Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase

Lactobacillus plantarum produces peptidoglycan precursors ending in D-lactate instead of D-alanine, making the bacterium intrinsically resistant to vancomycin. The ligase Ddl of L. plantarum plays a central role in this specificity by synthesizing D-alanyl-D-lactate depsipeptides that are added to the precursor peptide chain by the enzyme MurF. Here we show that L. plantarum also encodes a D-Ala-D-Ala dipeptidase, Aad, which eliminates D-alanyl-D-alanine dipeptides that are produced by the Ddl ligase, thereby preventing their incorporation into the precursors. Although D-alanine-ended precursors can be incorporated into the cell wall, inactivation of Aad failed to suppress growth defects of L. plantarum mutants deficient in d-lactate-ended precursor synthesis.     

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.     

Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.     

Conditional transgenesis using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCrexR26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.     

Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes

In chronic inflammatory diseases, such as rheumatoid arthritis, inflammation acts as an independent cardiovascular risk factor and the use of anti-inflammatory drugs, such as anti-tumor necrosis factor alpha (anti-TNFα), may decrease this risk. The phagocytosis of oxidized low density lipoproteins (LDLs) accumulated in the subendothelium by mononuclear cells influences atherosclerosis and depends on CD36 expression. We investigated the role of TNFα and adalimumab, a human anti-TNFα monoclonal antibody widely used in human pathology, in CD36 expression in human monocytes. Human monocytes were prepared by adherence from whole-blood buffy-coat fractions from healthy donors. CD36 expression was assessed by RT-PCR and flow cytometry, with various TNFα or adalimumab concentrations. Implication of peroxisome proliferator-activated receptor (PPAR)γ in the regulation of CD36 expression was assessed using specific inhibitor or gel shift assays. The impact of redox signaling was investigated using quantification of reactive oxygen species, antioxidant and a NADPH oxidase inhibitor. The F(ab')2 fragment of adalimumab was isolated and its effect was analyzed. TNFα inhibits both CD36 membrane expression and mRNA expression. This inhibition involves a reduction in PPARγ activation. In contrast, adalimumab increases both CD36 membrane expression and mRNA expression. This induction is independent of the Fc portion of adalimumab and involves redox signaling via NADPH oxidase activation. CD36 expression on human monocytes is inhibited by TNFα and independently increased by adalimumab. These data highlight that pro-inflammatory cytokines and their specific neutralization influence the expression of cellular receptors implicated in atherosclerosis. Further studies are needed to investigate the clinical implications of these results in accelerated atherosclerosis observed in rheumatoid arthritis.     

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4)

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)(4 )presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin alpha(V)beta(3) with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)(4)-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)(4)-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)(4)-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.     

Spontaneous duplications in diploid Saccharomyces cerevisiae cells

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.     

Evaluation of the influence of protein precipitation prior to on-line SPE-LC-API/MS procedures using multivariate data analysis

Matrix effects on mass spectrometry (MS) response were investigated with three atmospheric pressure ionization (API) sources after on-line solid-phase extraction (SPE) of human plasma. On-line SPE was evaluated with one restricted access material (RAM), two large particle supports (LPS) and one monolith. A sample protein precipitation (PP) with acetonitrile (2:1) and a direct injection were tested. Principal component analysis (PCA) was performed to simplify data presentation and interpretation. Protein precipitation was found to be mandatory for reducing signal modification. Regarding sensitivity towards matrix effects after PP, atmospheric pressure photoionization (APPI) was globally the least sensitive ionization mode while electrospray ionization ESI was the most sensitive.     

Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA

Tomato, an essential crop in terms of economic importance and nutritional quality, is also used as a model species for all fleshy fruits and for genomics of Solanaceae. Tomato fruit quality at harvest is a direct function of its metabolite content, which in turn is a result of many physiological changes during fruit development. The aim of the work reported here was to develop a global approach to characterize changes in metabolic profiles in two interdependent tissues from the same tomato fruits. Absolute quantification data of compounds in flesh and seeds from 8 days to 45 days post anthesis (DPA) were obtained through untargeted (proton nuclear magnetic resonance, ¹H-NMR) and targeted metabolic profiling (liquid chromatography with diode array detection (LC-DAD) or gas chromatography with flame ionization detection (GC-FID)). These data were analyzed with chemometric approaches. Kohonen self organizing maps (SOM) analysis of these data allowed us to combine multivariate (distribution of samples on Kohonen SOMs) and univariate information (component plane representation of metabolites) in a single analysis. This strategy confirmed published data and brought new insights on tomato flesh and seed composition, thus demonstrating its potential in metabolomics. The compositional changes were related to physiological processes occurring in each tissue. They pointed to (i) some parallel changes at early stages in relation to cell division and transitory storage of carbon, (ii) metabolites participating in the fleshy trait and (iii) metabolites involved in the specific developmental patterns of the seeds.