I and II and fibrillin 1-containing microfibrills. These findings suggest that the imbalance in the glycosaminoglycan content in developing tissues might interfere with normal deposition of other extracellular matrix components and ultimately contribute to the development of the phenotype observed in these patients. Furthermore, we ruled out the contribution of intrinsic platelet factors to the bleeding diathesis observed in some affected individuals. © 2012 Wiley Periodicals, Inc.

Regulation of the Gac/Rsm pathway in "Pseudomonas fluorescens" CHA0

SUMMARY : Two-component systems are key mediators implicated in the response of numerous bacteria to a wide range of signals and stimuli. The two-component system comprised of the sensor kinase GacS and the response regulator GacA is broadly distributed among γ-proteobacteria bacteria and fulfils diverse functions such as regulation of carbon storage and expression of virulence. In Pseudomonas fluorescens, a soil bacterium which protects plants from root-pathogenic fungi and nematodes, the GacS/GacA two-component system has been shown to be essential for the production of secondary metabolites and exoenzymes required for the biocontrol activity of the bacterium. The regulatory cascade initiated by GacS/GacA consists of two translational repressor proteins, RsmA and RsmE, as well as three GacAcontrolled small regulatory RNAs RsmX, RsmY and RsmZ, which titrate RsmA and RsmE to allow the expression of biocontrol factors. Genetic analysis revealed that two additional sensor kinases termed RetS and Lads were involved as negative and positive control elements, respectively, in the Gac/Rsm pathway in P. fluoresens CHAO. Furthermore, it could be proposed that RetS and Lads interact with GacS, thereby modulating the expression of antibiotic compounds and hydrogen cyanide, as well as the rpoS gene encoding the stress and stationary phase sigma factor σ. Temperature was found to be an important environmental cue that influences the Gac/Rsm network. Indeed, the production of antibiotic compounds and hydrogen cyanide was reduced at 35°C, by comparison with the production at 30°C. RetS was identified to be involved in this temperature control. The small RNA RsmY was confirmed to be positively regulated by GacA and RsmA/RsmE. Two essential regions were identified in the rsmY promoter by mutational analysis, the upstream activating sequence (UAS) and the linker sequence. Although direct experimental evidence is still missing, several observations suggest that GacA may bind to the UAS, whereas the linker region would be recognized by intermediate RsmA/RsmEdependent repressors and/or activators. In conclusion, this work has revealed new elements contributing to the function of the signal transduction mechanisms in the Gac/Rsm pathway. RESUME : Les systèmes ä deux composants sont des mécanismes d'une importance notoire que beaucoup de bactéries utilisent pour faire face et répondre aux stimuli environnementaux. Le système à deux composants comprenant le senseur GacS et le régulateur de réponse GacA est très répandu chez les γ-protéobactéries et remplit des fonctions aussi diverses que la régulation du stockage de carbone ou l'expression de la virulence. Chez Pseudomonas fluorescens CHAO, une bactérie du sol qui protège les racines des plantes contre des attaques de champignons et nématodes pathogènes, le système à deux composants GacS/GacA est essentiel à la production de métabolites secondaires et d'exoenzymes requis pour l'activité de biocontrôle de la bactérie. La cascade régulatrice initiée pas GacS/GacA fait intervenir deux protéines répresseur de traduction, RsmA et RsmE, ainsi que trois petits ARNs RsmX, RsmY et RsmZ, dont la production est contrôlée par GacA. Ces petits ARNs ont pour rôle de contrecarrer l'action des protéines répressseur de la traduction, ce qui permet l'expression de facteurs de biocontrôle. Des analyses génétiques ont révélé la présence de deux senseurs supplémentaires, appelés Rets et Lads, qui interviennent dans la cascade Gac/Rsm de P. fluorescens. L'impact de ces senseurs est, respectivement, négatif et positif. Ces interactions ont apparenunent lieu au niveau de GacS et permettent une modulation de l'expression des antibiotiques et de l'acide cyanhydrique, ainsi que du gène rpoS codant pour le facteur sigma du stress. La température s'est révélée être un facteur environnemental important qui influence la cascade Gac/Rsm. Il s'avère en effet que la production d'antibiotiques ainsi que d'acide cyanhydrique est moins importante à 35°C qu'à 30°C. L'implication du senseur Rets dans ce contrôle par la température a pu être démontrée. La régulation positive du petit ARN RsmY par GacA et RsmA/RsmE a pu être confirmée; par le biais d'une analyse mutationelle, deux régions essentielles ont pu être mises en évidence dans la région promotrice de rsmY. Malgré le manque de preuves expérimentales directes, certains indices suggèrent que GacA puisse directement se fixer sur une des deux régions (appelée UAS), tandis que la deuxième région (appelée linker) serait plutôt reconnue par des facteurs intermédiaires (activateurs ou répresseurs) dépendant de RsmA/RsmE. En conclusion, ce travail a dévoilé de nouveaux éléments permettant d'éclairer les mécanismes de transduction des signaux dans la cascade Gac/Rsm.

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans.

Gastric lipase (HGL) contributes significantly to fat digestion. However, little is known about its neurohormonal regulation in humans. We studied the role of CCK and cholinergic mechanisms in the postprandial regulation of HGL and pancreatic lipase (HPL) secretion in six healthy subjects. Gastric emptying of a mixed meal and outputs of HGL, pepsin, acid, and HPL were determined with a double-indicator technique. Three experiments were performed in random order: intravenous infusion of 1) placebo, 2) low-dose atropine (5 micrograms.kg-.h-1), and 3) the CCK-A receptor antagonist loxiglumide (22 mumol.kg-.h-1). Atropine decreased postprandial outputs of HGL, pepsin, gastric acid, and HPL (P < 0.03) while slowing gastric emptying (P < 0.05). Loxiglumide markedly increased the secretion of HGL, pepsin, and acid while distinctly reducing HPL outputs and accelerating gastric emptying (P < 0.03). Plasma CCK and gastrin levels increased during loxiglumide infusion (P < 0.03). Atropine enhanced gastrin but not CCK release. Postprandial HGL, pepsin, and acid secretion are under positive cholinergic but negative CCK control, whereas HPL is stimulated by cholinergic and CCK mechanisms. We conclude that CCK and cholinergic mechanisms have an important role in the coordination of HGL and HPL secretion to optimize digestion of dietary lipids in humans.