Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Dynamics of transportan in bicelles is surface charge dependent

In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide ¹⁵N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S ² is on average 0.11 ± 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of ¹H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s10858-006-9008-y and is accessible for authorized users.     

The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death

Patients affected by Sj?gren's syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sj?gren's syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sj?gren's syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.     

Environmental Assessment of Freight Transportation in the U.S. (11 pp)

Goal, Scope and Background This study provides a life cycle inventory of air emissions (CO2, NOx, PM10, and CO) associated with the transportation of goods by road, rail, and air in the U.S. It includes the manufacturing, use, maintenance, and end-of-life of vehicles, the construction, operation, maintenance, and end-of-life of transportation infrastructure, as well as oil exploration, fuel refining, and fuel distribution. Methods The comparison is performed using hybrid life cycle assessment (LCA), a combination of process-based LCA and economic input-output analysis-based LCA (EIO-LCA). All these components are added by means of a common functional unit of grams of air pollutant per ton-mile of freight activity. Results and Discussion Results show that the vehicle use phase is responsible for approximately 70% of total emissions of CO2 for all three modes. This confirms that tailpipe emissions underestimate total emissions of freight transportation as infrastructure, pre-combustion, as well as vehicle manufacturing and end-of-life account for a sizeable share of total emissions. Differences between tailpipe emissions and total system wide emissions can range from only 4% for road transportation's CO emissions to an almost ten-fold difference for air transportation's PM10 emissions. Conclusion Rail freight has the lowest associated air emissions, followed by road and air transportation. Depending on the pollutant, rail is 50-94% less polluting than road. Air transportation is rated the least efficient in terms of air emissions, partly due to the fact that it carries low weight cargo. It emits 35 times more CO2 than rail and 18 times more than road transportation on a ton-mile basis. It is important to consider infrastructure, vehicle manufacturing, and pre-combustion processes, whose life-cycle share is likely to increase as new tailpipe emission standards are enforced. Recommendation and Outlook Emission factors, fuel efficiency, and equipment utilization contribute the most to uncertainty in the results. Further studies are necessary to address all variables that influence these parameters, such as road grade, vehicle speed, and vehicle weight. A focus on regional variation, end-of-life processes, fuel refining processes, terminals, as well as more accurate infrastructure allocation between freight and passenger transportation would strengthen the model.     

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista)

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.     

Quantitative trait loci for grain moisture at harvest and field grain drying rate in maize (Zea mays, L.)

Hybrids with low grain moisture (GM) at harvest are specially required in mid- to short-season environments. One of the most important factors determining this trait is field grain drying rate (FDR). To produce hybrids with low GM at harvest, inbred lines can be obtained through selection for either GM or FDR. Thus, a single-cross population (181 F _2:3-generation plants) of two divergent inbred lines was evaluated to locate QTL affecting GM at harvest and FDR as a starting point for marker assisted selection (MAS). Moisture measurements were made with a hand-held moisture meter. Detection of QTL was facilitated with interval mapping in one and two dimensions including an interaction term, and a genetic linkage map of 122 SSR loci covering 1,557.8 cM. The markers were arranged in ten linkage groups. QTL mapping was made for the mean trait performance of the F _2:3 population across years. Ten QTL and an interaction were associated with GM. These QTL accounted for 54.8 and 65.2% of the phenotypic and genotypic variation, respectively. Eight QTL and two interactions were associated with FDR accounting for 35.7 and 45.2% of the phenotypic and genotypic variation, respectively. Two regions were in common between traits. The interaction between QTL for GM at harvest had practical implications for MAS. We conclude that MAS per se will not be an efficient method for reducing GM at harvest and/or increasing FDR. A selection index including both molecular marker information and phenotypic values, each appropriately weighted, would be the best selection strategy.