Making DNA without iron: induction of a manganese-dependent ribonucleotide reductase in response to iron starvation

Ribonucleotide reductases supply cells with their deoxyribonucleotides. Three enzyme types are known, classes I, II and III. Class II enzymes are anaerobic whereas class I enzymes are aerobic, and so class I and II enzymes are often produced by the same organism under opposing oxygen regimes. Escherichia coli contains two types of class I enzyme (Ia and Ib) with the Fe-dependent Ia enzyme (NrdAB) performing the major role aerobically, leaving the purpose of the Ib enzyme (NrdEF) unclear. Several papers have recently focused on the class Ib enzymes showing that they are Mn (rather than Fe) dependent and suggesting that the E. coli NrdEF may function under redox-stress conditions. A paper published in this issue of Molecular Microbiology from James Imlay's group confirms that this unexplained NrdEF Ib enzyme is Mn-dependent, but shows that it does not substitute for NrdAB during redox stress. Instead, a role during iron restriction is demonstrated. Thus, the purpose of NrdEF (and possibly other class Ib enzymes) is to enhance growth under aerobic, low-iron conditions, and to functionally replace the Fe-dependent NrdAB when iron is unavailable. This finding reveals a new mechanism by which bacteria adjust to life under iron deprivation.

Oxygen/Glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection

Blood pressure lowering effects of beetroot juice and novel beetroot enriched bread products in normotensive male subjects

A number of vegetables have a high nitrate content which after ingestion can be reduced to 36 nitrite by oral bacteria, and further to vasoprotective nitric oxide endogenously. Two separate 37 randomly controlled, single blind, cross-over, postprandial studies were performed in 38 normotensive volunteers. Ambulatory blood pressure was measured over a 24 h period 39 following consumption of either four doses of beetroot juice (BJ) 0 g, 100 g, 250 g and 500 g 40 (n = 18) or three bread products, control bread (0 g beetroot), red beetroot and white beetroot 41 enriched breads (n =14). Total urinary nitrate/nitrite (NOx) was measured at baseline, 2, 4 42 and 24 h post ingestion. BJ consumption significantly, and in a near dose dependent manner, 43 lowered systolic (P <0.01) and diastolic BP (P <0.001) over a period of 24 h, compared to 44 water control. Furthermore, bread products enriched with 100 g red or white beetroot lowered 45 systolic and diastolic BP over a period of 24 h (red beetroot enriched bread, P <0.05), with no 46 statistical differences between varieties. Total urinary NOx significantly increased following 47 consumption of 100 g (P<0.01), 250 g (P <0.001) and 500 g BJ (P <0.001) and after red 48 beetroot bread (P <0.05), but did not reach significance for white beetroot bread compared to 49 the no beetroot condition. These studies demonstrated significant hypotensive effects of a low 50 dose (100 g) of beetroot which was unaffected by processing, or the presence of betacyanins. 51 This data strengthens the evidence for cardioprotective BP lowering effects of dietary nitrate-52 rich vegetables.

Intestinal injury and endotoxemia in children undergoing surgery for congenital heart disease

RATIONALE: Children with congenital heart disease are at risk of gut barrier dysfunction and translocation of gut bacterial antigens into the bloodstream. This may contribute to inflammatory activation and organ dysfunction postoperatively. OBJECTIVES: To investigate the role of intestinal injury and endotoxemia in the pathogenesis of organ dysfunction after surgery for congenital heart disease. METHODS: We analyzed blood levels of intestinal fatty acid binding protein and endotoxin (endotoxin activity assay) alongside global transcriptomic profiling and assays of monocyte endotoxin receptor expression in children undergoing surgery for congenital heart disease. MEASUREMENTS AND MAIN RESULTS: Levels of intestinal fatty acid binding protein and endotoxin were greater in children with duct-dependent cardiac lesions. Endotoxemia was associated with severity of vital organ dysfunction and intensive care stay. We identified activation of pathogen-sensing, antigen-processing, and immune-suppressing pathways at the genomic level postoperatively and down-regulation of pathogen-sensing receptors on circulating immune cells. CONCLUSIONS: Children undergoing surgery for congenital heart disease are at increased risk of intestinal mucosal injury and endotoxemia. Endotoxin activity correlates with a number of outcome variables in this population, and may be used to guide the use of gut-protective strategies.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.

