Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase

A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N:-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.     

Characterization of phytoplankton assemblages in a tropical coastal environment using Kohonen self‐organizing map

This study was aimed at understanding the main abiotic environmental factors controlling the distribution patterns of abundance and composition of phytoplankton (size less than 10 μm) assemblages in the coastal waters of south‐eastern Côte d'Ivoire. Data were collected during two cruises, in January (low‐water period) and October (high‐water period) of 2014. A total of 67 species were identified and assigned to Bacillariophyceae (49%), Cyanophyceae (21%), Chlorophyceae (13%), Euglenophyceae (10%), Dinophyceae (4%) and Chrysophyceae (3%). Three biotic zones (I, IIA and IIB) were distinguishable on a Kohonen self‐organizing map after an unsupervised learning process. The diatom genera Eunotia sp., Navicula sp. and Actinoptychus senarius are significantly associated with I, IIA and IIB biotic zones, respectively. A clear seasonal cum salinity trend was apparent in phytoplankton distribution patterns. Turbidity and nitrate levels were the main abiotic factors controlling phytoplankton distribution in I, the upland tidal regions of the lagoon. In regions along the lagoon–sea continuum, phosphate and turbidity exert the most control during the low‐water season (IIA), while total dissolved solids control phytoplankton distribution during the high‐water season (IIB). These are climate‐sensitive parameters whose concentrations depend on prevailing hydroclimatic processes. Therefore, seasonality can have important consequences on phytoplankton community and inadvertently the productivity of these systems.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.     

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or the photosynthetic reaction center to provide a temporary driving force for understanding transport and chemiosmotic coupling (6, 7, 19, 35). However, light-driven reactions have not been used in metabolic engineering to alter microbial physiology and production of chemicals. The recent discovery of proteorhodopsin (PR) in ocean microorganisms and the ease with which this membrane protein can be functionally expressed by recombinant bacteria have made possible many engineering strategies previously not available (1, 16). In this paper, we describe progress toward the goal of integrating light-driven reactions with biocatalysis.In contrast to the situation for established industrial microorganisms, such as Escherichia coli, our current understanding of less-studied algal and phototrophic bacteria may limit metabolic engineering strategies which require genetic manipulation. Metabolic engineering strategies using photosynthetic bacteria have focused largely on methods to increase hydrogen production, and improvements rely mainly on engineering of nitrogenase and hydrogenase to produce H₂. Algae appear to be suited to large-scale cultivation for lipid production, but so far little has been done to engineer these organisms (36). In principle, platform microbial hosts capable of producing a diverse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.To demonstrate the feasibility of transferring a light-driven process into a nonphotosynthetic bacterium, we chose to study proteorhodopsin (PR) first because it is one of the simplest mechanisms for harnessing the energy from light. The proteorhodopsins are a group of transmembrane proteins that use the light-induced isomerization of retinal, the oxidative cleavage product of the carotenoid β-carotene, either to initiate signaling pathways or to catalyze the transfer of ions across cell membranes (8). PR was discovered by metagenomic analysis of marine samples (1) and is related to the well-studied bacteriorhodopsin of archaea (33) and rhodopsin (34), a eukaryotic light-sensing protein. The membrane potential generated by light-driven proton pumping by PR has been confirmed to drive ATP synthesis in a heterologous system (25). However, bacteria expressing heterologous PR were shown not to benefit from this pumping activity, as no significant increases in growth rates were observed (9). This led to the suggestion that PR may benefit the organism only under starvation conditions. In agreement with this hypothesis, Gomez-Consarnau et al. (10) have reported that the light-dependent growth rates of a marine flavobacterium that has a native PR are increased only when the organism is cultured under energy-limited conditions.Studies of both native and recombinant systems in which rhodopsins are expressed have generated light-dependent membrane potentials. In membrane vesicles isolated from a native host, the light-dependent membrane potential generated by bacteriorhodopsin provides the driving force for ATP synthesis (35) and uptake of leucine and glutamate (20, 22). More recently, studies of recombinant systems have coupled the membrane potential to other transport processes. In one example, the membrane potential-dependent export of specific toxic molecules increased when E. coli cells expressing both an archaeal rhodopsin and a specific efflux pump were exposed to light (17). In another experiment, starved E. coli cells expressing PR increased the swimming motion of their flagella when they were illuminated (44). Based upon measurements of flagellar motion as a function of light intensity and azide concentration, the proton motive force generated by PR was estimated to be −0.2 V, a value similar to the value for aerobic respiration in E. coli (42).As a nonphotosynthetic host for recombinant PR expression, we chose the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1, which is genetically tractable for engineering and is able to use a variety of terminal electron acceptors, including insoluble metal oxides (11, 30). Key to the ability of this bacterium to reduce metal oxides is a multicomponent extracellular respiratory pathway that transports electrons from menaquinol to cytochromes in the outer membrane. This pathway is composed of a cytoplasmic membrane tetraheme protein (CymA), a periplasmic decaheme protein (MtrA), an integral outer membrane protein (MtrB), and a decaheme lipoprotein (MtrC) that is associated with MtrB (14, 37, 40). The ability of S. oneidensis to reduce extracellular metal oxides has made it possible to harvest electrons from this organism by coupling it to an electrode which serves as the electron acceptor (21). The electron flow to the outer surface allows respiration rates to be measured directly by electrochemistry.In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demonstrated that there was integration of a light-driven process into the metabolism of a previously nonphotosynthetic organism that resulted in a useful output. We show here that PR allows cells to survive for extended periods in stationary phase and that the presence of light results in an increase in electricity generation. A possible physiological model to explain these effects is discussed.     

