Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.     

Social inequalities and mortality in europe - results from a large multi-national cohort

Background

Socio-economic inequalities in mortality are observed at the country level in both North America and Europe. The purpose of this work is to investigate the contribution of specific risk factors to social inequalities in cause-specific mortality using a large multi-country cohort of Europeans.

Methods

A total of 3,456,689 person/years follow-up of the European Prospective Investigation into Cancer and Nutrition (EPIC) was analysed. Educational level of subjects coming from 9 European countries was recorded as proxy for socio-economic status (SES). Cox proportional hazard model''s with a step-wise inclusion of explanatory variables were used to explore the association between SES and mortality; a Relative Index of Inequality (RII) was calculated as measure of relative inequality.

Results

Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest (HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78). The risk reduction was attenuated by 7% in men and 3% in women by the introduction of smoking and to a lesser extent (2% in men and 3% in women) by introducing body mass index and additional explanatory variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women). Social inequalities were highly statistically significant for all causes of death examined in men. In women, social inequalities were less strong, but statistically significant for all causes of death except for cancer-related mortality and injuries.

Discussion

In this European study, substantial social inequalities in mortality among European men and women which cannot be fully explained away by accounting for known common risk factors for chronic diseases are reported.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.Bacterial lipoproteins (Lpps)¹ are a subset of membrane proteins that are covalently modified with a lipidic moiety at their N-terminal cysteine residue. It is commonly reported that Lpps of Gram-positive bacteria are processed by two key enzymes; the prolipoprotein diacylglyceryl transferase (Lgt) and the lipoprotein signal peptidase (Lsp). The Lgt enzyme recognizes a so-called lipobox motif in the C-terminal region of the signal peptide of a premature lipoprotein and transfers a diacylglyceryl moiety to the cysteine residue of the lipobox (1), (2). Subsequently, the Lsp enzyme cleaves the signal peptide resulting in a mature Lpp (3), (4). Nevertheless, recent reports have suggested that N-acylation occurs in bacteria that lack the Gram-negative homologous apolipoprotein N-acyltransferase (Lnt) gene responsible for this modification (5, 6), and that Lpp N-terminal could also be modified with an acetyl group in some Gram-positive (7).Lpps have been described as virulence factors because they play critical roles in membrane stabilization, nutrient uptake, antibiotic resistance, bacterial adhesion to host cells, protein maturation and secretion and many of them still have unknown function (8). Several studies have suggested that bacterial Lpps are pathogen-associated molecular patterns (PAMPs) sensed by the mammalian host through Toll-like receptor 2 (TLR2) heterodimerized with TLR1 or TLR6 to induce innate immunity activation and to control adaptive immunity (9–12). TLR2 plays a critical role in the host response to the Gram-positive bacteria Staphylococcus aureus (13) and Streptococcus agalactiae (14). Although TLR2 has been considered a receptor for various structurally unrelated PAMPs, recent studies have suggested that, via their lipid moiety, bacterial Lpps function as the major, if not the sole, ligand molecules responsible for TLR2 activation (15). Although Gram-negative Lpps have been widely studied, little information is available for Gram-positive Lpps (16) and the ways they are released into the bacterial extracellular compartment and reach the host immune system remain unclear.We focused our attention on Lpps release by Streptococcus pyogenes. This Gram-positive bacterium is an important human pathogen that causes a wide range of diseases from superficial and self-limiting infection, e.g. pharyngitis and impetigo, to more systemic or invasive diseases like necrotizing fasciitis and septicemia (17). Understanding the role of bacterial Lpps in mediating innate and acquired immunity can be instrumental for the therapy and prophylaxis of human S. pyogenes infections. In this study, we showed that in S. pyogenes Lpps are released into the growth medium within vesicle-like structures in minute amounts. Conditions weakening the bacterial cell wall, such as the addition of sublethal concentrations of penicillin to the bacterial growth medium enhanced this phenomenon and allowed the recovery of sufficient material to enable an in-depth characterization. Proteomic analysis of the vesicles revealed that they were almost exclusively constituted of Lpps. A total of 28 Lpps were identified, representing more than 72% of the Lpps predicted from the genome of the strain under investigation. In addition, multiple transmembrane domain proteins were not found in abundance associated to the vesicles, indicating that vesicles were not representative of the bacterial membrane. We defined these vesicles as Lipoprotein-rich Membrane Vesicles (LMVs).Common characteristics are shared between the LMVs and the ExPortal described for the first time by Rosch and Caparon (18). This asymmetric and distinct membrane microdomain has been reported to be enriched in anionic phospholipids and acts in promoting the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and the accessory factors required for their maturation (19–21). An association between ExPortal and peptidoglycan synthesis has also been reported (22). Similarly, LMVs are enriched in anionic phosphatidylglycerol, enzymes involved in protein maturation/secretion and cell wall biogenesis, suggesting that LMVs might derive from the ExPortal. Finally, we showed that LMVs do not induce TLR2 activation, indicating that the Lpps did not act as PAMPs when integrated into the LMVs.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.