ABSTRACT
Respiratory distress syndrome increases the risk of death and bronchopulmonary dysplasia (BPD) in premature infants. Inhaled nitric oxide (iNO) may reduce these risks. Recent meta-analyses have suggested that iNO is effective only at doses higher than 5 ppm and in infants born to Black mothers. In a randomized, double-blinded, controlled trial, infants born before 32 0/7 weeks gestation, weighing <1500 g, and requiring respiratory support were assigned to receive iNO for either seven days (short iNO), or until 33 0/7 weeks PMA (long iNO). The primary outcome was death or BPD. A total of 273 patients were enrolled, of whom 83 receiving long iNO (61.5%) experienced the primary outcome, compared with 65 (47.1%) receiving short iNO (relative risk (RR) 1.37; 95% confidence interval (CI), 1.06-1.79; p = 0.017). This increase was due solely to increased BPD in infants weighing 750-999 g (RR 1.33, 95% CI 1.07-1.66, p = 0.009). However, there was no difference in the numbers of infants requiring supplemental oxygen at 40 weeks PMA. Among infants < 750 g, long-iNO-treated infants had a lower cumulative probability of death (χ2 5.12, p = 0.02). Long iNO increased the primary outcome in non-Black infants (RR 1.93, 95% CI 1.20-3.24) but not in Black infants. Understanding how maternal racial identity determines responses of premature infants to iNO may help narrow the gap in health outcomes between Black and non-Black infants.
ABSTRACT
Cultures of dissociated hippocampal neurons display a stereotypical development of network activity patterns within the first three weeks of maturation. During this process, network connections develop and the associated spiking patterns range from increasing levels of activity in the first two weeks to regular bursting activity in the third week of maturation. Characterization of network structure is important to examine the mechanisms underlying the emergent functional organization of neural circuits. To accomplish this, confocal microscopy techniques have been used and several automated synapse quantification algorithms based on (co)localization of synaptic structures have been proposed recently. However, these approaches suffer from the arbitrary nature of intensity thresholding and the lack of correction for random-chance colocalization. To address this problem, we developed and validated an automated synapse quantification algorithm that requires minimal operator intervention. Next, we applied our approach to quantify excitatory and inhibitory synaptogenesis using confocal images of dissociated hippocampal neuronal cultures captured at 5, 8, 14 and 20 days in vitro, the time period associated with the development of distinct neuronal activity patterns. As expected, we found that synaptic density increased with maturation, coinciding with increasing spiking activity in the network. Interestingly, the third week of the maturation exhibited a reduction in excitatory synaptic density suggestive of synaptic pruning that coincided with the emergence of regular bursting activity in the network.
ABSTRACT
Preterm infants born before 32 weeks gestation have increased risks for neurodevelopmental impairment at two years of age. How brain function differs between preterm infants with normal or impaired development is unknown. However, abnormal spontaneous motor behavior at 12-15 weeks post-term age is associated with neurodevelopmental impairment. We imaged brain blood oxygen level-dependent signals at term-equivalent age in 62 infants born at <32 weeks gestation and explored whether resting state functional connectivity (rsFC) differed with performances on the General Movement Assessment (GMA) at 12-15 weeks, and Bayley III scores at two years of corrected age. Infants with aberrant general movements exhibited decreased rsFC between the basal ganglia and regions in parietal and frontotemporal lobes. Infants with normal Bayley III cognitive scores exhibited increased rsFC between the basal ganglia and association cortices in parietal and occipital lobes compared with cognitively impaired children. Infants with normal motor scores exhibited increased rsFC between the basal ganglia and visual cortices, compared with children with motor impairment. Thus, the presence of abnormal general movements is associated with region-specific differences in rsFC at term. The association of abnormal long-term neurodevelopmental outcomes with decreased rsFC between basal ganglia and sub-score specific cortical regions may provide biomarkers of neurodevelopmental trajectory and outcome.
ABSTRACT
Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.
