Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of ΔpepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of ΔpepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress.     

N-formyl peptide receptors internalize but do not recycle in the absence of arrestins

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.     

Epidemiology of typhoid carriers among blood donors and patients with biliary, gastrointestinal and other related diseases

Enteric fever due to Salmonella Typhi is a major public health problem. Typhoid carriers have high titres of Vi agglutinins in their sera. We worked out the baseline data for Vi agglutinins from 705 healthy blood donors (controls) by ELISA and compared it with 446 patients with biliary, gastrointestinal and other related diseases (cases). The samples were divided into five groups based on the disease condition of the patients from whom they were collected. Group A (n=196) consisted of patients with stones in the gall bladder/common bile duct and Group B (n=27) with gall bladder carcinoma. Group C (n=33) comprised patients with carcinoma of the pancreas/ampulla, obstructive jaundice and/or cholangiocarcinoma. Group D (n=112) had patients with acute/chronic pancreatitis, abdominal pain, intestinal obstruction, peritonitis, carcinoma oesophagus, chronic diarrhoea, gastrointestinal bleeding and dyspepsia. Group E (n=78) included patients with miscellaneous diseases. The mean absorbance value obtained for healthy subjects +3 standard deviations was taken as the cut-off value for a positive typhoid carrier. In Group A, 10.2% samples were positive; in Group B, 7.4%; in Group C, 12.0%; in Group D, 9.8% and in Group E, 9.0%. There was a highly significant (P <0.001) increase in the presence of Vi agglutinins in the cases compared to the controls. High prevalence of typhoid carriers occurs in patients with biliary, gastrointestinal and other related diseases. Vi serology employing highly purified Vi antigen offers a practical and cost-effective way of screening for S. Typhi carriers.     

Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H(2)O(2)), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H(2)O(2) levels), whereas for long periods it showed a pro-oxidant activity (increase in ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.     

MOCAT: A Metagenomics Assembly and Gene Prediction Toolkit

MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.     

New ternary copper(II) complexes of l-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of α-amino acid having polypyridyl bases of general formulation [Cu(l-ala)(B)(H₂O)](X) (1-4), where l-ala is l-alanine, B is an N,N-donor heterocyclic base, viz. 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2′,3′-f]quinoxaline (dpq, 4), and X = / are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN₃O₂ coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near −100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 ? 1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.