The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.     

Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

Localization and domain characterization of Arabidopsis golgin candidates

Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.     

NPY regulates human endocardial endothelial cell function

Growing evidence suggests that endocardial endothelial cells (EECs) may play an important role in the regulation of cardiac function by releasing several cardioactive factors such as endothelin-1 (ET-1), Angiotensin II (Ang II) and nitric oxide (NO). In our laboratory, we demonstrated that similar to ET-1, EECs do possess different types of NPY receptors, specifically Y(1) and Y(2) receptors. Furthermore, activation of these receptors was found to increase the steady-state level of intracellular free Ca(2+) in EECs and the frequency of beating of cardiomyocytes. In addition, NPY was also found to be present in EECs, and an increase of steady-state intracellular free Ca(2+) induced the release of this peptide from these cells. Thus, similar to ET-1, NPY seems to be released from EECs and this peptide seems to regulate excitation-secretion of these cells as well as excitation-contraction coupling of ventricular cardiomyocytes.     

Absolute configuration of the creatonotines and callimorphines, two classes of arctiid-specific pyrrolizidine alkaloids

Arctiids which as larvae sequester pyrrolizidine alkaloids (PAs) from their food plants are known to synthesize insect-specific PAs by esterifying necine bases derived from plant PAs with necic acids of insect origin. There are two classes of insect PAs, the creatonotines and the callimorphines. The creatonotines contain as necic acids either 2-hydroxy-3-methylbutyric acid (creatonotine A) or 2-hydroxy-3-methylpentanoic acid (creatonotine B). The three known callimorphines contain 2-hydroxy-2-methylbutanoic acid whose hydroxyl group can be either free (deacetylcallimorphine) or acetylated (callimorphine) or propionylated (homocallimorphine). Insect PAs are assumed to play an important role in the recycling of plant derived necine bases and the processing by trans-esterification of PA monoesters that cannot be directly transmitted to the insect's pupal and adult life-stages. The absolute configuration of the insect-specific necic acids was elucidated in the context of the suggested role of the insect PAs as insect-made mimics of plant monoester PAs of the lycopsamine type. For this purpose all needed stereoisomers were synthesized and a gas chromatography-mass spectrometry (GC-MS) method was established that allows the enantioselective separation and assignment of the stereochemistry of all insect specific necic acids as their methyl esters. The method could also be applied to the GC-MS analysis of the intact alkaloids which were hydrolyzed during injection and converted into their methyl esters. Analysis of the creatonotines and callimorphines isolated from the polyphagous arctiids Estigmene acrea and Grammia geneura that were fed with pure PAs and defined PA mixtures revealed the following absolute configuration: the callimorphines and creatonotine A were present in 2'R configuration, whereas creatonotine B was found as mixture of (2'R, 3'S)- and (2'S, 3'S)-stereoisomers. The ratio of 2'S to 2'R was extremely variable ranging from 98% S to 94% R. The cause of the lack of stereospecificity is discussed particularly in respect of a possible epimerization of the hydroxyl group at C-2' in analogy to the known epimerization at C-3' of plant acquired PAs of the lycopsamine type.     

Petri net modelling of biological networks

Mathematical modelling is increasingly used to get insights into the functioning of complex biological networks. In this context, Petri nets (PNs) have recently emerged as a promising tool among the various methods employed for the modelling and analysis of molecular networks. PNs come with a series of extensions, which allow different abstraction levels, from purely qualitative to more complex quantitative models. Noteworthily, each of these models preserves the underlying graph, which depicts the interactions between the biological components. This article intends to present the basics of the approach and to foster the potential role PNs could play in the development of the computational systems biology.     

Operationalising emission and toxicity modelling of pesticides in LCA: the OLCA-Pest project contribution

The International Journal of Life Cycle Assessment - Current field emission modelling and toxicity characterisation of pesticides suffer from several shortcomings like mismatches between LCI...     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.