Preventing carbon contamination of optical devices for X-rays: the effect of oxygen on photon-induced dissociation of CO on platinum

Platinum is one of the most common coatings used to optimize mirror reflectivity in soft X-ray beamlines. Normal operation results in optics contamination by carbon-based molecules present in the residual vacuum of the beamlines. The reflectivity reduction induced by a carbon layer at the mirror surface is a major problem in synchrotron radiation sources. A time-dependent photoelectron spectroscopy study of the chemical reactions which take place at the Pt(111) surface under operating conditions is presented. It is shown that the carbon contamination layer growth can be stopped and reversed by low partial pressures of oxygen for optics operated in intense photon beams at liquidnitrogen temperature. For mirrors operated at room temperature the carbon contamination observed for equivalent partial pressures of CO is reduced and the effects of oxygen are observed on a long time scale.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

Substance P released by TRPV1-expressing neurons produces reactive oxygen species that mediate ethanol-induced gastric injury

Although neurokinin 1 receptor antagonists prevent ethanol (EtOH)-induced gastric lesions, the mechanisms by which EtOH releases substance P (SP) and SP damages the mucosa are unknown. We hypothesized that EtOH activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to release SP, which stimulates epithelial neurokinin 1 receptors to generate damaging reactive oxygen species (ROS). SP release was assayed in the mouse stomach, ROS were detected using dichlorofluorescein diacetate, and neurokinin 1 receptors were localized by immunofluorescence. EtOH-induced SP release was prevented by TRPV1 antagonism. High dose EtOH caused lesions, and TRPV1 or neurokinin 1 receptor antagonism and neurokinin 1 receptor deletion inhibited lesion formation. Coadministration of low, innocuous doses of EtOH and SP caused lesions by a TRPV1-independent but neurokinin 1 receptor-dependent process. EtOH, capsaicin, and SP stimulated generation of ROS by superficial gastric epithelial cells expressing neurokinin 1 receptors by a neurokinin 1 receptor-dependent mechanism. ROS scavengers prevented lesions induced by a high EtOH dose or a low EtOH dose plus SP. Gastric lesions are caused by an initial detrimental effect of EtOH, which is damaging only if associated with TRPV1 activation, SP release from sensory nerves, stimulation of neurokinin 1 receptors on epithelial cells, and ROS generation.

Oxygen isotope ratios in the shell of Mytilus edulis: archives of glacier meltwater in Greenland?

Melting of the Greenland Ice Sheet (GrIS) is accelerating and will contribute significantly to global sea level rise during the 21st century. Instrumental data on GrIS melting only cover the last few decades, and proxy data extending our knowledge into the past are vital for validating models predicting the influence of ongoing climate change. We investigated a potential meltwater proxy in Godthåbsfjord (West Greenland), where glacier meltwater causes seasonal excursions with lower oxygen isotope water (δ18Ow) values and salinity. The blue mussel (Mytilus edulis) potentially records these variations, because it precipitates its shell calcite in oxygen isotopic equilibrium with ambient seawater. As M. edulis shells are known to occur in raised shorelines and archaeological shell middens from previous Holocene warm periods, this species may be ideal in reconstructing past meltwater dynamics. We investigate its potential as a palaeo-meltwater proxy. First, we confirmed that M. edulis shell calcite oxygen isotope (δ18Oc) values are in equilibrium with ambient water and generally reflect meltwater conditions. Subsequently we investigated if this species recorded the full range of δ18Ow values occurring during the years 2007 to 2010. Results show that δ18Ow values were not recorded at very low salinities (< ~ 19), because the mussels appear to cease growing. This implies that Mytilus edulis δ18Oc values are suitable in reconstructing past meltwater amounts in most cases, but care has to be taken that shells are collected not too close to a glacier, but rather in the mid-region or mouth of the fjord. The focus of future research will expand on the geographical and temporal range of the shell measurements by sampling mussels in other fjords in Greenland along a south–north gradient, and by sampling shells from raised shorelines and archaeological shell middens from prehistoric settlements in Greenland.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays

Background Cortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat (‘artificial animal’) applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown. Results Consequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion. Conclusions We suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

Effect of hypobaric hypoxia, simulating conditions during long-haul air travel, on coagulation, fibrinolysis, platelet function, and endothelial activation

CONTEXT: The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane cabin create an increased risk compared with seated immobility at ground level. OBJECTIVE: To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis. DESIGN, SETTING, AND PARTICIPANTS: A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September 2003 to November 2005. Participants were screened for factor V Leiden G1691A and prothrombin G20210A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis. INTERVENTIONS: Individuals were exposed alternately (> or =1 week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of 2438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level). MAIN OUTCOME MEASURES: Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation. RESULTS: Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, -0.30 to 0.30 ng/mL); -0.02 [corrected] nmol/L for prothrombin fragment 1 + 2 (95% CI, -0.03 to 0.01 nmol/L); 1.38 ng/mL for D-dimer (95% CI, -3.63 to 9.72 ng/mL); and -2.00% for endogenous thrombin potential (95% CI, -4.00% to 1.00%). CONCLUSION: Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.

CalDAG-GEFI is at the nexus of calcium-dependent platelet activation.

The importance of the second messengers calcium (Ca(2+)) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca(2+) sensor(s). We here identify Ca(2+) and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca(2+)-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A(2) (TxA(2)) release. CalDAG-GEFI-dependent TxA(2) generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca(2+)-induced integrin activation, TxA(2) generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI- and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.