Correlation of prostaglandin-induced mitochondrial calcium release with contraction in bovine intrapulmonary vein

The effects of prostaglandins A₂, A₁, F_2α, E₂, E₁, F_1β and an analog of PGH₂ upon calcium release from mitochondria isolated from bovine intrapulmonary vein and contraction of helical strips of the same tissue were determined. The order of activity of the prostaglandins for calcium release was similar to that for contraction with the exception of the PGH₂ analog. It is suggested that prostaglandin A₂, F_2α, E₂ and A₁ induced release of mitochondrial calcium may influence the contractile state of bovine intrapulmonary vein. However, the PGH₂ analog has a subcellular mechanism other than or in addition to mitochondrial calcium release and is different from the other prostaglandins.     

Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX.

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.     

Biosynthesis of paf-acether. X. Phorbol myristate acetate-induced paf-acether biosynthesis and acetyltransferase activation in human neutrophils

We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.     

Synthesis of paf-acether from exogenous precursors by the prokaryote Escherichia coli

Paf-acether (paf) is a potent mediator of inflammatory diseases and septic shock. Using normal-phase HPLC, a paf-like activity was found in culture supernatants from E. coli. Prokaryotic paf exhibited the same biological and physico-chemical properties as eukaryotic cells and synthetic paf. Further, reverse-phase HPLC indicates that paf generated by bacteria is predominantly of the hexadecyl and octadecyl species. When cultures were supplemented with lyso-paf, a dramatic increase in paf production was observed. The purity and molecular structure of bacterial paf were further characterized by mass spectral analysis. These results could be of importance considering the pathogenetic role of enterobacteria. Further, it appears that the competence to form and release paf is an early phylogenetic development.     

Reduced number of homogalacturonan domains in pectins of an Arabidopsis mutant enhances the flexibility of the polymer

Pectins are a family of highly complex multifunctional cell wall polysaccharides. Little is known on the relation between pectin structure, hydrodynamic properties, and cellular function. In this study, we took advantage of the Arabidopsis pectin mutant quasimodo2 (qua2), which specifically lacks half of its homogalacturonan blocks, to study the relationship between the amount of homogalacturonan blocks and the hydrodynamic properties of pectins. It was first shown that, in qua2 pectins, homogalacturonans had maintained the same size as those in the wild type. The persistence lengths of isolated homogalacturonan and rhamnogalacturonan-I blocks were then measured and it was shown that homogalacturonan was over 4-fold more rigid than rhamnogalacturonan-I. WT and qua2 pectins were next compared and it appeared that the specific reduction of the number of homogalacturonan blocks leads to an increased flexibility of qua2 pectins. These results show for the first time how mutant pectins can be used to demonstrate the opposite influence of rhamnogalacturonan-I and homogalacturonan blocks on the hydrodynamic properties of pectins.     

NMR assignment of PN2-3, a tubulin interaction subdomain of the CPAP protein

We report the NMR assignment of the PN2-3 subdomain of the CPAP protein. It has been previously shown that this motif interacts with tubulin, inhibits microtubule nucleation from the centrosome and depolymerizes taxol-stabilized microtubules. Marie-Jeanne Clément and Philippe Savarin contributed equally.     

Epistatic determinism of durum wheat resistance to the wheat spindle streak mosaic virus

Key message

The resistance of durum wheat to the Wheat spindle streak mosaic virus (WSSMV) is controlled by two main QTLs on chromosomes 7A and 7B, with a huge epistatic effect.

Abstract

Wheat spindle streak mosaic virus (WSSMV) is a major disease of durum wheat in Europe and North America. Breeding WSSMV-resistant cultivars is currently the only way to control the virus since no treatment is available. This paper reports studies of the inheritance of WSSMV resistance using two related durum wheat populations obtained by crossing two elite cultivars with a WSSMV-resistant emmer cultivar. In 2012 and 2015, 354 recombinant inbred lines (RIL) were phenotyped using visual notations, ELISA and qPCR and genotyped using locus targeted capture and sequencing. This allowed us to build a consensus genetic map of 8568 markers and identify three chromosomal regions involved in WSSMV resistance. Two major regions (located on chromosomes 7A and 7B) jointly explain, on the basis of epistatic interactions, up to 43% of the phenotypic variation. Flanking sequences of our genetic markers are provided to facilitate future marker-assisted selection of WSSMV-resistant cultivars.

     

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5′UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.     

Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic pentasaccharide fragments using NMR and molecular modeling

As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.     

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.