Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.     

Wolbachia transfer from Rhagoletis cerasi to Drosophila simulans: investigating the outcomes of host-symbiont coevolution

Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.     

Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery system in the gut

New strategies to prevent or treat diseases have been focusing on innovative approaches, such as the oral administration of living recombinant micro-organisms delivering active compounds in the digestive environment. The survival rate and the ability of two recombinant Saccharomyces cerevisiae strains (WppV(5)H(6) and WppGSTV(5)H(6)) to initiate the synthesis and secrete either a model peptide (peptide-V(5)H(6), MW: 5.6 kDa) or a model protein (glutathione-S-transferase-V(5)H(6), MW: 31.5 kDa) were studied in a gastric-small intestinal system simulating human digestive conditions. The WppV(5)H(6) and WppGSTV(5)H(6) strains respectively showed 83.1%+/-9.6 (n=3) and 95.3%+/-22.7 (n=4) survival rates in the model upper digestive tract after 270 min of digestion. The secretion products were detected as early as 90 min after the yeast intake/gene induction in each compartment of the in vitro system, but mostly in the jejunum and ileum. The GST-V(5)H(6) concentrations in the digestive medium reached 15 ng ml(-1), close to values measured in batch cultures. These results open up new opportunities for the set up of drug delivery systems based on engineered yeasts secreting compounds directly in the digestive tract. The main potential medical applications include the development of oral vaccines, the correction of metabolic disorders and the in situ production of biological mediators.     

Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.     

Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein

It is commonly assumed that the physiological isoform of prion protein, PrP(C), is cleaved during its normal processing between residues 111/112, whereas the pathogenic isoform, PrP(Sc), is cleaved at an alternate site in the octapeptide repeat region around position 90. Here we demonstrated both in cultured cells and in vivo, that PrP(C) is subject to a complex set of post-translational processing with the molecule being cleaved upstream of position 111/112, in the octapeptide repeat region or at position 96. PrP has therefore two main cleavage sites that we decided to name alpha and beta. Cleavage of PrP(C) at these sites leads us to re-evaluate the function of both N- and C-terminus fragments thus generated.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Erk2 signaling and early embryo stem cell self-renewal

Differentiation of the mammalian blastocyst generates two distinct cell lineages: the trophectoderm, which contributes to the trophoblast layers of the placenta, and the inner cell mass, which forms the embryo. We and others recently demonstrated that the MAP kinase ERK2 is essential for trophoblast development. Erk2 mutant embryos fail to form extra-embryonic ectoderm and the ectoplacental cone, suggesting a role for ERK2 activation in the proliferation of trophoblast stem (TS) cells. Previous studies have documented that ERK1/2 activity is dispensable for proliferation of embryonic stem (ES) cells and rather interferes with self-renewal. Thus, signaling by the ERK1/2 MAP kinase pathway appears to be critical for the regulation of self-renewal and propagation of early embryo stem cell populations.     

Multiple co-inertia analysis: a tool for assessing synchrony in the temporal variability of aquatic communities

Multitable techniques are rarely used for investigating patterns in ecological data surveys despite their ability to deal with the spatial and/or temporal stability of assemblages. Based on a covariance optimisation criterion, Multiple Co-inertia analysis (MCOA) enables the simultaneous ordination of several tables. Such analysis allows the representation of the stable vs. unstable part of the assemblage structure in comparison to a reference derived from each table. We used MCOA on multiple time series of invertebrate sampling to show that synchrony in the temporal variability of communities can establish between geographically distant locations despite the spatial and temporal plasticity of the faunistic responses to long-term changes in environmental conditions.     

Pharmacomodulation of a sulfamide 5-HT6 receptor ligand

A series of N-omega-aminoalkyl- or N-omega-amidinoalkyl-2,4,6-triisopropyl benzenesulfonamides has been synthesized and their respective affinity indices on 5-HT6 receptor determined. This evaluation clearly showed that the compounds possessing an arylpiperazine moiety or an amidine function exhibited good affinity for the model.