Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.     

Effects of NaCl on water relations and cell wall elasticity and composition of red-osier dogwood (Cornus stolonifera) seedlings

Red-osier dogwood ( Cornus stolonifera Michx, Syn. Cornus sericea ), a species relatively well adapted to moderately saline conditions compared with other boreal species, was used to test the effects of NaCl on plant water relations, cell wall elasticity, and cell wall composition of seedlings. Three month-old seedlings were treated hydroponically with 0, 25, and 50 m m NaCl for 21 days. The osmotic potential at full turgor, osmotic potential at turgor loss, pressure potential at full turgor, and relative water content at turgor loss of red-osier dogwood shoot tissue were not significantly affected by the NaCl treatments. Cell wall elasticity of the shoot tissues did not change following NaCl treatments, suggesting that elastic adjustment did not play a role in the adaptation mechanism. Hemicellulose content of the cell wall increased in salt treated seedlings. The primary sugar found in the cell wall hemicellulose fraction was xylose. In the pectin fraction arabinose and galacturonic acid were the main sugars. Sodium chloride stress did not alter the sugar composition of the hemicellulose fraction; however, NaCl did increase the amount of rhamnose in the pectin fraction. The results of this study suggest that at moderate salinity red-osier dogwood does not make any osmotic or elastic adjustments in the shoot tissue, but some changes in the cell wall composition do occur. These changes could contribute to the decrease in growth recorded in red-osier dogwood during NaCl stress.     

Extensive phenotypic variation in early flowering mutants of Arabidopsis

Flowering time, the major regulatory transition of plant sequential development, is modulated by multiple endogenous and environmental factors. By phenotypic profiling of 80 early flowering mutants of Arabidopsis, we examine how mutational reduction of floral repression is associated with changes in phenotypic plasticity and stability. Flowering time measurements in mutants reveal deviations from the linear relationship between the number of leaves and number of days to bolting described for natural accessions and late flowering mutants. The deviations correspond to relative early bolting and relative late bolting phenotypes. Only a minority of mutants presents no detectable phenotypic variation. Mutants are characterized by a broad release of morphological pleiotropy under short days, with leaf characters being most variable. They also exhibit changes in phenotypic plasticity across environments for florigenic-related responses, including the reaction to light and dark, photoperiodic behavior, and Suc sensitivity. Morphological pleiotropy and plasticity modifications are differentially distributed among mutants, resulting in a large diversity of multiple phenotypic changes. The pleiotropic effects observed may indicate that floral repression defects are linked to global developmental perturbations. This first, to our knowledge, extensive characterization of phenotypic variation in early flowering mutants correlates with the reports that most factors recruited in floral repression at the molecular genetic level correspond to ubiquitous regulators. We discuss the importance of functional ubiquity for floral repression with respect to robustness and flexibility of network biological systems.     

Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303

phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.     

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.     

The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.     

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

The development of potent non-peptidic PTP-1B inhibitors

The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).