Seed germination ecology of Viola calaminaria,an endangered metallophyte with a narrow distribution

Viola calaminaria is an endangered metallophyte endemic to a small area close to the border between Belgium, Germany and the Netherlands, where it grows on rock outcrops rich in heavy metals (zinc, lead and cadmium). Because V. calaminaria reproduces mainly by seeds, it is of crucial importance to understand its germination requirements. Germination percentage and speed at constant (11–25°C) and alternating (23/09°C) temperatures were investigated in five large populations. Germination percentage was positively correlated to seed weight. Germination was low (<25%) at 11 and 16°C, intermediate (around 65%) between 20 and 25°C and the highest (93%) at the alternating temperature regime (23/09°C). V. calaminaria is a slow germinator requiring 41 days on average to germinate at 23/09°C and considerably more at 20 to 25°C (105 days on average). Our results also highlighted that the species is desiccation tolerant and can therefore be safely conserved under standard seed bank conditions.     

Grapevine MATE-Type Proteins Act as Vacuolar H+-Dependent Acylated Anthocyanin Transporters

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H⁺-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.     

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS)

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.     

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance

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Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the P-glycoprotein

Morphine may affect the properties of the blood-brain barrier (BBB) by modifying the expression of certain BBB markers. We have determined the effect of chronic morphine treatment on the expression and function of some BBB markers in the rat. The mRNAs of 19 selected genes encoding caveolins, endothelial transporters, receptors and tight junctions proteins in the total RNA of isolated cortex microvessels were assayed by quantitative RT-PCR (qRT-PCR). The expression of genes Mdr1a, Mrp1, Bcrp, Glut-1 and Occludin, was slightly increased, while that of Flk-1 was decreased in microvessels from morphine-treated rats. The expression of the Mrd1a and Mdr1b genes encoding the P-glycoprotein (P-gp) also increased in the whole hippocampus and cortex of morphine-treated rats. The Mdr1a gene induction (1.38-fold) observed by qRT-PCR was also confirmed using in situ hybridization technique (1.40-fold). Immunoblotting revealed an increase in P-gp expression in the hippocampus (1.8-fold) and cortex (1.36-fold) of morphine-treated rats, but no effect in isolated microvessels. In contrast, morphine treatment increased by 1.48-fold the expression of P-gp in a large vessel-enriched fraction. The integrity of the BBB, measured by in situ brain perfusion of [(14)C]-sucrose, and the activity of P-gp at the BBB, measured with the P-gp substrate [(3)H]-colchicine, were not modified by morphine. Immunohistofluorescence experiments revealed that P-gp expression is restricted to large vessels and microvessels in control rats and that morphine treatment did not induce the expression of P-gp in the brain parenchyma (astrocytes or neurons). Taken together, our results showed that chronic morphine treatment does not significantly alter BBB integrity or P-gp activity. The impact of morphine-mediated P-gp induction observed in large vessels remains to be determined in terms of brain disposition of drugs that are P-gp substrates.     

Endocranial morphology of Palaeocene Plesiadapis tricuspidens and evolution of the early primate brain

Expansion of the brain is a key feature of primate evolution. The fossil record, although incomplete, allows a partial reconstruction of changes in primate brain size and morphology through time. Palaeogene plesiadapoids, closest relatives of Euprimates (or crown-group primates), are crucial for understanding early evolution of the primate brain. However, brain morphology of this group remains poorly documented, and major questions remain regarding the initial phase of euprimate brain evolution. Micro-CT investigation of the endocranial morphology of Plesiadapis tricuspidens from the Late Palaeocene of Europe—the most complete plesiadapoid cranium known—shows that plesiadapoids retained a very small and simple brain. Plesiadapis has midbrain exposure, and minimal encephalization and neocorticalization, making it comparable with that of stem rodents and lagomorphs. However, Plesiadapis shares a domed neocortex and downwardly shifted olfactory-bulb axis with Euprimates. If accepted phylogenetic relationships are correct, then this implies that the euprimate brain underwent drastic reorganization during the Palaeocene, and some changes in brain structure preceded brain size increase and neocortex expansion during evolution of the primate brain.     

Accumulation of Free Oligosaccharides and Tissue Damage in Cytosolic α-Mannosidase (Man2c1)-deficient Mice

Free Man_7–9GlcNAc₂ is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man₅GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man_8–9GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man_8–9GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman''s capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.     

New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl^P(O-C₆H₄-4-Cl)₂. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.