Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medflyCeratitis capitata

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.     

Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody‐mediated rejection

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody‐mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non‐selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients’ clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

A rapid and efficient method for the synthesis of selectively S-Trt or S-Mmt protected Cys-containing peptides

Selective removal of protecting groups under different cleavage mechanisms could be an asset in peptide synthesis, since it provides the feasibility to incorporate different functional groups in similar reactive centres. However, selective protection/deprotection of orthogonal protecting groups in peptides is still challenging, especially for Cys-containing peptides, where protection of the cysteine side-chain is mandatory since the nucleophilic thiol can be otherwise alkylated, acylated or oxidized. Herein, we established a protocol for the synthesis of Cys-selective S-Trt or S-Mmt protected Cys-containing peptides, in a rapid way. This was achieved by, simply fine-tuning the carbocation scavenger in the final acidolytic release of the peptide from the solid support in the classic SPPS.     

Temporal and transient expression of olive enoyl-ACP reductase gene during flower and fruit development.

Enoyl-ACP reductase is a catalytic component of the fatty acid synthetase (FAS) type II system in plants that is involved in the de novo fatty acid biosynthesis in plastids. A cDNA encoding an enoyl-ACP reductase responsible for the removal of the trans-unsaturated double bonds to form saturated acyl-ACP has been isolated from a library made from ripening fruits of Olea europaea L. The predicted protein contains 393 amino acid residues including a consensus chloroplast specific transit peptide. A strong homology was observed when olive enoyl-ACP reductase aligned with other plant sequences. Southern hybridization analysis revealed that enoyl-ACP reductase is encoded by a single gene in olives. Northern hybridization showed a transient expression of the enoyl-ACP reductase (ENR) gene at early stages of drupe (5-7 weeks after flowering, WAF), embryo and endosperm (13-16 WAF) while in mesocarp (13-19 WAF) the expression remained at high levels. In situ hybridization showed particularly prominent expression in the palisade and vascular tissue of young leaves, the tapetum, developing pollen grains and vascular tissue of anthers and to less extent in the embryo sac and transmitting tissue of the carpel. The distinctive spatial and temporal regulation of the ENR gene is consistent with major roles, not only in thylakoid membrane formation and fatty acid deposition, but also in the provision of precursor molecules for the biosynthesis of oxilipins that are important in plant tissues involved in transportation and reproduction.     

A phylogenomic resolution for the taxonomy of Aegean green lizards

Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next-generation sequencing and double-digest restriction-site-associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome-wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.