In‐depth protein profiling of the postsynaptic density from mouse hippocampus using data‐independent acquisition proteomics

Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion‐mobility enhanced data‐independent label‐free LC‐MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions, and PSD derived from mouse hippocampus including TOP3‐based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49 491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replicates. Identified proteins play pivotal roles in neurological and synaptic processes providing a rich resource for studies on hippocampal PSD function as well as on the pathogenesis of neuropsychiatric disorders. All MS data have been deposited in the ProteomeXchange with identifier PXD000590 ( http://proteomecentral.proteomexchange.org/dataset/PXD000590 ).     

Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.     

Molecular Mechanism for the Dual Alcohol Modulation of Cys-loop Receptors

Cys-loop receptors constitute a superfamily of pentameric ligand-gated ion channels (pLGICs), including receptors for acetylcholine, serotonin, glycine and γ-aminobutyric acid. Several bacterial homologues have been identified that are excellent models for understanding allosteric binding of alcohols and anesthetics in human Cys-loop receptors. Recently, we showed that a single point mutation on a prokaryotic homologue (GLIC) could transform it from a channel weakly potentiated by ethanol into a highly ethanol-sensitive channel. Here, we have employed molecular simulations to study ethanol binding to GLIC, and to elucidate the role of the ethanol-enhancing mutation in GLIC modulation. By performing 1-µs simulations with and without ethanol on wild-type and mutated GLIC, we observed spontaneous binding in both intra-subunit and inter-subunit transmembrane cavities. In contrast to the glycine receptor GlyR, in which we previously observed ethanol binding primarily in an inter-subunit cavity, ethanol primarily occupied an intra-subunit cavity in wild-type GLIC. However, the highly ethanol-sensitive GLIC mutation significantly enhanced ethanol binding in the inter-subunit cavity. These results demonstrate dramatic effects of the F(14′)A mutation on the distribution of ligands, and are consistent with a two-site model of pLGIC inhibition and potentiation.     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development

By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.     

Fenofibrate activates the biochemical pathways and the de novo expression of genes related to lipid handling and uncoupling protein-3 functions in liver of normal rats

Fibrates (anti-hyperlipidemic agents) enhance the mRNA expression of uncoupling protein 2 (UCP2) in the liver and that of uncoupling protein 3 (UCP3) in skeletal muscle in standard-diet-fed rats and induce a de novo expression of UCP3 (mRNA and protein) in the liver of high-fat-fed rats. Here, we report that in the liver of normal rats, fenofibrate induces a de novo expression of UCP3 and a 6-fold increase in UCP2 mRNA, whereas UCP2 protein was not detectable. Indeed, we evidenced an ORF in UCP2 exon 2 potentially able to inhibit the expression of the protein. Fenofibrate increases the expression and activity of hepatic enzymes and cofactors involved in lipid handling and UCP3 activity and, as is the case for UCP3, induces other muscle-specific genes (e.g., Carnitine palmitoyl transferase 1b and Ubiquinone biosynthesis protein COQ7 homolog). In addition, we demonstrated that in mitochondria from fenofibrate-treated rats a palmitoyl-carnitine-induced GDP-sensitive uncoupling takes place, involving UCP3 rather than other uncouplers (i.e., UCP2 and Adenine Nucleotide Translocase). Thus, the liver of fenofibrate-treated standard-diet- fed rat is a useful model for investigations of the biochemical functions of UCP3 and allowed us to demonstrate that fenofibrate programs a gene-expression pattern able to modulate lipid handling and UCP3 activation.     

A tracheomycosis as a tool for studying the impact of stem xylem dysfunction on leaf water status and gas exchange in Citrus aurantium L.

Permanent xylem blockage is a common result of attacks by herbivores and fungi. The mitosporic fungus Phoma tracheiphila (Petri) Kantschaveli et Gikachvili, is the agent of a Citrus tracheomycosis (“malsecco disease”) causing xylem impairment and leading to leaf shedding and plant dieback. In the present study, this pathogen was used for monitoring the effects of increasing levels of stem hydraulic resistance (R _stem) on leaf water status and gas exchange. In this view, measurements are reported of changes in the hydraulic resistance of infected stems (R _stem) of C. aurantium (sour orange) during progressive and irreversible xylem blockage with parallel measurements of leaf water potential and conductance to water vapour. Leaves were highly responsive to increasing R _stem as due to fungal infection, with substantial stomatal closure and drop in water potential.     

Hydraulic connections of leaves and fruit to the parent plant in Capsicum frutescens (hot pepper) during fruit ripening

Background and Aims

The hydraulic architecture and water relations of fruits and leaves of Capsicum frutescens were measured before and during the fruiting phase in order to estimate the eventual impact of xylem cavitation and embolism on the hydraulic isolation of fruits and leaves before maturation/abscission.

Methods

Measurements were performed at three different growth stages: (1) actively growing plants with some flowers before anthesis (GS1), (2) plants with about 50 % fully expanded leaves and immature fruits (GS2) and (3) plants with mature fruits and senescing basal leaves (GS3). Leaf conductance to water vapour as well as leaf and fruit water potential were measured. Hydraulic measurements were made using both the high-pressure flow meter (HPFM) and the vacuum chamber (VC) technique.

Key Results

The hydraulic architecture of hot pepper plants during the fruiting phase was clearly addressed to favour water supply to growing fruits. Hydraulic measurements revealed that leaves of GS1 plants as well as leaves and fruit peduncles of GS2 plants were free from significant xylem embolism. Substantial increases in leaf petiole and fruit peduncle resistivity were recorded in GS3 plants irrespective of the hydraulic technique used. The higher fraction of resistivity measured using the VC technique compared with the HPFM technique was apparently due to conduit embolism.

Conclusions

The present study is the first to look at the hydraulics of leaves and fruits during growth and maturation through direct, simultaneous measurements of water status and xylem efficiency of both plant regions at different hours of the day.