Subject(s)
Biocompatible Materials/pharmacology , Methacrylates/chemistry , Phosphorylcholine/analogs & derivatives , Polymers/chemistry , Biocompatible Materials/chemistry , Biomimetics/methods , Cell Death/drug effects , Cell Membrane/drug effects , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Methacrylates/pharmacology , Phosphorylcholine/chemistry , Phosphorylcholine/pharmacology , Polymers/pharmacologyABSTRACT
Endotracheal intubation and invasive mechanical ventilation have been mainstays in respiratory care of neonates with respiratory distress syndrome. Together with antenatal steroids and surfactant, this approach has accounted for significant reductions in neonatal mortality. However, with the increased survival of very low birthweight infants, the incidence of bronchopulmonary dysplasia (BPD), the primary respiratory morbidity of prematurity, has also increased. Arrest of alveolar growth and development and the abnormal development of the pulmonary vasculature after birth are the primary causes of BPD. However, invasive ventilation-associated lung inflammation and airway injury have long been believed to be important contributors. In fact, discontinuing invasive ventilation in favor of noninvasive respiratory support has been considered the single best approach that neonatologists can implement to reduce BPD. In this review, we present and discuss the mechanisms, efficacy, and long-term outcomes of the four main approaches to noninvasive respiratory support of the preterm infant currently in use: nasal continuous positive airway pressure, high-flow nasal cannula, nasal intermittent mandatory ventilation, and neurally adjusted ventilatory assist. We show that noninvasive ventilation can decrease rates of intubation and the need for invasive ventilation in preterm infants with respiratory distress syndrome. However, none of these noninvasive approaches decrease rates of BPD. Accordingly, noninvasive respiratory support should be considered for clinical goals other than the reduction of BPD.
Subject(s)
Respiration, Artificial/methods , Respiratory Distress Syndrome, Newborn/therapy , Humans , Infant, Newborn , Infant, PrematureABSTRACT
Here we report a gain in function for mutant (mt) superoxide dismutase I (SOD1), a cause of familial amyotrophic lateral sclerosis (FALS), wherein small soluble oligomers of mtSOD1 acquire a membrane toxicity. Phosphatidylglycerol (PG) lipid domains are selectively targeted, which could result in membrane damage or "toxic channels" becoming active in the bilayer. This PG-selective SOD1-mediated membrane toxicity is largely reversible in vitro by a widely-available FDA-approved surfactant and membrane-stabilizer P188. Treatment of G93ASOD1 transgenic mice with P188 significantly delayed symptoms onset, extended survival and decreased motoneuron death. The use of P188 or an analogue, which targets mtSOD1 misfolding-induced membrane toxicity, may provide a new direction for ALS treatment.
Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Cell Membrane/physiology , Mutation/physiology , Poloxamer/therapeutic use , Superoxide Dismutase-1/genetics , Amyotrophic Lateral Sclerosis/pathology , Animals , Cell Membrane/drug effects , Cell Membrane/pathology , Humans , Male , Mice , Mice, Transgenic , Mutation/drug effects , Poloxamer/pharmacology , Surface-Active Agents/pharmacology , Surface-Active Agents/therapeutic useABSTRACT
Global brain ischemia can lead to widespread neuronal death and poor neurologic outcomes in patients. Despite detailed understanding of the cellular and molecular mechanisms mediating neuronal death following focal and global brain hypoxia-ischemia, treatments to reduce ischemia-induced brain injury remain elusive. One pathway central to neuronal death following global brain ischemia is mitochondrial dysfunction, one consequence of which is the cascade of intracellular events leading to mitochondrial outer membrane permeabilization. A novel approach to rescuing injured neurons from death involves targeting cellular membranes using a class of synthetic molecules called Pluronics. Pluronics are triblock copolymers of hydrophilic poly[ethylene oxide] (PEO) and hydrophobic poly[propylene oxide] (PPO). Evidence is accumulating to suggest that hydrophilic Pluronics rescue injured neurons from death following substrate deprivation by preventing mitochondrial dysfunction. Here, we will review current understanding of the nature of interaction of Pluronic molecules with biological membranes and the efficacy of F-68, an 80% hydrophilic Pluronic, in rescuing neurons from injury. We will review data indicating that F-68 reduces mitochondrial dysfunction and mitochondria-dependent death pathways in a model of neuronal injury in vitro, and present new evidence that F-68 acts directly on mitochondria to inhibit mitochondrial outer membrane permeabilization. Finally, we will present results of a pilot, proof-of-principle study suggesting that F-68 is effective in reducing hippocampal injury induced by transient global ischemia in vivo. By targeting mitochondrial dysfunction, F-68 and other Pluronic molecules constitute an exciting new approach to rescuing neurons from acute injury.
