The long-chain fatty acid sensor, PsrA, modulates the expression of rpoS and the type III secretion exsCEBA operon in Pseudomonas aeruginosa

The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5 β-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC . DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N -(butyryl)- l -homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa .     

The structure of a putative S‐formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C—O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.     

Structural and functional characteristics of photosynthetic apparatus of chlorophyll-containing grape vine tissue

Photosynthesis in tissues under periderm of woody stems and shoots of perennial plants occurs in environment that is very different from the internal environment of leaf chloroplasts. These tissues are characterized by high CO₂ and low O₂ concentrations, more acidic surroundings, besides that only light which have passed through periderm reaches photosynthetic antennas. In contrast to leaves of deciduous plants chlorenchyma tissues of wintering plant organs are exposed to temperature fluctuations during all seasons, that is why the photosynthetic apparatus of woody stems has to be able to adapt to a wide range of environmental temperatures. In order to reveal unique features, which enable photosynthetic apparatus of chlorenchyma cells in woody plant organs to implement biological functions under different light and temperature conditions, we studied photosynthetic tissues of stem cortex in grapevine (Vitis vinifera L.) under normal conditions and after exposure to suboptimal temperatures and high light intensity. Comparative analysis of photosynthetic pigment composition and low-temperature chlorophyll fluorescence emission spectrum of leaves, young shoots and chlorenchyma of lignified shoots revealed relatively high level of chlorophyll b and carotenoids, and high photosystem II (PSII) to photosystem I (PSI) ratio in woody shoots. Analysis of parameters of variable chlorophyll fluorescence revealed high PSII activity in grapevine shoot cortex and demonstrated improved freeze tolerance and higher sensitivity to light of photosynthetic apparatus in grape vine in comparison to leaves. It was shown for the first time that photosynthetic apparatus in chlorenchyma cells of vine undergoes so-called “state-transition”–fast rearrangements leading to redistribution of energy between photosystems. Analysis of fatty acid (FA) compositions of lipids in examined tissues showed that the FA unsaturation index in green tissue of vine is lower than in leaves. A distinct feature of FA compositions of lipids in vine cortex was relatively high level of linoleic acid.     

Long-term results of chronic occlusion recanalization in patients with coronary heart disease

The study was undertaken to assess the long-term results of recanalization of chronically occluded coronary arteries, by applying drug-eluting stents to patients with coronary heart disease. The study enrolled 585 patients with one-vessel occlusive lesion of one of three great coronary arteries (TIMI 0; occlusion duration, > or = 3 months): 321 patients who underwent successful recanalization of chronic occlusion and further implantation of drug-eluting stents and 264 patients who received drug therapy (a control group). The short- and long-term results of recanalization were investigated. The follow-up averaged 1095 +/- 36 days; reexaminations were made after 1, 2, and 3 years. The direct success rate of recanalization of chronically occluded coronary arteries was 84.9% (321/378). The results of a 3-year follow-up showed the efficiency and expediency of endovascular recanalization of chronic occlusions: the invasively treated patients had the symptoms of angina pectoris and heart failure significantly less frequently, showed higher exercise tolerance and a less need for antianginal therapy, and had a better long-term prognosis.     

Purinergic Membrane Receptors as Targets for the Effect of Purotoxin 1, a Component of Venom of Spiders from the <Emphasis Type="Italic">Geolycosa</Emphasis> Genus

We examined effects of purotoxin 1 (PT1), a component of the venom of Geolycosa spiders, on a few voltageand ligand-operated ion channels present in the plasma membrane of sensory neurons from the rat dorsal root ganglia (DRGs). Purotoxin 1 in a 100 nM concentration evoked no changes in ion currents through voltage-operated sodium, potassium, and calcium channels in the membranes of isolated sensory neurons. This agent was also found to be ineffective with respect to capsaicin-sensitive receptor-channel complexes (TRPV1). Testing of the effects of PT1 on purinergic receptor-channel complexes P2X3, P2X2, and P2X2/3 showed that this toxin is a highly selective blocker of exclusively P2X3 receptors. The selectivity of action of PT1 demonstrated in our experiments shows that it is a unique agent, which opens up new prospects in the studies of structural/functional peculiarities of receptor-channel complexes P2X3 as a peripheral link of the nociception system.