Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Effect of groundwater composition on arsenic detection by bacterial biosensors.

A luminescent bacterial biosensor was used to quantify bioavailable arsenic in artificial groundwater. Its light production above the background emission was proportional to the arsenite concentration in the toxicologically relevant range of 0 to 0.5 mu M. Effects of the inorganic solutes phosphate, Fe(II) and silicate on the biosensor signal were studied. Phosphate at a concentration of 0.25 g L-1 phosphate slightly stimulated the light emission, but much less than toxicologically relevant concentrations of the much stronger inducer arsenite. No effect of phosphate was oberved in the presence of arsenite. Freshly prepared sodium silicate solution at a concentration of 10 g L-1 Si reduced the arsenite-induced light production by roughly 37%, which can be explained by transient polymerization leading to sequestration of some arsenic. After three days of incubation, silicate did not have this effect anymore, probably because depolymerization occurred. In the presence of 0.4 g L-1 Fe(II), the arsenite-induced light emission was reduced by up to 90%, probably due to iron oxidation followed by arsenite adsorption on the less soluble Fe(III) possibly along with some oxidation to the stronger adsorbing As(V). Addition of 100 mu M EDTA was capable of releasing all arsenic from the precipitate and to transform it into the biologically measurable, dissolved state. The biosensor also proved valuable for monitoring the effectiveness of an arsenic removal procedure based on water filtration through a mixture of sand and iron granules.

Dietary proteins and atherosclerosis.

More than one hundred years ago the "protein hypothesis" of the pathogenesis of atherosclerosis and its association with cardiovascular disease was put forward on the basis of animal experiments; however, it has so far never been verified in humans. This theory was soon replaced by the "lipid hypothesis", which was confirmed in humans as of 1994. Epidemiological ecological studies in the 1960 s showed significant associations between dietary animal protein and mortality from cardiovascular disease. However, animal protein intake was also significantly correlated with saturated fatty acid and cholesterol intake. In the last decades two prospective cohort studies demonstrated a decreased cardiovascular risk in women during high- versus low-protein intake when adjusting for other dietary factors (e. g., saturated fats) and other cardiovascular risk factors. A direct cholesterol lowering effect of proteins has not been shown. Despite earlier research indicating that soy protein has cardioprotective effects as compared to other proteins, these observations have not been confirmed by randomized placebo-controlled trials. However, most experts recommend the consumption of foods rich in plant proteins as alternatives to meat and dairy products rich in saturated fat and containing cholesterol. There are no scientific arguments to increase the daily protein intake to more than 20 % of total energy intake as recommended by the guidelines, in order to improve cardiovascular health.

Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

Pathogenesis-related proteins and the jasmonate signaling pathway

Résumé Etant une importante source d'énergie, les plantes sont constamment attaquées par des pathogènes. Ne pouvant se mouvoir, elles ont développé des systèmes de défense sophistiqués afin de lutter contre ces prédateurs. Parmi ces systèmes, les voies de signalisation mettant en jeu des éliciteurs endog8nes tels que les jasmonates permettent d'induire la production de protéines de défense telles que les protéines dites "liées à la pathogénèse". Les gènes codant pour ces protéines appartiennent à des familles multigéniques. Le premier but de cette thèse est d'évaluer le nombre de ces gènes dans le génome d'Arabidopsis thaliana et d'estimer la part de ce système de défense, dépendant de la voie de signalisation des jasmonates. Nous avons défini un cluster de seulement 1S gènes sur 266, "liés à la pathogénèse", exclusivement régulés par les jasmonates. De multiples membres des familles des lectines de type jacaline et des inhibiteurs de trypsines semblent dépendre du jasmonate. Présente dans tous les systèmes immunitaires des eucaryotes, la famille des défensines est une famille très intéressante. Chez Arabidopsis thaliana, 317 protéines similaires aux défensines ont été définies, cependant seulement 15 défensines (PDF) sont bien annotées. Ces 15 défensines sont séparées en deux groupes dont un semble avoir évolué plus récemment. Le second but de cette thèse est d'étudier ce groupe de défensines à l'aide de la bioinformatique et des techniques de biologie moléculaire (gêne rapporteur, PCR en temps réel). Nous avons montré que ce groupe contenait une défensine acide intéressante, PDF1.5, qui semblait avoir subi une sélection positive. Cette protéine n'avait encore jamais été étudiée. Contrairement à ce que nous pensions, nous avons établi que cette protéine pouvait avoir une activité biologique liée à la défense. Ce travail de thèse a permis de préciser le nombre de gènes "liées à la pathogénèse" induits par la voie des jasmonates et d'apporter des éléments de réponse sur la question de la redondance des gènes de défense. En conclusion, même si de nombreuses familles de gènes intervenant dans la défense sont bien définies chez Arabidopsis, il reste encore de nombreuses études à faire sur chacun de ces membres. Abstract Being an important source of energy, plants are constantly attacked by herbivores and pathogens. As sessile organisms, they have developed sophisticated defense responses to cope with attack. Among these responses, signalling pathways, using endogenous elicitors including jasmonates (JA), allow the plant to induce the production of defense proteins such as pathogenesis-related (PR) proteins. The genes encoding these proteins belong to multigenic families. The first goal of this thesis was to evaluate the number of PR genes in the genome of Arabidopsis thaliana and estimate how much of this plant defense system was dependent on the jasmonate signaling pathway in leaves. Surprisingly a cluster of only 1S genes out of 2ó6 PR genes was exclusively regulated by JA. Multiple members of the jacalin lectin and trypsin inhibitor gene families were shown to be regulated by JA. Present in all eukaryotic immune systems, defensins are an attractive PR family to study. In Arabidopsis thaliana, 317 defensin-related proteins have been found but just 1S defensins (i.e. PDF family) are well annotated. These defensins are split into 2 groups. One of these groups may have appeared and diversified recently. The second goal of this thesis was to study this defensin gene group combining bioinformatic, reporter gene and quantitative PCR techniques. We have shown that this group contains an interesting acidic defensin, PDF1.S, which seems to have undergone positive selection. No information was known on this protein. We have established that this protein may have a biological activity in plant defense. This thesis allowed us to define the number of PR genes induced by the jasmonate pathway and gave initial leads to explain the redundancy of the PR genes in the genome of Arabidopsis. In conclusion, even if many defense gene families are already defined in the Arabidopsis genome, much work remains to be done on individual members.

