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1.  Increased Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Expression Mediates Intrinsic Airway Smooth Muscle Hypercontractility in Asthma 
Rationale: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood.
Objectives: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility.
Methods: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility.
Measurements and Main Results: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress–induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin.
Conclusions: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.
doi:10.1164/rccm.201107-1281OC
PMCID: PMC3402550  PMID: 22108207
asthma; airway smooth muscle; airway hyperresponsiveness; NOX4; SOD2
2.  PERK Activation at Low Glucose Concentration Is Mediated by SERCA Pump Inhibition and Confers Preemptive Cytoprotection to Pancreatic β-Cells 
Molecular Endocrinology  2010;25(2):315-326.
A decrease in the ATP/energy status of the cell in response to a decrease in glucose concentration results in SERCA pump inhibition, the efflux of Ca2+ from the ER and the activation of PERK, which plays an important role in both pancreatic β-cell function and survival.
Protein kinase R-like ER kinase (PERK) is activated at physiologically low glucose concentrations in pancreatic β-cells. However, the molecular mechanisms by which PERK is activated under these conditions and its role in β-cell function are poorly understood. In this report, we investigated, in dispersed rat islets of Langerhans and mouse insulinoma-6 (MIN6) cells, the relationship between extracellular glucose concentration, the free endoplasmic reticulum (ER) calcium concentration ([Ca2+]ER) measured directly using an ER targeted fluorescence resonance energy transfer-based calcium sensor, and the activation of PERK. We found that a decrease in glucose concentration leads to a concentration-dependent reduction in [Ca2+]ER that parallels the activation of PERK and the phosphorylation of its substrate eukaryotic initiation factor-2α. We provide evidence that this decrease in [Ca2+]ER is caused by a decrease in sarcoplasmic/ER Ca2+-ATPase pump activity mediated by a reduction in the energy status of the cell. Importantly, we also report that PERK-dependent eukaryotic initiation factor-2α phosphorylation at low glucose concentration plays a significant role in 1) the regulation of both proinsulin and global protein synthesis, 2) cell viability, and 3) conferring preemptive cytoprotection against ER stress. Taken together, these results provide evidence that a decrease in the ATP/energy status of the cell in response to a decrease in glucose concentration results in sarcoplasmic/ER Ca2+-ATPase pump inhibition, the efflux of Ca2+ from the ER, and the activation of PERK, which plays an important role in both pancreatic β-cell function and survival.
doi:10.1210/me.2010-0309
PMCID: PMC3070211  PMID: 21193559
3.  Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic β-cells 
The Journal of endocrinology  2007;192(1):179-187.
In pancreatic β-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0·5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1·5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, β-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining β-cell integrity and survival during conditions of nutritional stress.
doi:10.1677/joe.1.06898
PMCID: PMC1831533  PMID: 17210755
4.  Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic β-cells 
The Journal of Endocrinology  2007;192(1):179-187.
In pancreatic β-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0·5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1·5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, β-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining β-cell integrity and survival during conditions of nutritional stress.
doi:10.1677/joe.1.06898
PMCID: PMC1831533  PMID: 17210755
5.  Identification of Domains and Residues within the ɛ Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bɛ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation 
Molecular and Cellular Biology  2000;20(11):3965-3976.
Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bɛ). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bɛ in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bɛ (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bɛ (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bɛ without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bɛ disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bɛ alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bɛ alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bɛ and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bɛ is an activation domain that responds to eIF2B complex formation.
PMCID: PMC85753  PMID: 10805739
6.  [Ca2+]i oscillations in ASM: Relationship with persistent airflow obstruction in asthma 
Respirology (Carlton, Vic.)  2014;19(5):763-766.
The cause of airway smooth muscle (ASM) hypercontractility in asthma is not fully understood. The relationship of spontaneous intracellular calcium oscillation frequency in ASM to asthma severity was investigated. Oscillations were increased in subjects with impaired lung function abolished by extracellular calcium removal, attenuated by caffeine and unaffected by verapamil or nitrendipine. Whether modulation of increased spontaneous intracellular calcium oscillations in ASM from patients with impaired lung function represents a therapeutic target warrants further investigation.
doi:10.1111/resp.12318
PMCID: PMC4190647  PMID: 24850215
airway hyperresponsiveness; airway smooth muscle; asthma; calcium; oscillations

Results 1-6 (6)