cAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.     

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation

Background  

High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts.     

Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Background

Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray? genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.

Results

Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.

Conclusions

We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.

     

Olfactory sensitivity in tsetse flies: a daily rhythm

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U- shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.      

AgdbNet – antigen sequence database software for bacterial typing

Background  

Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences.     

Hypochlorous acid alters bronchial epithelial cell membrane properties and prevention by extracellular glutathione.

In chronic inflammatory diseases of the airways, such as cystic fibrosis, hypochlorous acid (HOCl) generated by neutrophils is involved in airway injury. We examined the effects of HOCl on 16HBE14o- bronchial epithelial cells by bolus addition or by generation with glucose oxidase plus myeloperoxidase. HOCl produced both carbonyl formation of a discreet number of proteins and modification of surface targets that were recognized by an antibody raised against HOCl-modified protein. Bolus or enzymatically generated HOCl decreased transepithelial resistance, but surprisingly bolus HOCl also increased short-circuit current. Glutathione in lung epithelial lining fluid is an excellent scavenger of HOCl; however, glutathione content is lower in cystic fibrosis epithelial lining fluid due to deficient glutathione transport to the apical side of bronchial-tracheal epithelial cells (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). We found that alteration of the GSH content of apical fluid above 16HBE14o- cells was protective because all HOCl-induced changes were delayed or eliminated by exogenous glutathione within the physiological range. Extrapolating this to cystic fibrosis suggests that HOCl can alter cell function without destruction but that elevating glutathione could be protective.