The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis.

Background: Sulfate and phosphate are both vital macronutrients required for plant growth and development. Despite evidence for interaction between sulfate and phosphate homeostasis, no transcriptional factor has yet been identified in higher plants that affects, at the gene expression and physiological levels, the response to both elements. This work was aimed at examining whether PHR1, a transcription factor previously shown to participate in the regulation of genes involved in phosphate homeostasis, also contributed to the regulation and activity of genes involved in sulfate inter-organ transport. Results: Among the genes implicated in sulfate transport in Arabidopsis thaliana, SULTR1;3 and SULTR3;4 showed up-regulation of transcripts in plants grown under phosphate-deficient conditions. The promoter of SULTR1;3 contains a motif that is potentially recognizable by PHR1. Using the phr1 mutant, we showed that SULTR1;3 up regulation following phosphate deficiency was dependent on PHR1. Furthermore, transcript up regulation was found in phosphate-deficient shoots of the phr1 mutant for SULTR2;1 and SULTR3;4, indicating that PHR1 played both a positive and negative role on the expression of genes encoding sulfate transporters. Importantly, both phr1 and sultr1;3 mutants displayed a reduction in their sulfate shoot-to-root transfer capacity compared to wild-type plants under phosphate-deficient conditions. Conclusions: This study reveals that PHR1 plays an important role in sulfate inter-organ transport, in particular on the regulation of the SULTR1;3 gene and its impact on shoot-to-root sulfate transport in phosphate-deficient plants. PHR1 thus contributes to the homeostasis of both sulfate and phosphate in plants under phosphate deficiency. Such a function is also conserved in Chlamydomonas reinhardtii via the PHR1 ortholog PSR1.

Bacterial biosensors for measuring availability of environmental pollutants

Abstract: Traditionally, pollution risk assessment is based on the measurement of a pollutant's total concentration in a sample. The toxicity of a given pollutant in the environment, however, is tightly linked to its bioavailability, which may differ significantly from the total amount. Physico-chemical and biological parameters strongly influence pollutant fate in terms of leaching, sequestration and biodegradation. Bacterial sensorreporters, which consist of living micro-organisms genetically engineered to produce specific output in response to target chemicals, offer an interesting alternative to monitoring approaches. Bacterial sensor-reporters detect bioavailable and/or bioaccessible compound fractions in samples. Currently, a variety of environmental pollutants can be targeted by specific biosensor-reporters. Although most of such strains are still confined to the lab, several recent reports have demonstrated utility of bacterial sensing-reporting in the field, with method detection limits in the nanomolar range. This review illustrates the general design principles for bacterial sensor-reporters, presents an overview of the existing biosensor-reporter strains with emphasis on organic compound detection. A specific focus throughout is on the concepts of bioavailability and bioaccessibility, and how bacteria-based sensing-reporting systems can help to improve our basic understanding of the different processes at work.

Amoebae as a tool to isolate new bacterial species, to discover new virulence factors and to study the host-pathogen interactions.

Amoebae are unicellular protozoan present worldwide in several environments mainly feeding on bacteria. Some of them, the amoebae-resistant bacteria (ARBs), have evolved mechanisms to survive and replicate inside amoebal species. These mainly include legionella, mycobacteria and Chlamydia-related bacteria. Amoebae can provide a replicative niche, can act as reservoir for bacteria whereas the cystic form can protect the internalized bacteria. Moreover, the amoebae represent a Trojan horse for ARBs to infect animals. The long interaction between amoebae and bacteria has likely selected for bacterial virulence traits leading to the adaptation towards an intracellular lifestyle, and some ARBs have acquired the ability to infect mammals. This review intends to highlight the important uses of amoebae in several fields in microbiology by describing the main tools developed using amoebal cells. First, amoebae such as Acanthamoeba are used to isolate and discover new intracellular bacterial species by two main techniques: the amoebal co-culture and the amoebal enrichment. In the second part, taking Waddlia chondrophila as example, we summarize some important recent applications of amoebae to discover new bacterial virulence factors, in particular thanks to the amoebal plaque assay. Finally, the genetically tractable Dictyostelium discoideum is used as a model organism to study host-pathogen interactions, in particular with the development of several approaches to manipulate its genome that allowed the creation of a wide range of mutated strains largely shared within the Dictyostelium community.

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development.

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton.