Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma–style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis

A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation.

Most MPN patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells we developed a novel strategy (KISMET) which utilises the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional siRNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the MAPK pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by MPL-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34-cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.Leukemia accepted article preview online, 14 October 2016. doi:10.1038/leu.2016.280.Work in the Green lab is supported by Leukemia and Lymphoma Research, Cancer Research UK, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre and the Leukemia & Lymphoma Society of America. WW is supported by the Austrian Science Foundation (J 3578-B21). CGA is supported by Kay Kendall Leukaemia Fund clinical research fellowship. UM is supported by a Cancer Research UK Clinician Scientist Fellowship. Work in the Huntly lab is supported by the European Research Council, the MRC (UK), Bloodwise, the Cambridge NIHR funded BRC, KKLF and a WT/MRC Stem Cell centre grant. Work in the Green and Huntly Labs is supported by core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140/z/12/z) and Wellcome Trust-MRC Cambridge Stem Cell Institute (097922/Z/11/Z)

Major agricultural changes required to mitigate phosphorus losses under climate change

Phosphorus losses from land to water will be impacted by climate change and land management for food production, with detrimental impacts on aquatic ecosystems. Here we use a unique combination of methods to evaluate the impact of projected climate change on future phosphorus transfers, and to assess what scale of agricultural change would be needed to mitigate these transfers. We combine novel high-frequency phosphorus flux data from three representative catchments across the UK, a new high-spatial resolution climate model, uncertainty estimates from an ensemble of future climate simulations, two phosphorus transfer models of contrasting complexity and a simplified representation of the potential intensification of agriculture based on expert elicitation from land managers. We show that the effect of climate change on average winter phosphorus loads (predicted increase up to 30% by 2050s) will be limited only by large-scale agricultural changes (e.g., 20–80% reduction in phosphorus inputs)

Theoretical description of hydrogen bonding in oxalic acid dimer and trimer based on the combined extended-transition-state energy decomposition analysis and natural orbitals for chemical valence (ETS-NOCV)

In the present study we have analyzed hydrogen bonding in dimer and trimer of oxalic acid, based on a recently proposed charge and energy decomposition scheme (ETS-NOCV). In the case of a dimer, two conformations, α and β, were considered. The deformation density contributions originating from NOCV’s revealed that the formation of hydrogen bonding is associated with the electronic charge deformation in both the σ—(Δρσ) and π-networks (Δρπ). It was demonstrated that σ-donation is realized by electron transfer from the lone pair of oxygen on one monomer into the empty \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho_{H - O}^*$$\end{document} orbital of the second oxalic acid fragment. In addition, a covalent contribution is observed by the density transfer from hydrogen of H-O group in one oxalic acid monomer to the oxygen atom of the second fragment. The resonance assisted component (Δρπ), is based on the transfer of electron density from the π—orbital localized on the oxygen of OH on one oxalic acid monomer to the oxygen atom of the other fragment. ETS-NOCV allowed to conclude that the σ(O---HO) component is roughly eight times as important as π (RAHB) contribution in terms of energetic estimation. The electrostatic factor (ΔEelstat) is equally as important as orbital interaction term (ΔEorb). Finally, comparing β-dimer of oxalic acid with trimer we found practically no difference concerning each of the O---HO bonds, neither qualitative nor quantitative

Water safety plans and climate change mitigation

[Excerpt] Definition Quality water at affordable prices for all is a key condition for the promotion of public health, environmental sustainability, and quality and safety of life. In a context of growing external uncertainties arising from changes in the climate and the environment, ensuring these conditions is an upward concern and is of utmost relevance to increase scientific research on the impacts of climate change on water quality modification and in minimization/mitigation strategies

Calnexin is necessary for T cell transmigration into the central nervous system

In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) and induces Ca²⁺release from endoplasmic reticulum (ER) stores.</p> <p>Methods</p> <p>In the present study, we monitored the cytosolic Ca²⁺transients using the UV light photolysis technique to uncage caged Ca²⁺or caged IP₃into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca²⁺receptors present in the ER, and measured their Ca²⁺dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).</p> <p>Results</p> <p>We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP₃R) dependent-Ca²⁺response following both correcting treatments compared to uncorrected cells in such a way that Ca²⁺responses (CF+treatment <it>vs </it>wild-type cells) were normalized. This normalization of the Ca²⁺rate does not affect the activity of Ca²⁺-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP₃R1, we observed a decrease of the implication of IP₃R1 in the Ca²⁺response in CF corrected cells. We observed a similar Ca²⁺mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP₃R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.</p> <p>Conclusion</p> <p>These results suggest reversal of the IP₃R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP₃R Ca²⁺release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.</p