R-domain interactions with distal regions of CFTR lead to phosphorylation and activation

Cystic fibrosis is caused by the aberrant function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. We examined whether intramolecular binding interactions involving the regulatory (R) domain contributed to CFTR regulation and function. When the R-domain (amino acids 596-836) was coexpressed with Delta1-836 CFTR (a carboxyl hemi-CFTR beginning immediately after the R-domain), strong binding between the two polypeptides was exhibited. The R-domain that co-immunoprecipitated with Delta1-836 exhibited a slower mobility on SDS-PAGE that resulted from phosphorylation of the protein. A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with Delta1-836. Moreover, coexpression of M837X and Delta1-836 led to enhanced halide permeability in living cells. The activity, unlike in full-length CFTR, was present without forskolin activation, but still sensitive to the PKA inhibitor, Rp-8-CPT-cAMPS. This PKA inhibition of activity was found to be dependent on the carboxy region of the R-domain, amino acids 723-836. Our results indicate that the R-domain binds CFTR residues after amino acid 836 and that this binding facilitates phosphorylation and CFTR activation. We have also characterized a subdomain within CFTR (residues 723-837) that is necessary for PKA-dependent constitutive activation. Finally, these experiments demonstrate that constitutive CFTR activity can be accomplished by at least two mechanisms: (1) direct modulation of the R-domain to abrogate PKA regulation and (2) modifications that increase R-domain susceptibility to steady-state phosphorylation through PKA.     

Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway

ATP and itsmetabolites stimulate Clsecretion in human epithelium in vitro and in vivo. The specificpurinergic receptor subtypes that govern these effects have beendifficult to separate, in part due to multiple parallel pathways forCl secretion in respiratoryand intestinal epithelia. In a simplified model using COS-7 cells, wedemonstrate acquisition of an ATP-, ADP-, AMP-, and adenosine(ADO)-regulated halide permeability specifically following expressionof wild-type (wt) cystic fibrosis transmembrane conductance regulator(CFTR). This halide permeability is blocked by theP₁ purinergic receptor antagonist8-phenyl theophylline, sensitive to the protein kinase A inhibitorH-89, and associated with a modest, dose-dependent increase in cellularcAMP concentration. Phorbol esters poorly activate halide permeabilitycompared with ADO, and ADO-stimulated efflux was not affected bytreatment with the protein kinase C inhibitor bisindolylmaleimide I. The A₂ ADO receptor (AR) agonists5'-N-ethylcarboxamide adenosineand ADO were strong activators, whereas theA₁ AR agonistR-phenylisopropyladenosine failed toactivate halide permeability. Metabolic conversion of ADO nucleotidesby surface ecto-5'-nucleotidase to more active (lessphosphorylated) forms contributes to anion transport activation inthese cells. Immunoprecipitation withanti-A_2B AR antibody identified a31-kDa protein in both COS-7 and human bronchial epithelial cells.Together, these findings indicate that ADO and its nucleotides arecapable of activating wtCFTR-dependent halide permeability throughA_2B AR and that this AR subtype ispresent in human bronchial epithelium. We also present data showingthat this pathway can activate clinically significant mutant CFTRmolecules such as R117H.     

The predictive value of transcranial duplex sonography for the clinical diagnosis in undiagnosed parkinsonian syndromes: comparison with SPECT scans

Background  

Transcranial duplex sonography (TCD) of the substantia nigra has emerged as a promising, non-invasive tool to diagnose idiopathic Parkinson's disease (IPD). However, its diagnostic accuracy in patients with undefined parkinsonism remains to be determined.     

Fertilization effects with P on accumulated leaf area duration,biomass and yield of three cultivars of maize in Toluca,Mexico

The effect of six phosphorus levels (0, 40, 80, 120, 160 and 200 kg/ha) on the duration of cumulative leaf area, biomass and agronomic yield was determined in the maize cultivars: Amarillo Almoloya, Cacahuacintle and Condor in 2010 and 2011. Such cultivars were sown in the Cerrillo Piedras Blancas Mexico. A completely randomized complete block design with factorial arrangement was utilized. High phosphorus levels (120, 160 and 200 kg/ha) positively affected the duration of cumulative leaf area; greatest values were obtained in Cacahuacintle. A greater duration of accumulated leaf area contributes to determine high values of biomass accumulation and grain yield in this cultivar. Leaf area duration appeared to be a useful tool for evaluating different genotypes in a given environment.     

Dexamethasone induces gelsolin synthesis and altered morphology in L929 cells

When L929 cells are exposed to 5 μg/ml dexamethasone, synthesis of a 90,000 M(r) polypeptide is induced within 12 h. Flattening of the cells begins at about this time and progresses to become quite prominent after 48 h of exposure. Two-dimensional PAGE and partial proteolytic fingerprints identify the 90,000 M(r) polypeptide as gelsolin, a Ca(++)-dependent inhibitor of actin polymerization. Thus, this system provides evidence that gelsolin may have a role in regulating cell shape in response to physiological agents such as glucocorticoids.     

Potential for epidemic take-off from the primary outbreak farm via livestock movements

Background

We consider the potential for infection to spread in a farm population from the primary outbreak farm via livestock movements prior to disease detection. We analyse how this depends on the time of the year infection occurs, the species transmitting, the length of infectious period on the primary outbreak farm, location of the primary outbreak, and whether a livestock market becomes involved. We consider short infectious periods of 1 week, 2 weeks and 4 weeks, characteristic of acute contagious livestock diseases. The analysis is based on farms in Scotland from 1 January 2003 to 31 July 2007.

Results

The proportion of primary outbreaks from which an acute contagious disease would spread via movement of livestock is generally low, but exhibits distinct annual cyclicity with peaks in May and August. The distance that livestock are moved varies similarly: at the time of the year when the potential for spread via movements is highest, the geographical spread via movements is largest. The seasonal patterns for cattle differ from those for sheep whilst there is no obvious seasonality for pigs. When spread via movements does occur, there is a high risk of infection reaching a livestock market; infection of markets can amplify disease spread. The proportion of primary outbreaks that would spread infection via livestock movements varies significantly between geographical regions.

Conclusions

In this paper we introduce a set-up for analysis of movement data that allows for a generalized assessment of the risk associated with infection spreading from a primary outbreak farm via livestock movements, applying this to Scotland, we assess how this risk depends upon the time of the year, species transmitting, location of the farm and other factors.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.