Uncoupling of Elastin Complex Receptor during In Vitro Aging Is Related to Modifications in Its Intrinsic Sialidase Activity and the Subsequent Lactosylceramide Production

Degradation of elastin leads to the production of elastin-derived peptides (EDP), which exhibit several biological effects, such as cell proliferation or protease secretion. Binding of EDP on the elastin receptor complex (ERC) triggers lactosylceramide (LacCer) production and ERK1/2 activation following ERC Neu-1 subunit activation. The ability for ERC to transduce signals is lost during aging, but the mechanism involved is still unknown. In this study, we characterized an in vitro model of aging by subculturing human dermal fibroblasts. This model was used to understand the loss of EDP biological activities during aging. Our results show that ERC uncoupling does not rely on Neu-1 or PPCA mRNA or protein level changes. Furthermore, we observe that the membrane targeting of these subunits is not affected with aging. However, we evidence that Neu-1 activity and LacCer production are altered. Basal Neu-1 catalytic activity is strongly increased in aged cells. Consequently, EDP fail to promote Neu-1 catalytic activity and LacCer production in these cells. In conclusion, we propose, for the first time, an explanation for ERC uncoupling based on the age-related alterations of Neu-1 activity and LacCer production that may explain the loss of EDP-mediated effects occurring during aging.     

The Impact of the Invasive Alien Plant,Impatiens glandulifera,on Pollen Transfer Networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward.     

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.     

The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A–sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.     

Iron absorption and distribution in TNFΔARE/+ mice,a model of chronic inflammation

Hemizygous TNF^ΔARE/+ mice are a murine model for chronic inflammation. We utilized these animals to study iron-kinetics and corresponding protein expression in an iron-deficient and iron-adequate setting. ⁵⁹Fe-absorption was determined in ligated duodenal loops in vivo. Whole body distribution of i.v. injected ⁵⁹Fe was analysed, and the organ specific expression of ferroportin, transferrin receptor-1, hepcidin and duodenal DMT-1 was quantified by real-time PCR and Western blotting.Duodenal ⁵⁹Fe-lumen-to-body transport was not affected by the genotype. Duodenal ⁵⁹Fe-retention was increased in TNF^ΔARE/+ mice, suggesting higher ⁵⁹Fe-losses with defoliated enterocytes. Iron-deficiency increased duodenal ⁵⁹Fe-lumen-to-body transport, and higher duodenal ⁵⁹Fe-tissue retention went along with higher duodenal DMT-1, ferroportin, and liver hepcidin expression. TNF^ΔARE/+ mice significantly increase their ⁵⁹Fe-content in inflamed joints and ilea, and correspondingly reduce splenic ⁵⁹Fe-content. Leukocyte infiltrations in the joints suggest a substantial shift of iron-loaded RES cells to inflamed tissues as the underlying mechanism. This finding was paralleled by increased non-haem iron content in joints and reduced haemoglobin and haematocrit concentrations in TNF^ΔARE/+ mice.In conclusion, erythropoiesis in inflamed TNF^ΔARE/+ mice could be iron-limited due to losses with exfoliated iron-loaded enterocytes and/or to increased iron-retention in RES cells that shift from the spleen to inflamed tissues.     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process

Background

Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.

Results

The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native GPD1 promoter in the yeast genome by previously generated well-characterized TEF promoter mutant versions in a gpd2 Δ background. Two TEF promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as TEFmut7 and TEFmut2. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in TEFmut7 and 88% in TEFmut2. The overall ethanol production yield on glucose was improved from 0.43 g g^-1 in the wild type to 0.44 g g^-1 measured in TEFmut7 and 0.45 g g^-1 in TEFmut2. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for TEFmut7 and TEFmut2 respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L^-1), growth-inhibiting ethanol concentration (87 ± 3 g L^-1) and volumetric ethanol productivity (2.1 ± 0.15 g l^-1 h^-1) measured in wild-type remained virtually unchanged in the engineered strains.

Conclusions

This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" GPD1 expression in a gpd2 Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains gpd1 Δ or gpd2 Δ. Although glycerol reduction is known to be even higher in a gpd1 Δ gpd2 Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.     

Steady-state mycophenolate mofetil pharmacokinetic parameters enable prediction of systemic lupus erythematosus clinical flares: an observational cohort study

Introduction  

The aim of this study was to determine whether mycophenolate mofetil (MMF) pharmacokinetics (PK) under combined MMF and prednisone remission-maintenance therapy can predict systemic lupus erythematosus (SLE) clinical flares.