Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

The Neural/Immune Gene Ontology: clipping the Gene Ontology for neurological and immunological systems

Background  

The Gene Ontology (GO) is used to describe genes and gene products from many organisms. When used for functional annotation of microarray data, GO is often slimmed by editing so that only higher level terms remain. This practice is designed to improve the summarizing of experimental results by grouping high level terms and the statistical power of GO term enrichment analysis.     

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling

α-Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, αS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.     

N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress

Oxidative stress plays an important role in the progression of neurodegenerative and age-related diseases, causing damage to proteins, DNA, and lipids. A novel thiol N-acetylcysteine amide (AD4), the amide form of N-acetylcysteine (NAC) and a Cu(2+) chelator, was assessed for its antioxidant and protective effects using human red blood cells (RBCs) as a model. AD4 was shown by flow cytometry to inhibit tert.-butylhydroxyperoxide (BuOOH)-induced intracellular oxidation in RBCs stained with the oxidant-sensitive probe 2',7'-dichlorofluorescein diacetate. In addition, AD4 retarded BuOOH-induced thiol depletion and hemoglobin oxidation. Restoration of the thiol-depleted RBCs by externally applied AD4 was significantly greater compared with NAC and, unlike NAC, was accompanied by hemoglobin protection from oxidation. In a cell-free system we have demonstrated that AD4 reacted with oxidized glutathione (GSSG) to generate reduced glutathione (GSH). The formation of GSH was determined enzymatically using GSH peroxidase and by HPLC. Based on these results a thiol-disulfide exchange between AD4 and GSSG is proposed as the mechanism underlying the antioxidant effects of AD4 on BuOOH-treated RBCs. Together, these studies demonstrate that AD4 readily crosses cell membranes, replenishes intracellular GSH, and, by incorporating into the redox machinery, defends the cell from oxidation. These results provide further evidence for the efficient membrane permeation of AD4 over NAC, and support the possibility that it could be explored for treatment of neurodegeneration and other oxidation-mediated disorders.     

Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain

The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.     

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.