Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.     

Gastric inflammatory markers and interleukins in patients with functional dyspepsia treated with astaxanthin

The chronic active inflammation caused by Helicobacter pylori is dominated by neutrophils, macrophages, lymphocytes and plasma cells. Several interleukins are involved in the inflammatory process. The aim of this study was to investigate the effect of astaxanthin on gastric inflammation in patients with functional dyspepsia. Forty-four consecutive patients were included, and biopsies were examined for IL-4, IL-6, IL-8, IL-10, interferon-gamma, CD4, CD8, CD14, CD19, CD25 and CD30. Patients were randomized: 21 patients were treated with 40 mg of astaxanthin daily, and 23 patients were treated with a placebo. There was a significant decrease in gastric inflammation in H. pylori-positive patients from both groups. There were no significant changes in the density of H. pylori or in any of the interleukins during or after treatment. There was a significant up-regulation of CD4 and down-regulation of CD8 in patients with H. pylori treated with astaxanthin. Astaxanthin had an effect on the inflammation and on the density of H. pylori in mice in a study where the diet could be standardized without antioxidants (Bennedsen et al., 1999). These dietary conditions are impossible in studies involving humans, and may be due to the minor effect when the host have access to antioxidants in their diet.     

The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium

In being both, a modifier of cellular immune effector pathways and an essential nutrient for microbes, iron is a critical determinant in host-pathogen interaction. Here, we investigated the metabolic changes of macrophage iron homeostasis and immune function following the infection of RAW264.7 murine macrophages with Salmonella typhimurium. We observed an enhanced expression of the principal iron export protein, ferroportin 1, and a subsequent increase of iron efflux in Salmonella-infected phagocytes. In parallel, the expression of haem oxygenase 1 and of the siderophore-binding peptide lipocalin 2 was markedly enhanced following pathogen entry. Collectively, these modulations reduced both the cytoplasmatic labile iron and the ferritin storage iron pool within macrophages, thus restricting the acquisition of iron by intramacrophage Salmonella. Correspondingly, limitation of macrophage iron decreased microbial survival, whereas iron supplementation impaired immune response pathways in Salmonella-infected macrophages (nitric oxide formation and tumour necrosis factor-alpha production) and promoted intracellular bacterial proliferation. Our findings suggest that the enhancement of ferroportin 1-mediated iron efflux, the upregulation of the haem-degrading enzyme haem oxygenase 1 and the induction of lipocalin 2 following infection concordantly aim at withholding iron from intracellular S. typhimurium and to increase antimicrobial immune effector pathways thus limiting pathogen proliferation.     

Early onset pauciarticular arthritis is the major risk factor for naproxen-induced pseudoporphyria in juvenile idiopathic arthritis

Pseudoporphyria (PP) is characterized by skin fragility, blistering and scarring in sun-exposed skin areas without abnormalities in porphyrin metabolism. The phenylpropionic acid derivative group of nonsteroidal anti-inflammatory drugs, especially naproxen, is known to cause PP. Naproxen is currently one of the most prescribed drugs in the therapy of juvenile idiopathic arthritis (JIA). The prevalence of PP was determined in a 9-year retrospective study of children with JIA and associated diseases. In addition, we prospectively studied the incidence of PP in 196 patients (127 girls and 69 boys) with JIA and associated diseases treated with naproxen from July 2001 to March 2002. We compared these data with those from a matched control group with JIA and associated diseases not treated with naproxen in order to identify risk factors for development of PP. The incidence of PP in the group of children taking naproxen was 11.4%. PP was particularly frequent in children with the early-onset pauciarticular subtype of JIA (mean age 4.5 years). PP was associated with signs of disease activity, such as reduced haemoglobin (<11.75 g/dl), and increased leucocyte counts (>10,400/μl) and erythocyte sedimentation rate (>26 mm/hour). Comedications, especially chloroquine intake, appeared to be additional risk factors. The mean duration of naproxen therapy before the onset of PP was 18.1 months, and most children with PP developed their lesions within the first 2 years of naproxen treatment. JIA disease activity seems to be a confounding factor for PP. In particular, patients with early-onset pauciarticular JIA patients who have significant inflammation appear to be prone to developing PP upon treatment with naproxen.     

A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.     

