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In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Background Alzheimer’s disease (AD) is the most common form of dementia, the number of affected individuals is rising, with significant impacts for healthcare systems. Current symptomatic treatments delay, but do not halt, disease progression. Genetic evidence points to aggregation and deposition of amyloid-β (Aβ) in the brain being causal for the neurodegeneration and dementia typical of AD. Approaches to target Aβ via inhibition of γ-secretase or passive antibody therapy have not yet resulted in substantial clinical benefits. Inhibition of BACE1 (β-secretase) has proven a challenging concept, but recent BACE1inhibitors can enter the brain sufficiently well to lower Aβ. However, failures with the first clinical BACE1 inhibitors have highlighted the need to generate compounds with appropriate efficacy and safety profiles, since long treatment periods are expected to be necessary in humans. Results Treatment with NB-360 , a potent and brain penetrable BACE-1 inhibitor can completely block the progression of Aβ deposition in the brains of APP transgenic mice, a model for amyloid pathology. We furthermore show that almost complete reduction of Aβ was achieved also in rats and in dogs, suggesting that these findings are translational across species and can be extrapolated to humans. Amyloid pathology may be an initial step in a complex pathological cascade; therefore we investigated the effect of BACE-1 inhibition on neuroinflammation, a prominent downstream feature of the disease. NB-360 stopped accumulation of activated inflammatory cells in the brains of APP transgenic mice. Upon chronic treatment of APP transgenic mice, patches of grey hairs appeared. Conclusions In a rapidly developing field, the data on NB-360 broaden the chemical space and expand knowledge on the properties that are needed to make a BACE-1 inhibitor potent and safe enough for long-term use in patients. Due to its excellent brain penetration, reasonable oral doses of NB-360 were sufficient to completely block amyloid-β deposition in an APP transgenic mouse model. Data across species suggest similar treatment effects can possibly be achieved in humans. The reduced neuroinflammation upon amyloid reduction by NB-360 treatment supports the notion that targeting amyloid-β pathology can have beneficial downstream effects on the progression of Alzheimer’s disease.

Brominated flame retardants (BFRs) are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA) and decabromodiphenyl ether (DBPE), all are cytotoxic at low micromolar concentrations (LC(50) being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively). They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+)] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+)] levels appear to occur through a mechanism involving microsomal Ca(2+)-ATPase inhibition and this maybe responsible for Ca(2+)-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42) processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is synthesized principally in the stomach and is released in response to fasting. Ghrelin is structurally related to motilin and, together, they represent a novel family of gut–brain regulatory peptides. In addition to having a powerful effect on the secretion of growth hormone, ghrelin stimulates energy production and signals directly to the hypothalamic regulatory nuclei that control energy homeostasis. The study of ghrelin has extended our understanding of how growth is controlled, and has shown that the stomach is an important component of this system. Key Points The identification of growth hormone secretagogues led to the discovery of a new receptor with homology to the motilin receptor. Subsequent studies led to the identification of its endogenous ligand — ghrelin. Ghrelin can indeed stimulate the release of growth hormone from the pituitary in a way that is independent of the action of growth-hormone-releasing hormone. In addition to its effect on the release of growth hormone, ghrelin is an important regulator of food intake. It is released from the stomach in response to fasting, and increases feeding behaviour by acting on the arcuate nucleus of the hypothalamus. The action of ghrelin is opposite to that of another important regulator of food intake — leptin. Leptin is released from adipose tissue, and its plasma levels decrease in response to fasting. This molecule also acts on the arcuate nucleus of the hypothalamus, where it has an anorexigenic effect. The discovery of ghrelin has several clinical implications. It can be used to stimulate the release of growth hormone in cases of human deficiency, by acting on the endogenous oscillators that control pulsatile hormone release. It can also be used for the regulation of body weight by stimulating food intake, particularly in conditions accompanied by cachexia, such as cancer and AIDS.

The proglucagon-derived peptide family consists of three highly related peptides, glucagon and the glucagon-like peptides GLP-1 and GLP-2. Although the biological activity of glucagon as a counter-regulatory hormone has been known for almost a century, studies conducted over the past decade have now also elucidated important roles for GLP-1 as an antidiabetic hormone, and for GLP-2 as a stimulator of intestinal growth. In contrast to pancreatic glucagon, the GLPs are synthesized in the intestinal epithelial L cells, where they are subject to the influences of luminal nutrients, as well as to a variety of neuroendocrine inputs. In this review, we will focus on the complex integrative mechanisms that regulate the secretion of these peptides from L cells, including both direct and indirect regulation by ingested nutrients.Key words: GLP-1, GLP-2, intestine, secretion, nutrients, neural.

