Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.     

SAR studies of pyridazinone derivatives as novel glucan synthase inhibitors

A novel series of pyridazinone analogs has been developed as potent β-1,3-glucan synthase inhibitors through structure-activity relationship study of the lead 5-[4-(benzylsulfonyl)piperazin-1-yl]-4-morpholino-2-phenyl-pyridazin-3(2H)-one (1). The effect of changes to the core structure is described in detail. Optimization of the sulfonamide moiety led to the identification of important compounds with much improved systematic exposure while retaining good antifungal activity against the fungal strains Candida glabrata and Candida albicans.     

Organization and polymorphism of rabbit immunoglobulin heavy chain genes

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.     

Biochemical localization of the alkaline phosphatase of Bacillus licheniformis as a function of culture age.

Biochemical localization of the enzyme as a function of age of cell culture showed the alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity of Bacillus licheniformis MC14 predominantly in the particulate cell fraction in early- and mid-log cells. However, in late-log and stationary cells, increasing amounts of activity were found in the soluble fraction of lysed cells. Upon protoplast formation of these cells, the activity was released into the soluble fraction. No alkaline phosphatase activity was found in either the cytoplasmic fraction or in the cell medium during any phase of cell growth. The soluble fraction released on protoplast formation that contained alkaline phosphatase activity showed immunological cross-reactivity with antibody to the purified heat--salt-solubilized membrane alkaline phosphatase (F. M. Hulett-Cowling and L. L. Campbell, 1971). Theparticulate membrane fraction containing a firmly associated alkaline phosphatase also showed similar cross-reactivity. Further, the effectiveness of nonionic detergents, ionic detergents, bile salts, and various concentrations of magnesium and sodium as solubilizing agents for this membrane-bound alkaline phosphatase was investigated. Hexadecyl pyridinium chloride (0.03 M) and magnesium and sodium salts (above 0.2 M) were effective solubilizing agents. The substrate specificities of the various fractions were determined and compared to the substrate specificities of the purified membrane alkaline phosphatase.     

Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

Introduction

Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages.

Methods and results

Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes.

Conclusion

Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key source of inflammatory mediators in OSA.     

Effects of topical anesthesia on upper airway resistance during wake-sleep transitions.

Deformation of the upper airway (UA) by negative transmural pressure alters the activity of UA mechanoreceptors, causing a reflex increase in UA muscle activity. Topical anesthesia of the UA mucosa, which greatly reduces this reflex response, causes an increase in UA resistance during stage 2 sleep. We hypothesized that topical anesthesia of the UA mucosa would predispose to UA instability at sleep onset and, therefore, examined the effect of UA anesthesia on pharyngeal resistance (Rph) in stage 1 sleep. Eleven normal, healthy volunteers were instrumented to record standard polysomnographic variables, respiratory airflow, and UA pressure at the nasal choanae and the epiglottis. Subjects were permitted to sleep until stable stage 2 sleep was reached and were then awoken. This procedure was repeated three times to obtain reproducible wake-sleep transitions. The UA mucosa was then anesthetized with 10% lidocaine to the oropharynx and laryngopharynx, and the pharyngeal mechanics were studied during the subsequent wake-sleep transition. Three subjects were excluded because of failure to resume sleep postanesthesia. Rph was significantly higher after anesthesia during stage 1 sleep [2.88 +/- 0.77 cmH(2)O.l(-1).s (mean +/- SE)] compared with control (0.95 +/- 0.35 cmH(2)O.l(-1).s; P < 0.05), but there was no difference during wakefulness. Furthermore, there was a significant rise in Rph at wake-to-sleep transitions and a significant fall in Rph at sleep-to-wake transitions after anesthesia (P < 0.05) but not in the control condition. We conclude that sensory receptors in the UA mucosa contribute to the maintenance of UA patency at wake-sleep transition in normal humans.     

Making change last: applying the NHS institute for innovation and improvement sustainability model to healthcare improvement

The implementation of evidence-based treatments to deliver high-quality care is essential to meet the healthcare demands of aging populations. However, the sustainable application of recommended practice is difficult to achieve and variable outcomes well recognised. The NHS Institute for Innovation and Improvement Sustainability Model (SM) was designed to help healthcare teams recognise determinants of sustainability and take action to embed new practice in routine care. This article describes a formative evaluation of the application of the SM by the National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care for Northwest London (CLAHRC NWL).Data from project teams’ responses to the SM and formal reviews was used to assess acceptability of the SM and the extent to which it prompted teams to take action. Projects were classified as ‘engaged,’ ‘partially engaged’ and ‘non-engaged.’ Quarterly survey feedback data was used to explore reasons for variation in engagement. Score patterns were compared against formal review data and a ‘diversity of opinion’ measure was derived to assess response variance over time.Of the 19 teams, six were categorized as ‘engaged,’ six ‘partially engaged,’ and seven as ‘non-engaged.’ Twelve teams found the model acceptable to some extent. Diversity of opinion reduced over time. A minority of teams used the SM consistently to take action to promote sustainability but for the majority SM use was sporadic. Feedback from some team members indicates difficulty in understanding and applying the model and negative views regarding its usefulness.The SM is an important attempt to enable teams to systematically consider determinants of sustainability, provide timely data to assess progress, and prompt action to create conditions for sustained practice. Tools such as these need to be tested in healthcare settings to assess strengths and weaknesses and findings disseminated to aid development. This study indicates the SM provides a potentially useful approach to measuring teams’ views on the likelihood of sustainability and prompting action. Securing engagement of teams with the SM was challenging and redesign of elements may need to be considered. Capacity building and facilitation appears necessary for teams to effectively deploy the SM.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Design of a bovine low-density SNP array optimized for imputation

The Illumina BovineLD BeadChip was designed to support imputation to higher density genotypes in dairy and beef breeds by including single-nucleotide polymorphisms (SNPs) that had a high minor allele frequency as well as uniform spacing across the genome except at the ends of the chromosome where densities were increased. The chip also includes SNPs on the Y chromosome and mitochondrial DNA loci that are useful for determining subspecies classification and certain paternal and maternal breed lineages. The total number of SNPs was 6,909. Accuracy of imputation to Illumina BovineSNP50 genotypes using the BovineLD chip was over 97% for most dairy and beef populations. The BovineLD imputations were about 3 percentage points more accurate than those from the Illumina GoldenGate Bovine3K BeadChip across multiple populations. The improvement was greatest when neither parent was genotyped. The minor allele frequencies were similar across taurine beef and dairy breeds as was the proportion of SNPs that were polymorphic. The new BovineLD chip should facilitate low-cost genomic selection in taurine beef and dairy cattle.