Subthalamic nucleus deep brain stimulation in elderly patients – analysis of outcome and complications

Background  

There is an ongoing discussion about age limits for deep brain stimulation (DBS). Current indications for DBS are tremor-dominant disorders, Parkinson's disease, and dystonia. Electrode implantation for DBS with analgesia and sedation makes surgery more comfortable, especially for elderly patients. However, the value of DBS in terms of benefit-risk ratio in this patient population is still uncertain.     

2-Cysteine Peroxiredoxins and Thylakoid Ascorbate Peroxidase Create a Water-Water Cycle That Is Essential to Protect the Photosynthetic Apparatus under High Light Stress Conditions

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H₂O₂)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H₂O₂, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H₂O₂- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.Plants are frequently exposed to different abiotic stresses, including high light (HL), UV irradiation, heat, cold, and drought. A component common to these stresses is the rapid formation of reactive oxygen species (ROS) as the result of metabolic dysbalances. A major ROS produced under moderate light (ML) and, in particular, HL photooxidative stress conditions was shown to be singlet oxygen, ¹O₂, that is produced in illuminated chloroplasts predominantly at the PSII (Triantaphylidès et al., 2008). Most of the singlet oxygen is quenched by carotenoids and tocopherols or reacts with galactolipids in thylakoid membranes, yielding galactolipid hydroperoxides (Zoeller et al., 2012; Farmer and Mueller, 2013). In addition, superoxide radicals, O₂·⁻, are produced predominantly at the PSI and rapidly dismutate to hydrogen peroxide (H₂O₂) either spontaneously or because of being catalyzed by superoxide dismutase. Hence, lipid peroxides and H₂O₂ are produced close to the photosystems and may damage thylakoid proteins. In this context, 2-Cys peroxiredoxin (PRX) enzymes have been implicated in the reductive detoxification of lipid peroxides and H₂O₂ (König et al., 2002).During photosynthesis, light energy absorbed by PSII is used to split water molecules, and the electrons are channeled from PSII through PSI to ferredoxin (Fd). As a result, electrons flow from water to Fd. The main electron sink reaction is the Fd NADP oxidoreductase-catalyzed production of NADPH that functions as an electron donor to reduce carbon dioxide to sugars. Under HL conditions, excessive excitation energy is dissipated into heat, which was indicated by nonphotochemical quenching of chlorophyll fluorescence. In addition, excessive photosynthetic electrons can be donated from PSI to O₂, yielding O₂·⁻ (Miyake, 2010). This process, the Mehler reaction, creates an alternative electron sink and electron flow. Superoxide anion radicals, O₂·⁻, can be dismutated to O₂ and H₂O₂ by a thylakoid-attached copper/zinc superoxide dismutase (Cu/ZnSOD; Rizhsky et al., 2003). H₂O₂ can then be reduced to water by peroxidases. As a result, O₂ molecules originating from the water-splitting process at PSII are reduced to water by electrons originating from PSI. This process is termed the water-water cycle (WWC) that is thought to protect the photosynthetic apparatus from excessive light and alleviate photoinhibition.In the classical WWC, the Mehler-ascorbate peroxidase (MAP) pathway, ascorbate peroxidases (APXs) have been considered as key enzymes in the reductive detoxification of H₂O₂ in chloroplasts (Kangasjärvi et al., 2008). APXs reduce H₂O₂ to water and oxidize ascorbate to monodehydroascorbate radicals. NADPH functions as an electron donor to regenerate ascorbate by monodehydroascorbate radical reductase. There are two functional APX homologs in plastids: a 33-kD stromal ascorbate peroxidase (sAPX) and a 38-kD thylakoid ascorbate peroxidase (tAPX). The latter tAPX is thought to reside close to the site of H₂O₂ generation at PSI. Surprisingly, knockout-tAPX mutants as well as double mutants lacking both the tAPX and the sAPX exhibited no visible symptoms of stress after long-term (1–14 d) HL (1.000 µmol photons m⁻² s⁻¹) exposure (Giacomelli et al., 2007; Kangasjärvi et al., 2008; Maruta et al., 2010). Moreover, the photosynthetic efficiency of PSII (as judged by the maximum photochemical efficiency of PSII in the dark-adapted state [F_v/F_m]), H₂O₂ production, antioxidant levels (ascorbate, glutathione, and tocopherols), protein oxidation, and anthocyanin accumulation were similar between light-stressed mutant and wild-type plants. Hence, other H₂O₂ detoxification mechanisms can efficiently compensate for the lack of the sAPX and tAPX detoxification system.In addition to APX, glutathione peroxidases and PRXs may reduce H₂O₂ to water. It has been postulated that, in the chloroplast, two highly homologous thylakoid-associated 2-Cys peroxiredoxins (2CPs), 2CPA and 2CPB, can create an alternative ascorbate-independent WWC (Dietz et al., 2006). In support of this concept, HL stress-acclimated tapx sapx double-mutant plants showed increased levels of 2-Cys PRX compared with wild-type plants (Kangasjärvi et al., 2008). Because the two plastidial 2CPA and 2CPB dynamically interact with the stromal side of thylakoid membranes and are capable of reducing peroxides, 2-Cys PRX enzymes may be involved in both H₂O₂ detoxification and reduction of lipid peroxides in thylakoids (König et al., 2002).The reaction mechanism of 2-Cys PRX is highly conserved and involves a Cys residue, which becomes transiently oxidized to sulphenic acid (termed the peroxidatic Cys residue), thereby reducing H₂O₂ to water. The sulphenic acid is subsequently attacked by a second Cys residue, termed resolving Cys residue, yielding an intermolecular disulfide bridge and water (Dietz, 2011).At high peroxide concentrations, the peroxidase function of 2-Cys PRX becomes inactivated through overoxidation, and excess H₂O₂ may function as a redox signal (Puerto-Galán et al., 2013). It has been postulated that 2-Cys PRXs function as a floodgate that allows H₂O₂ signaling only under oxidative stress conditions (Wood et al., 2003; Dietz, 2011; Puerto-Galán et al., 2013). In addition to its function as peroxidase, 2-Cys PRX may also serve as proximity-based thiol oxidases and chaperones (König et al., 2013).The genome of Arabidopsis (Arabidopsis thaliana) contains two 2CP genes. To study 2-Cys PRX function, transgenic plants with reduced 2-Cys PRX levels were generated by antisense suppression (Baier et al., 2000) as well as crossing of transfer DNA (T-DNA) insertion mutants (Pulido et al., 2010). The T-DNA insertion double mutant was shown to contain less than 5% of the wild-type content of 2CPA and no 2CPB. Hence, full knockout lines lacking both 2-Cys PRXs have not yet been established. Under standard growth conditions, 2-Cys PRX double mutants (similar to plastid APX-deficient plants) also did not show a photooxidative stress phenotype that might be because of compensation by alternative H₂O₂ reduction systems (Pulido et al., 2010). Because of the lack of a clear phenotype of the 2-Cys PRX double-knockdown mutant under ML conditions, the physiological functions of 2CPA and 2CPB remain to be elucidated.The main aim of this study was to identify the physiological function of 2CPA and 2CPB under HL stress conditions, when the WWC is of particular importance in protecting the photosynthetic apparatus from photooxidative damage. We investigated mutants completely deficient in 2-Cys PRX (2cpa 2cpb) or tAPX (tapx) and in addition, 2cpa 2cpb tapx triple knockout plants to study the extent of the functional overlap between these enzymes. Results suggest that 2-Cys PRXs are involved in a 2-Cys PRX-dependent WWC that seems to be more important in protecting the photosynthetic apparatus than the tAPX-dependent WWC, the MAP cycle.     

Brotgetreide

Bread cereals Wheat and spelt are primarily used as bread cereals together with rye. To increase the worldwide wheat production and achieve cultivation goals faster, the very large bread wheat genome is currently analyzed intensively. Wheat is hexaploid and contains three genomes side by side which do not hybridize. The progenitors of einkorn (diploid) and emmer (tetraploid) have been the ancestors of today's wheat and spelt. Spelt is less demanding than wheat but requires an extra stage of husk removal before milling. In Germany, spelt is nowadays a modern bread cereal again. As it has a higher ratio of essential amino acids, the protein part of rye is more valuable for nutrition than that of wheat. Climatic conditions as well as poorer soils in Northern Germany are more suitable for rye than for wheat. Therefore rye has been a typical German bread cereal since medieval times.