Subject(s)
Mitochondria/drug effects , Neurons/drug effects , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Propylene Glycols/chemistry , Propylene Glycols/pharmacology , Animals , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Gerbillinae , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Male , Mice , Mitochondria/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Neurons/metabolism , Pilot Projects , Polyethylene Glycols/metabolism , Propylene Glycols/metabolism , Rats , Rats, Sprague-Dawley , Substrate Specificity/drug effects , Substrate Specificity/physiologyABSTRACT
The mechanism for the earliest response of central neurons to hypoxia-an increase in voltage-gated sodium current (INa)-has been unknown. Here, we show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons (CGN) and that SUMOylation of NaV1.2 channels increases INa. The time-course for SUMOylation of single NaV1.2 channels at the cell surface and changes in INa coincide, and both are prevented by mutation of NaV1.2-Lys38 or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the voltage-dependence of channel activation so that depolarizing steps evoke larger sodium currents. Given the recognized role of INa in hypoxic brain damage, the SUMO pathway and NaV1.2 are identified as potential targets for neuroprotective interventions.
Subject(s)
Hypoxia , NAV1.2 Voltage-Gated Sodium Channel/metabolism , Neurons/physiology , Sumoylation , Animals , CHO Cells , Cerebellum/cytology , Cricetulus , Electrophysiological Phenomena , Humans , RatsABSTRACT
SPARC is a matrix protein that mediates interactions between cells and the microenvironment. In cancer, SPARC may either promote or inhibit tumor growth depending upon the tumor type. In neuroblastoma, SPARC is expressed in the stromal Schwannian cells and functions as a tumor suppressor. Here, we developed a novel in vivo model of stroma-rich neuroblastoma using non-tumorigenic SHEP cells with modulated levels of SPARC, mixed with tumorigenic KCNR cells. Tumors with stroma-derived SPARC displayed suppressed growth, inhibited angiogenesis and increased lipid accumulation. Based on the described chaperone function of SPARC, we hypothesized that SPARC binds albumin complexed with fatty acids and transports them to tumors. We show that SPARC binds albumin with Kd=18.9±2.3 uM, and enhances endothelial cell internalization and transendothelial transport of albumin in vitro. We also demonstrate that lipids induce toxicity in neuroblastoma cells and show that lipotoxicity is increased when cells are cultured in hypoxic conditions. Studies investigating the therapeutic potential of SPARC are warranted.
Subject(s)
Lipid Metabolism/drug effects , Neuroblastoma/metabolism , Osteonectin/genetics , Osteonectin/metabolism , Palmitic Acid/pharmacology , Serum Albumin, Bovine/metabolism , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Genetic Therapy , Human Umbilical Vein Endothelial Cells , Humans , Mice , Models, Biological , Neuroblastoma/genetics , Neuroblastoma/therapy , Palmitic Acid/chemistry , Serum Albumin, Bovine/chemistryABSTRACT
The National Health and Nutrition Examination Survey (NHANES) is a population survey implemented by the Centers for Disease Control and Prevention (CDC) to monitor the health of the United States whose data is publicly available in hundreds of files. This Data Descriptor describes a single unified and universally accessible data file, merging across 255 separate files and stitching data across 4 surveys, encompassing 41,474 individuals and 1,191 variables. The variables consist of phenotype and environmental exposure information on each individual, specifically (1) demographic information, physical exam results (e.g., height, body mass index), laboratory results (e.g., cholesterol, glucose, and environmental exposures), and (4) questionnaire items. Second, the data descriptor describes a dictionary to enable analysts find variables by category and human-readable description. The datasets are available on DataDryad and a hands-on analytics tutorial is available on GitHub. Through a new big data platform, BD2K Patient Centered Information Commons (http://pic-sure.org), we provide a new way to browse the dataset via a web browser (https://nhanes.hms.harvard.edu) and provide application programming interface for programmatic access.
Subject(s)
Databases, Factual , Nutrition Surveys , Centers for Disease Control and Prevention, U.S. , Environmental Exposure , Humans , United StatesABSTRACT
The goal of this work was to define the contributions of intrinsic and synaptic mechanisms toward spontaneous network-wide bursting activity, observed in dissociated rat hippocampal cell cultures. This network behavior is typically characterized by short-duration bursts, separated by order of magnitude longer interburst intervals. We hypothesize that while short-timescale synaptic processes modulate spectro-temporal intraburst properties and network-wide burst propagation, much longer timescales of intrinsic membrane properties such as persistent sodium (Nap) currents govern burst onset during interburst intervals. To test this, we used synaptic receptor antagonists picrotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP) to selectively block GABAA, AMPA, and NMDA receptors and riluzole to selectively block Nap channels. We systematically compared intracellular activity (recorded with patch clamp) and network activity (recorded with multielectrode arrays) in eight different synaptic connectivity conditions: GABAA + NMDA + AMPA, NMDA + AMPA, GABAA + AMPA, GABAA + NMDA, AMPA, NMDA, GABAA, and all receptors blocked. Furthermore, we used mixed-effects modeling to quantify the aforementioned independent and interactive synaptic receptor contributions toward spectro-temporal burst properties including intraburst spike rate, burst activity index, burst duration, power in the local field potential, network connectivity, and transmission delays. We found that blocking intrinsic Nap currents completely abolished bursting activity, demonstrating their critical role in burst onset within the network. On the other hand, blocking different combinations of synaptic receptors revealed that spectro-temporal burst properties are uniquely associated with synaptic functionality and that excitatory connectivity is necessary for the presence of network-wide bursting. In addition to confirming the critical contribution of direct excitatory effects, mixed-effects modeling also revealed distinct combined (nonlinear) contributions of excitatory and inhibitory synaptic activity to network bursting properties.
Subject(s)
Action Potentials/physiology , Hippocampus/cytology , Nerve Net/physiology , Neurons/physiology , Action Potentials/drug effects , Animals , Biophysics , Cells, Cultured , Drug Combinations , Electric Stimulation , Embryo, Mammalian , Models, Neurological , Nerve Net/drug effects , Neurons/drug effects , Neurotransmitter Agents/pharmacology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Spectrum AnalysisABSTRACT
Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.
Subject(s)
Atlases as Topic , Receptors, Cytoplasmic and Nuclear/physiology , Signal Transduction/physiology , Animals , Datasets as Topic , Humans , Information Dissemination , InternetABSTRACT
Perinatal hypoxic-ischemic brain injury is a common problem with potentially devastating impact on neurodevelopmental outcomes. While therapeutic hypothermia, the first available treatment for this disease, reduces the risk of death or major neurodevelopmental disability, the risk of major neurologic morbidity following HI remains significant. Basic research has identified cellular mechanisms that mediate neuronal death. This article reviews the cellular processes induced that lead to brain injury following HI, and identify treatments currently under investigation for potential translation to clinical trials.
ABSTRACT
Alveolar hypoxia elicits increases in mitochondrial reactive oxygen species (ROS) signaling in pulmonary arterial (PA) smooth muscle cells (PASMCs), triggering hypoxic pulmonary vasoconstriction. Mice deficient in sirtuin (Sirt) 3, a nicotinamide adenine dinucleotide-dependent mitochondrial deacetylase, demonstrate enhanced left ventricular hypertrophy after aortic banding, whereas cells from these mice reportedly exhibit augmented hypoxia-induced ROS signaling and hypoxia-inducible factor (HIF)-1 activation. We therefore tested whether deletion of Sirt3 would augment hypoxia-induced ROS signaling in PASMCs, thereby exacerbating the development of pulmonary hypertension (PH) and right ventricular hypertrophy. In PASMCs from Sirt3 knockout (Sirt3(-/-)) mice in the C57BL/6 background, we observed that acute hypoxia (1.5% O2; 30 min)-induced changes in ROS signaling, detected using targeted redox-sensitive, ratiometric fluorescent protein sensors (roGFP) in the mitochondrial matrix, intermembrane space, and the cytosol, were indistinguishable from Sirt3(+/+) cells. Acute hypoxia-induced cytosolic calcium signaling in Sirt3(-/-) PASMCs was also indistinguishable from Sirt3(+/+) cells. During sustained hypoxia (1.5% O2; 16 h), Sirt3 deletion augmented mitochondrial matrix oxidant stress, but this did not correspond to an augmentation of intermembrane space or cytosolic oxidant signaling. Sirt3 deletion did not affect HIF-1α stabilization under normoxia, nor did it augment HIF-1α stabilization during sustained hypoxia (1.5% O2; 4 h). Sirt3(-/-) mice housed in chronic hypoxia (10% O2; 30 d) developed PH, PA wall remodeling, and right ventricular hypertrophy that was indistinguishable from Sirt3(+/+) littermates. Thus, Sirt3 deletion does not augment hypoxia-induced ROS signaling or its consequences in the cytosol of PASMCs, or the development of PH. These findings suggest that Sirt3 responses may be cell type specific, or restricted to certain genetic backgrounds.
Subject(s)
Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/metabolism , Hypoxia/complications , Hypoxia/metabolism , Sirtuin 3/deficiency , Animals , Calcium Signaling , Female , Hypertension, Pulmonary/pathology , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Muscle/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Reactive Oxygen Species/metabolism , Sirtuin 3/genetics , Sirtuin 3/physiology , Vasoconstriction/physiologyABSTRACT
The ability to adapt to acute and chronic hypoxia is critical for cellular survival. Two established functional responses to hypoxia include the regulation of gene transcription by HIF (hypoxia-inducible factor), and the constriction of pulmonary arteries in response to alveolar hypoxia. The mechanism of O2 sensing in these responses is not established, but some studies implicate hypoxia-induced mitochondrial ROS (reactive oxygen species) signalling. To further test this hypothesis, we expressed PRDX5 (peroxiredoxin-5), a H2O2 scavenger, in the IMS (mitochondrial intermembrane space), reasoning that the scavenging of ROS in that compartment should abrogate cellular responses triggered by the release of mitochondrial oxidants to the cytosol. Using adenoviral expression of IMS-PRDX5 (IMS-targeted PRDX5) in PASMCs (pulmonary artery smooth muscle cells) we show that IMS-PRDX5 inhibits hypoxia-induced oxidant signalling in the IMS and cytosol. It also inhibits HIF-1α stabilization and HIF activity in a dose-dependent manner without disrupting cellular oxygen consumption. IMS-PRDX5 expression also attenuates the increase in cytosolic [Ca(2+)] in PASMCs during hypoxia. These results extend previous work by demonstrating the importance of IMS-derived ROS signalling in both the HIF and lung vascular responses to hypoxia.
Subject(s)
Mitochondrial Membranes/enzymology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Peroxiredoxins/biosynthesis , Pulmonary Artery/enzymology , Reactive Oxygen Species/metabolism , Signal Transduction , Animals , Calcium/metabolism , Cell Hypoxia , Gene Expression Regulation, Enzymologic/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Peroxiredoxins/genetics , Pulmonary Artery/pathology , RatsABSTRACT
Pluronic F-68, an 80% hydrophilic member of the Pluronic family of polyethylene-polypropylene-polyethylene tri-block copolymers, protects non-neuronal cells from traumatic injuries and rescues hippocampal neurons from excitotoxic and oxidative insults. F-68 interacts directly with lipid membranes and restores membrane function after direct membrane damage. Here, we demonstrate the efficacy of Pluronic F-68 in rescuing rat hippocampal neurons from apoptosis after oxygen-glucose deprivation (OGD). OGD progressively decreased neuronal survival over 48 h in a severity-dependent manner, the majority of cell death occurring after 12 h after OGD. Administration of F-68 for 48 h after OGD rescued neurons from death in a dose-dependent manner. At its optimal concentration (30 µm), F-68 rescued all neurons that would have died after the first hour after OGD. This level of rescue persisted when F-68 administration was delayed 12 h after OGD. F-68 did not alter electrophysiological parameters controlling excitability, NMDA receptor-activated currents, or NMDA-induced increases in cytosolic calcium concentrations. However, F-68 treatment prevented phosphatidylserine externalization, caspase activation, loss of mitochondrial membrane potential, and BAX translocation to mitochondria, indicating that F-68 alters apoptotic mechanisms early in the intrinsic pathway of apoptosis. The profound neuronal rescue provided by F-68 after OGD and the high level of efficacy with delayed administration indicate that Pluronic copolymers may provide a novel, membrane-targeted approach to rescuing neurons after brain ischemia. The ability of membrane-active agents to block apoptosis suggests that membranes or their lipid components play prominent roles in injury-induced apoptosis.
Subject(s)
Apoptosis/drug effects , Hippocampus/cytology , Neurons/cytology , Neurons/drug effects , Polyethylene Glycols/pharmacology , Propylene Glycols/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/physiology , Caspase 1/metabolism , Caspase Inhibitors/pharmacology , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cytochromes c/metabolism , Enzyme Activation/drug effects , Enzyme Activation/physiology , Female , Fetus/cytology , Glucose/metabolism , Glucose/pharmacology , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mitochondria/metabolism , Neurons/physiology , Oxygen/metabolism , Oxygen/pharmacology , Patch-Clamp Techniques , Pregnancy , Primary Cell Culture , Rats , Rats, Sprague-Dawley , bcl-2-Associated X Protein/metabolismABSTRACT
Current efforts to understand antibiotic resistance on the whole genome scale tend to focus on known genes even as high throughput sequencing strategies uncover novel mechanisms. To identify genomic variations associated with antibiotic resistance, we employed a modified genome-wide association study; we sequenced genomic DNA from pools of E. coli clinical isolates with similar antibiotic resistance phenotypes using SOLiD technology to uncover single nucleotide polymorphisms (SNPs) unanimously conserved in each pool. The multidrug-resistant pools were genotypically similar to SMS-3-5, a previously sequenced multidrug-resistant isolate from a polluted environment. The similarity was evenly spread across the entire genome and not limited to plasmid or pathogenicity island loci. Among the pools of clinical isolates, genomic variation was concentrated adjacent to previously reported inversion and duplication differences between the SMS-3-5 isolate and the drug-susceptible laboratory strain, DH10B. SNPs that result in non-synonymous changes in gyrA (encoding the well-known S83L allele associated with fluoroquinolone resistance), mutM, ligB, and recG were unanimously conserved in every fluoroquinolone-resistant pool. Alleles of the latter three genes are tightly linked among most sequenced E. coli genomes, and had not been implicated in antibiotic resistance previously. The changes in these genes map to amino acid positions in alpha helices that are involved in DNA binding. Plasmid-encoded complementation of null strains with either allelic variant of mutM or ligB resulted in variable responses to ultraviolet light or hydrogen peroxide treatment as markers of induced DNA damage, indicating their importance in DNA metabolism and revealing a potential mechanism for fluoroquinolone resistance. Our approach uncovered evidence that additional DNA binding enzymes may contribute to fluoroquinolone resistance and further implicate environmental bacteria as a reservoir for antibiotic resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli/drug effects , Fluoroquinolones/pharmacology , Genotype , DNA, Bacterial/genetics , Escherichia coli/genetics , Microbial Sensitivity Tests , Polymorphism, Single NucleotideABSTRACT
RATIONALE: The role of reactive oxygen species (ROS) signaling in the O(2) sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses. OBJECTIVES: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA). METHODS: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC). MEASUREMENTS AND MAIN RESULTS: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca(2+)](i). In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca(2+)](i) of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice. CONCLUSIONS: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.
Subject(s)
Electron Transport Complex III/metabolism , Hypoxia/metabolism , Mitochondria/metabolism , Pulmonary Circulation , Superoxides/metabolism , Animals , Cytosol/metabolism , Disease Models, Animal , Electron Transport Complex III/genetics , Hypoxia/genetics , Lung/blood supply , Lung/metabolism , Mice , Mice, Transgenic , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/metabolism , Reactive Oxygen Species/metabolismABSTRACT
The standing outward K(+) current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. We showed that SUMOylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to SUMOylation, and the TASK (two-P domain, acid-sensitive K(+)) channel subunits K2P3 or K2P9. The heterodimeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that SUMOylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.