Intercomparison of spectroradiometers and Sun photometers for the determination of the aerosol optical depth during the VELETA-2002 field campaign

[ 1] In July 2002 the VELETA-2002 field campaign was held in Sierra Nevada ( Granada) in the south of Spain. The main objectives of this field campaign were the study of the influence of elevation and atmospheric aerosols on measured UV radiation. In the first stage of the field campaign, a common calibration and intercomparison between Licor-1800 spectroradiometers and Cimel-318 Sun photometers was performed in order to assess the quality of the measurements from the whole campaign. The intercomparison of the Licor spectroradiometers showed, for both direct and global irradiances, that when the comparisons were restricted to the visible part of the spectrum the deviations were within the instruments' nominal accuracies which allows us to rely on these instruments for measuring physical properties of aerosols at the different measurement stations. A simultaneous calibration on AOD data was performed for the Cimel-318 Sun photometers. When a common calibration and methodology was applied, the deviation was lowered to much less than 0.01 for AOD. At the same time an intercomparison has been made between the AOD values given by the spectroradiometers and the Sun photometers, with deviations obtained from 0.01 to 0.03 for the AOD in the visible range, depending on the channel. In the UVA range, the AOD uncertainty was estimated to be around 0.02 and 0.05 for Cimel and Licor respectively. In general the experimental differences were in agreement with this uncertainty estimation. In the UVB range the AOD measurements should not be used due to maximum instrumental uncertainties

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis

An Amphipathic Alpha-Helix in the Prodomain of Cocaine and Amphetamine Regulated Transcript Peptide Precursor Serves as Its Sorting Signal to the Regulated Secretory Pathway

<div><p>Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1–41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. <i>In silico</i> modeling predicted an alpha-helix structure located between residues 24–37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCART_L34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCART_L34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.</p> </div

proCART-EGFPm and proCART_L34F-EGFPm colocalize with SgII-ir.

<p>(A) Schema depicting all CART-EGFPm fusion proteins used in the study. Positions of amino acid residues from proCART_1–89 fragments are indicated by the numbers alongside each fusion protein. (B) Fusion proteins were expressed in PC12 cells and cell lysates were analyzed by western blotting with anti-GFP antibody. Confocal images of PC12 cells transfected with proCART-EGFPm (C) or proCART_L34F-EGFPm (D). Aldehyde-fixed cells were subjected to immunofluorescence protocols using SgII antibody. (C y D) Left panel: EGFPm signal. Middle: SgII-ir signal. Right panel: Merge of both signals. Zoom from merge image showing spots with both signals. Scale bar: 2 μm; zoom: 0.25 μm.</p

proCART and proCART_L34F, but not proCART_MISS colocalized with NPY-DsRed.

<p>proCART species without EGFP were cotransfected in PC12 cells with NPY-DsREd expressing vector and detected with an antiCART specific polyclonal rabbit antibody. (A) proCART and (B) proCART_L34F showed a punctate pattern and both colocalized with NPY-DsRed. However, (C) proCART_MISS showed a perinuclear pattern and did not colocalize with NPY-DsRed. (D) RT-PCR for CART mRNA in rat primary cortical neurons, PC12 cells and rat midbrain at the level of the Edinger-Westphal nucleus. Total RNA was extracted with Trizol and 0.5 µg of each RNA was used for each RT-PCR. A set of specific primers (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059695#pone.0059695.s004" target="_blank">Table S1</a>) to amplify a 322 bp fragment of pre-proCART mRNA were used. Cyclophilin (Cyc) mRNA was also amplified as a control.</p

proCART-EGFPm and proCART_L34F-EGFPm are released upon barium stimulation, and proCART_MISS-EGFPm is secreted constitutively.

<p>(A) Basal secretion. The 48 h incubation media of 3 independent samples for proCART-EGFPm, proCART_L34F-EGFPm and proCART_MISS-EGFPm were immunoblotted with an anti-GFP antibody to determine the secretion in the absence of stimulus. Twenty μg of each cell lysate were immunoblotted with anti-GFP and anti-GAPDH antibodies. Secreted levels were plot as percentage of fusion protein released/total fusion protein expressed. (*P<0.0001; One-way ANOVA followed by Dunnet post-hoc test). (B) Stimulated secretion. PC12 cells transfected with either proCART-EGFPm, proCART_L34F-EGFPm or proCART_MISS-EGFPm and 48 h after, cells were incubated for 30 min in basal medium (− lanes) and thereafter for additional 30 min in 2 mM Ba²⁺-medium (+ lanes). proCART-EGFPm, proCART_L34F-EGFPm and proCART_MISS-EGFPm secreted in the respective media were analyzed by western blotting with anti-GFP antibody. In all cases immunoblots were also revealed with anti-SgII antibody to control for the presence of the normal response to barium-stimulation.</p

Subcellular distribution of CART-EGFPm fusion proteins.

<p>Confocal images of PC12 cells transfected with SigP-EGFPm (A), proCART_1–9-EGFPm (B), proCART_1–26-EGFPm (C), proCART_1–41-EGFPm (D) or proCART_MISS-EGFPm (E) expression vectors. Aldehyde-fixed cells were subjected to immunofluorescence protocols using SgII antibody. (A–E) Left panel: EGFPm signal. Middle: SgII-ir signal. Right panel: Merging of both signals. (F) Pearson's coefficient values for the colocalization of each of the fusion protein with SgII-ir (* P<0.001, One-way ANOVA followed by Dunnet's post-hoc test).</p

Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity

The release of protons (H) occurs via the Na/H exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1–dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 μmol/L), SR11302 (1 μmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 μmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 μmol/L, NHE3 inhibitor), concanamycin A (0.1 μmol/L, V-ATPases inhibitor), and Schering (10 μmol/L, H/K-ATPase inhibitor). [H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NHCl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (βi) and H flux (J) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), J (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1–dependent NHE1 activation in MDCK cells