Activation of chloride secretion in cystic fibrosis cells and tissues by the substituted imidazole SRI 2931

Recent interest in nucleotides and related agents as part of clinical trials in cystic fibrosis (CF) therapy have elicited efforts to identify novel compounds capable of activating transepithelial chloride (Cl(-)) transport in CF cells and tissues. From a library of nucleosides, bases, and other substituted heterocycles, 341 compounds were screened for their ability to activate anion transport in CF cells grown on permeable supports. One compound, SRI 2931, was found to confer prolonged and potent activity when administered to the apical surfaces of CF pancreatic epithelial cells, primary CF nasal epithelial cells, non-CF human colonic epithelial cells, and intact tissue taken from mouse models for CF. Concentrations of SRI 2931 (20 microM), which activated Cl(-) transport, had minimal effect on cell proliferation. SRI 2931 was not calcium (Ca(2+)) or cAMP dependent, suggesting important differences from conventional chloride secretagogues. The compound selectively released ATP from the apical, but not basolateral, surfaces of CF cells grown on permeable supports. The magnitude, longevity, and mechanism of action of the response provide a tool for dissecting pathways of epithelial ATP extracellular signaling and Cl(-) permeability.     

Common structural patterns in human genes

MOTIVATION: A graphical representation of the exon-intron structure of various genes, such as that presented by the National Center for Biotechnology Information Map Viewer, suggests a digital waveform or pattern that varies either in amplitude or frequency. This observation suggests that different genes may have portions of their total exon-intron structure in common. The existence of common structural patterns across unrelated genes suggests the repeated insertion of transposable elements throughout the human genome and/or a common structural function. RESULTS: We compared the exon-intron size patterns of a number of human genes and discovered numerous conserved arrangements with similarity at a high degree of stringency (>99%) across the otherwise unrelated and diverse genomic landscape. In our experimental analyses, more than 200 patterns of length 2 or greater at 99% stringency were found among the 72 genes we compared.     

Above‐ and belowground linkages in Sphagnum peatland: climate warming affects plant‐microbial interactions

Peatlands contain approximately one third of all soil organic carbon (SOC). Warming can alter above‐ and belowground linkages that regulate soil organic carbon dynamics and C‐balance in peatlands. Here we examine the multiyear impact of in situ experimental warming on the microbial food web, vegetation, and their feedbacks with soil chemistry. We provide evidence of both positive and negative impacts of warming on specific microbial functional groups, leading to destabilization of the microbial food web. We observed a strong reduction (70%) in the biomass of top‐predators (testate amoebae) in warmed plots. Such a loss caused a shortening of microbial food chains, which in turn stimulated microbial activity, leading to slight increases in levels of nutrients and labile C in water. We further show that warming altered the regulatory role of Sphagnum‐polyphenols on microbial community structure with a potential inhibition of top predators. In addition, warming caused a decrease in Sphagnum cover and an increase in vascular plant cover. Using structural equation modelling, we show that changes in the microbial food web affected the relationships between plants, soil water chemistry, and microbial communities. These results suggest that warming will destabilize C and nutrient recycling of peatlands via changes in above‐ and belowground linkages, and therefore, the microbial food web associated with mosses will feedback positively to global warming by destabilizing the carbon cycle. This study confirms that microbial food webs thus constitute a key element in the functioning of peatland ecosystems. Their study can help understand how mosses, as ecosystem engineers, tightly regulate biogeochemical cycling and climate feedback in peatlands     

Palaeoenvironment and taphonomy of a Late Jurassic (Late Tithonian) Lagerstätte from central Poland

A rich assemblage of exceptionally preserved marine and terrestrial fossils occurs in fine‐grained limestones in the upper part of the Late Tithonian (Middle Volgian) shallowing upward carbonate sequence in Central Poland. The richest horizon, a deposit known locally as the Corbulomima horizon, is named after the shallow burrowing suspension feeding bivalve Corbulomima obscura, moulds of which occur in densities of up to 500 per square metre on some bedding planes. The fauna in this bed also includes organic and phosphatic remains of a wide range of other creatures including the exuviae of limulids and decapods, disarticulated fish skeletons and rare isolated pterosaur bones and teeth. There are also perfectly preserved dragonfly wings and beetle exoskeletons. The average stable carbon and oxygen isotope values for ostracod shells and fine‐grained sediment from this horizon suggest precipitation of the calcium carbonate from warm seawater of normal marine salinity. The carbonate sediments overlying the fossiliferous horizon have been interpreted as nearshore to shoreface facies. These pass abruptly into coarse reworked intraclastic sediments interpreted as possible tsunami or storm surge over‐wash deposits. The clasts in this deposit have more positive oxygen isotope values than those in the underlying limestone, which may indicate that they were lithified in a slightly more evaporative, perhaps intertidal, setting. The succession terminates with silicified fine‐grained limestones likely to have formed in extremely shallow lagoonal environments. In contrast with the Solnhofen limestones of Lower Tithonian age in south‐central Germany the Corbulomima horizon is interpreted as a transitional deposit formed in a shallow marine setting by rapid burial with elements of both Konservat‐ and Konzentrat‐Lagerstätte preservation. □Konzentrat and Konservat‐Lagerstätte, Taphonomy, Palaeoenvironment, Paleogeography, Late Jurassic, Poland.     

Geographic and topographic determinants of local FMD transmission applied to the 2001 UK FMD epidemic

Background

Models of Foot and Mouth Disease (FMD) transmission have assumed a homogeneous landscape across which Euclidean distance is a suitable measure of the spatial dependency of transmission. This paper investigated features of the landscape and their impact on transmission during the period of predominantly local spread which followed the implementation of the national movement ban during the 2001 UK FMD epidemic. In this study 113 farms diagnosed with FMD which had a known source of infection within 3 km (cases) were matched to 188 control farms which were either uninfected or infected at a later timepoint. Cases were matched to controls by Euclidean distance to the source of infection and farm size. Intervening geographical features and connectivity between the source of infection and case and controls were compared.

Results

Road distance between holdings, access to holdings, presence of forest, elevation change between holdings and the presence of intervening roads had no impact on the risk of local FMD transmission (p > 0.2). However the presence of linear features in the form of rivers and railways acted as barriers to FMD transmission (odds ratio = 0.507, 95% CIs = 0.297,0.887, p = 0.018).

Conclusion

This paper demonstrated that although FMD spread can generally be modelled using Euclidean distance and numbers of animals on susceptible holdings, the presence of rivers and railways has an additional protective effect reducing the probability of transmission between holdings.

     

Design and evaluation of 5'-modified nucleoside analogs as prodrugs for an E. coli purine nucleoside phosphorylase mutant

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-alpha-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-alpha-L-talofuranosyl]-2-F-adenine, and 9-[alpha-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

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.