Proteomic analysis reveals differences between Vitis vinifera L. cv. Chardonnay and cv. Cabernet Sauvignon and their responses to water deficit and salinity

The impact of water deficit and salt stress on two important wine grape cultivars, Chardonnay and Cabernet Sauvignon, was investigated. Plants were exposed to increasing salinity and water deficit stress over a 16 d time period. Measurements of stem water potentials, and shoot and leaf lengths indicated that Chardonnay was more tolerant to these stresses than Cabernet Sauvignon. Shoot tips were harvested every 8 d for proteomic analysis using a trichloroacetic acid/acetone extraction protocol and two-dimensional gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue, quantified, and then 191 unique proteins were identified using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry. Peptide sequences were matched against both the NCBI nr and TIGR Vitis expressed sequence tag (EST) databases that had been implemented with all public Vitis sequences. Approximately 44% of the protein isoforms could be identified. Analysis of variance indicated that varietal difference was the main source of protein expression variation (40%). In stressed plants, reduction of the amount of proteins involved with photosynthesis, protein synthesis, and protein destination was correlated with the inhibition of shoot elongation. Many of the proteins up-regulated in Chardonnay were of unclassified or of unknown function, whereas proteins specifically up-regulated in Cabernet Sauvignon were involved in protein metabolism.     

Proteome analysis of grape skins during ripening

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.     

Neurotrophic effects of PACAP in the cerebellar cortex

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.     

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.     

Toxoplasma gondii triggers secretion of interleukin-12 but low level of interleukin-10 from the THP-1 human monocytic cell line

Previous studies using both in vitro and in vivo mouse models have demonstrated that a subtle balance between pro- and anti-inflammatory cytokines, among which interleukin-12 (IL-12) and interleukin-10 (IL-10), respectively is crucial to control Toxoplasma infection. However, the few studies performed with human cell lines highlighted important host-related differences in the immune response to Toxoplasma gondii. The goal of our work was thus to study the production of both IL-12 and IL-10 by the THP-1 human monocytic cell line in response to Toxoplasma. We demonstrated that infection by live parasites (RH strain) triggers secretion of IL-12, but low level of IL-10. IL-12 secretion appeared within 8 h, up to 48 h. We also showed that infection by live parasites is not mandatory since heat-killed parasites, crude tachyzoite lysate as well as excreted/secreted antigens induced significant, yet reduced production of IL-12.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).     

MareyMap: an R-based tool with graphical interface for estimating recombination rates

Comparing genetic and physical maps (the so-called Marey map approach) is still the most widely used approach to estimate genome-wide recombination rates. Remarkably, there is no available bioinformatics tool specifically devoted to Marey map approach. Here, we developed such a tool called MareyMap based on GNU R and Tcl/Tk. MareyMap offers a user-friendly graphical interface and includes useful features, such as data cleaning process, sophisticated interpolation methods to estimate local rates, possibility of complex queries, various range of import-export files. Moreover, MareyMap comes with ready-to-use maps for human, Drosophila, Caenorhabditis elegans and Arabidopsis. MareyMap has been made so that it can be easily upgraded with new data and interpolation methods. Availability: http://pbil.univ-lyon1.fr/software/mareymap/.