Mouse Delta Opioid Receptors are Located on Presynaptic Afferents to Hippocampal Pyramidal Cells

Delta opioid receptors participate in the control of chronic pain and emotional responses. Recent data have also identified their implication in drug-context associations pointing to a modulatory role on hippocampal activity. We used fluorescent knock-in mice that express a functional delta opioid receptor fused at its carboxy terminus with the green fluorescent protein in place of the native receptor to investigate the receptor neuroanatomical distribution in this structure. Fine mapping of the pyramidal layer was performed in hippocampal acute brain slices and organotypic cultures using fluorescence confocal imaging, co-localization with pre- and postsynaptic markers and correlative light-electron microscopy. The different approaches concurred to identify delta opioid receptors on presynaptic afferents to glutamatergic principal cells. In the latter, only scarce receptors were detected that were confined within the Golgi or vesicular intracellular compartments with no receptor present at the cell surface. In the mouse hippocampus, expression of functional delta opioid receptors is therefore mostly associated with interneurons emphasizing a presynaptic modulatory effect on the pyramidal cell firing rate.     

Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.     

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.     

Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly ¹³C, ¹⁵N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.     

An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.     

Rodent-Borne Hantaviruses in Cambodia, Lao PDR, and Thailand

In order to evaluate the circulation of hantaviruses present in southeast Asia, a large scale survey of small mammal species was carried out at seven main sites in the region (Cambodia, Lao People's Democratic Republic, and Thailand). Small scale opportunistic trapping was also performed at an eighth site (Cambodia). Using a standard IFA test, IgG antibodies reacting to Hantaan virus antigens were detected at six sites. Antibody prevalence at each site varied from 0 to 5.6% with antibodies detected in several rodent species (Bandicota indica, B. savilei, Maxomys surifer, Mus caroli, M. cookii, Rattus exulans, R. nitidius, R. norvegicus, and R. tanezumi). When site seroprevalence was compared with site species richness, seropositive animals were found more frequently at sites with lower species richness. In order to confirm which hantavirus species were present, a subset of samples was also subjected to RT-PCR. Hantaviral RNA was detected at a single site from each country. Sequencing confirmed the presence of two hantavirus species, Thailand and Seoul viruses, including one sample (from Lao PDR) representing a highly divergent strain of Seoul virus. This is the first molecular evidence of hantavirus in Lao PDR and the first reported L segment sequence data for Thailand virus.     

New drying process for lactic bacteria based on their dehydration behavior in liquid medium

This study describes the different stages of optimization in an original drying process for lactic acid bacteria that allows the retrieval of dried samples of Lactobacillus plantarum with maximum viability. The process involves the addition of casein powder to bacterial pellets, followed by mixing and then air-drying in a fluidized bed dryer. The effects on bacterial viability of the a(w) of the casein powder and the kinetics of a(w) variation in the fluidized bed dryer are considered. These parameters were first studied in a water-glycerol solution and the results were then applied to the drying process. Data from the study in liquid medium were reliable in the fluidized drying stage, insofar as optimal viability was achieved for similar dehydration times (16-50 min in liquid medium, and 30 min in the fluidized bed dryer). However, when the powder was mixed rapidly with bacteria, the level of destruction differed from that observed in liquid medium. Viability was up to 70% when the a(w) of water-glycerol was 0.55, whereas it was only 2.1% when the a(w) of the casein-bacterial mix was 0.64. The predictive capacity of dehydration in liquid medium is discussed with regard to the permeability of cells to external solutes. The new process allowed 100% survival of L. plantarum after complete drying (final a(w) < 0.2). However, when used for the desiccation of L. bulgaricus, these parameters achieved a viability of less than 10%.     

The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.