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1.  Genome-Wide Association Analysis of Blood Biomarkers in Chronic Obstructive Pulmonary Disease 
Rationale: A genome-wide association study (GWAS) for circulating chronic obstructive pulmonary disease (COPD) biomarkers could identify genetic determinants of biomarker levels and COPD susceptibility.
Objectives: To identify genetic variants of circulating protein biomarkers and novel genetic determinants of COPD.
Methods: GWAS was performed for two pneumoproteins, Clara cell secretory protein (CC16) and surfactant protein D (SP-D), and five systemic inflammatory markers (C-reactive protein, fibrinogen, IL-6, IL-8, and tumor necrosis factor-α) in 1,951 subjects with COPD. For genome-wide significant single nucleotide polymorphisms (SNPs) (P < 1 × 10−8), association with COPD susceptibility was tested in 2,939 cases with COPD and 1,380 smoking control subjects. The association of candidate SNPs with mRNA expression in induced sputum was also elucidated.
Measurements and Main Results: Genome-wide significant susceptibility loci affecting biomarker levels were found only for the two pneumoproteins. Two discrete loci affecting CC16, one region near the CC16 coding gene (SCGB1A1) on chromosome 11 and another locus approximately 25 Mb away from SCGB1A1, were identified, whereas multiple SNPs on chromosomes 6 and 16, in addition to SNPs near SFTPD, had genome-wide significant associations with SP-D levels. Several SNPs affecting circulating CC16 levels were significantly associated with sputum mRNA expression of SCGB1A1 (P = 0.009–0.03). Several SNPs highly associated with CC16 or SP-D levels were nominally associated with COPD in a collaborative GWAS (P = 0.001–0.049), although these COPD associations were not replicated in two additional cohorts.
Conclusions: Distant genetic loci and biomarker-coding genes affect circulating levels of COPD-related pneumoproteins. A subset of these protein quantitative trait loci may influence their gene expression in the lung and/or COPD susceptibility.
Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).
doi:10.1164/rccm.201206-1013OC
PMCID: PMC3622441  PMID: 23144326
biomarker; chronic obstructive pulmonary disease; genome-wide association study
2.  Probing S4 and S5 Segment Proximity in Mammalian Hyperpolarization Activated HCN Channels by Disulfide Bridging and Cd2+ Coordination 
We explored the structural basis of voltage sensing in HCN1 hyperpolarization-activated channels by examining the relative orientation of the voltage-sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd2+ bonds. As Cd2+ coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker Kv channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.
doi:10.1007/s00424-008-0613-3
PMCID: PMC2748781  PMID: 19034494
HCN clones; pacemaker; voltage gating; cysteine; cross-bridge
3.  Changes in Local S4 Environment Provide a Voltage-sensing Mechanism for Mammalian Hyperpolarization–activated HCN Channels 
The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (∼3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.
doi:10.1085/jgp.200308918
PMCID: PMC2217414  PMID: 14676285
voltage-gated K+ channels; HCN1 channel; cysteine; sulfhydryl reagents; ion channel gating
4.  A new class of bronchodilator improves lung function in COPD: a trial with GSK961081 
The European Respiratory Journal  2013;42(4):972-981.
GSK961081 is a bifunctional molecule demonstrating both muscarinic antagonist and β-agonist activities.
This was a 4-week, multicentre, randomised, double-blind, double-dummy, placebo and salmeterol controlled parallel group study. Doses ranging across three twice-daily doses and three once-daily doses were assessed in moderate and severe chronic obstructive pulmonary disease (COPD) patients. Trough forced expiratory volume in 1 s (FEV1) at day 29 was the primary end-point. At days 1 and 28, 12-h FEV1 spirometry was performed in all patients. A subset of patients underwent complete 24-h spirometry at day 28.
The study recruited 436 patients. GSK961081 showed statistically and clinically significant differences from placebo in all doses and regimens for trough FEV1 on day 29 (155–277 mL). The optimal total daily dose was 400 μg, either as 400 μg once daily or as 200 μg twice daily, with an improvement in day 29 trough FEV1 of 215 mL and 249 mL, respectively. Other efficacy end-points also showed improvement. No effects were observed on glucose, potassium, heart rate, blood pressure and no dose–response effect was seen on corrected QT elongation.
This study showed that GSK961081 is an effective bronchodilator in COPD and appeared to be safe and well tolerated.
Phase IIb study results showed the muscarinic antagonist–β-agonist GSK961081β is an effective bronchodilator in COPD http://ow.ly/lh7mU
doi:10.1183/09031936.00165712
PMCID: PMC3787816  PMID: 23429913
5.  Genetics of Sputum Gene Expression in Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(9):e24395.
Previous expression quantitative trait loci (eQTL) studies have performed genetic association studies for gene expression, but most of these studies examined lymphoblastoid cell lines from non-diseased individuals. We examined the genetics of gene expression in a relevant disease tissue from chronic obstructive pulmonary disease (COPD) patients to identify functional effects of known susceptibility genes and to find novel disease genes. By combining gene expression profiling on induced sputum samples from 131 COPD cases from the ECLIPSE Study with genomewide single nucleotide polymorphism (SNP) data, we found 4315 significant cis-eQTL SNP-probe set associations (3309 unique SNPs). The 3309 SNPs were tested for association with COPD in a genomewide association study (GWAS) dataset, which included 2940 COPD cases and 1380 controls. Adjusting for 3309 tests (p<1.5e-5), the two SNPs which were significantly associated with COPD were located in two separate genes in a known COPD locus on chromosome 15: CHRNA5 and IREB2. Detailed analysis of chromosome 15 demonstrated additional eQTLs for IREB2 mapping to that gene. eQTL SNPs for CHRNA5 mapped to multiple linkage disequilibrium (LD) bins. The eQTLs for IREB2 and CHRNA5 were not in LD. Seventy-four additional eQTL SNPs were associated with COPD at p<0.01. These were genotyped in two COPD populations, finding replicated associations with a SNP in PSORS1C1, in the HLA-C region on chromosome 6. Integrative analysis of GWAS and gene expression data from relevant tissue from diseased subjects has located potential functional variants in two known COPD genes and has identified a novel COPD susceptibility locus.
doi:10.1371/journal.pone.0024395
PMCID: PMC3174957  PMID: 21949713

Results 1-5 (5)