High Virulence of Wolbachia after Host Switching: When Autophagy Hurts

Wolbachia are widespread endosymbionts found in a large variety of arthropods. While these bacteria are generally transmitted vertically and exhibit weak virulence in their native hosts, a growing number of studies suggests that horizontal transfers of Wolbachia to new host species also occur frequently in nature. In transfer situations, virulence variations can be predicted since hosts and symbionts are not adapted to each other. Here, we describe a situation where a Wolbachia strain (wVulC) becomes a pathogen when transfected from its native terrestrial isopod host species (Armadillidium vulgare) to another species (Porcellio d. dilatatus). Such transfer of wVulC kills all recipient animals within 75 days. Before death, animals suffer symptoms such as growth slowdown and nervous system disorders. Neither those symptoms nor mortalities were observed after injection of wVulC into its native host A. vulgare. Analyses of wVulC''s densities in main organs including Central Nervous System (CNS) of both naturally infected A. vulgare and transfected P. d. dilatatus and A. vulgare individuals revealed a similar pattern of host colonization suggesting an overall similar resistance of both host species towards this bacterium. However, for only P. d. dilatatus, we observed drastic accumulations of autophagic vesicles and vacuoles in the nerve cells and adipocytes of the CNS from individuals infected by wVulC. The symptoms and mortalities could therefore be explained by this huge autophagic response against wVulC in P. d. dilatatus cells that is not triggered in A. vulgare. Our results show that Wolbachia (wVulC) can lead to a pathogenic interaction when transferred horizontally into species that are phylogenetically close to their native hosts. This change in virulence likely results from the autophagic response of the host, strongly altering its tolerance to the symbiont and turning it into a deadly pathogen.     

Interactor-mediated nuclear translocation and retention of protein phosphatase-1

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Effect of two different long-sprint training regimens on sprint performance and associated metabolic responses

The purpose of this study was to analyze 2 different long-sprint training programs (TPs) of equal total work load, completed either with short recovery (SR) or long recovery (LR) between sets and to compare the effects of 6 long-sprint training sessions (TSs) conducted over a 2-week period on a 300-m performance. Fourteen trained subjects performed 3 pretraining maximal sprints (50-, 100-, and 300-m), were paired according to their 300-m performance, and randomly allocated to an LR or SR group, which performed 6 TSs consisting of sets of 150, 200, or 250 m. The recovery in the LR group was double that of the SR group. During the third TS and the 300-m pretest and posttest, blood pH, bicarbonate concentration ([HCO??]), excess-base (EB), and lactate concentration were recorded. Compared with a similar TS performed with SR, the LR training tends to induce a greater alteration of the acid-base balance: pH: 7.09 ± 0.08 (LR) and 7.14 ± 0.05 (SR) (p = 0.10), [HCO??]: 7.8 ± 1.9 (LR) and 9.6 ± 2.7 (SR) (p = 0.04), and EB: -21.1 ± 3.8 (LR) and -17.7 ± 2.8 (SR) (p = 0.11). A significant improvement in the 300-m performance between pre-TP and post-TP (42.45 ± 2.64 vs. 41.52 ± 2.45, p = 0.01) and significant decreases in pH (p < 0.01), EB (p < 0.001) and increase in [La] (p < 0.001) have been observed post-TP compared with those pre-TP. Although sprint training with longer recovery induces higher metabolic disturbances, both sprint training regimens allow a similar 300-m performance improvement with no concomitant significant progress in the 50- and 100-m performance.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Horizontal Gene Transfer of “Prototype” Nramp in Bacteria

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in C, C, C. The proteins from C. tepidum (MntH B) and E. faecalis (MntH C1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH C gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the C genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii prototype Nramp and some MntH C (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other prototype Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that prototype Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection. Equally contributing authors (Etienne Richer and Pascal Courville).