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer’s disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer’s disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

The increased accumulation of iron in the brain in Alzheimer’s disease (AD) is well documented, and excess iron is strongly implicated in the pathogenesis of the disease. The adverse effects of accumulated iron in AD brain may include the oxidative stress, altered amyloid beta-metabolism and the augmented toxicity of metal-bound amyloid beta 42. In this study, we have shown that exogenously added iron in the form of ferric ammonium citrate (FAC) leads to considerable accumulation of amyloid precursor protein (APP) without a corresponding change in the concerned gene expression in cultured SHSY5Y cells during exposure up to 48 h. This phenomenon is also associated with increased β-secretase activity and augmented release of amyloid beta 42 in the medium. Further, the increase in β-secretase activity, in SHSY5Y cells, upon exposure to iron apparently involves reactive oxygen species (ROS) and NF-κB activation. The synthetic flavone negletein (5,6-dihydroxy-7-methoxyflavone), which is a known chelator for iron, can significantly prevent the effects of FAC on APP metabolism in SHSY5Y cells. Further, this compound inhibits the iron-dependent formation of ROS and also blocks the iron-induced oligomerization of amyloid beta 42 in vitro. In concentrations used in this study, negletein alone appears to have only marginal toxic effects on cell viability, but, on the other hand, the drug is capable of ameliorating the iron-induced loss of cell viability considerably. Our results provide the initial evidence of potential therapeutic effects of negletein, which should be explored in suitable animal models of AD.

Activation of TGR5 via bile acids or bile acid analogs leads to the release of glucagon-like peptide-1 (GLP-1) from intestine, increases energy expenditure in brown adipose tissue, and increases gallbladder filling with bile. Here, we present compound 18, a non-bile acid agonist of TGR5 that demonstrates robust GLP-1 secretion in a mouse enteroendocrine cell line yet weak GLP-1 secretion in a human enteroendocrine cell line. Acute administration of compound 18 to mice increased GLP-1 and peptide YY (PYY) secretion, leading to a lowering of the glucose excursion in an oral glucose tolerance test (OGTT), while chronic administration led to weight loss. In addition, compound 18 showed a dose-dependent increase in gallbladder filling. Lastly, compound 18 failed to show similar pharmacological effects on GLP-1, PYY, and gallbladder filling in Tgr5 knockout mice. Together, these results demonstrate that compound 18 is a mouse-selective TGR5 agonist that induces GLP-1 and PYY secretion, and lowers the glucose excursion in an OGTT, but only at doses that simultaneously induce gallbladder filling. Overall, these data highlight the benefits and potential risks of using TGR5 agonists to treat diabetes and metabolic diseases.

Obestatin Promotes Survival of Pancreatic β-Cells and Human Islets and Induces Expression of Genes Involved in the Regulation of β-Cell Mass and Function Riccarda Granata 1 2 , Fabio Settanni 1 2 , Davide Gallo 1 2 , Letizia Trovato 1 2 , Luigi Biancone 2 , Vincenzo Cantaluppi 2 , Rita Nano 3 , Marta Annunziata 1 2 , Pietro Campiglia 4 , Elisa Arnoletti 5 , Corrado Ghè 5 , Marco Volante 6 , Mauro Papotti 6 , Giampiero Muccioli 5 and Ezio Ghigo 2 1 Laboratory of Molecular and Cellular Endocrinology, Department of Internal Medicine, University of Turin, Turin, Italy 2 Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Turin, Italy 3 Department of Medicine, Transplant Unit, Scientific Institute San Raffaele, Vita-Salute University, Milan, Italy 4 Department of Pharmaceutical Sciences, University of Salerno, Fisciano (Salerno), Italy 5 Department of Anatomy, Pharmacology, and Forensic Medicine, University of Turin, Turin, Italy 6 Department of Clinical and Biological Sciences and San Luigi Hospital, University of Turin, Turin, Italy Address correspondence and reprint requests to Riccarda Granata, PhD, Laboratory of Molecular and Cellular Endocrinology, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Turin, Corso Dogliotti, 14-10126 Turin, Italy. E-mail: riccarda.granata{at}unito.it Abstract OBJECTIVE— Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of β-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS— β-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS— Obestatin showed specific binding on HIT-T15 and INS-1E β-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-γ/tumor necrosis factor-α/interleukin-1β treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys 3 ]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in β-cells and human islets. β-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased β-cell cAMP and activated extracellular signal–related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on β-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1 ) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element–binding protein phosphorylation; 2 ) stimulated insulin secretion and gene expression; and 3 ) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. CONCLUSIONS— These results indicate that obestatin promotes β-cell and human islet cell survival and stimulates the expression of main regulatory β-cell genes, identifying a new role for this peptide within the endocrine pancreas. BrdU, 5-bromo-2-deoxyuridine CREB, cAMP response element–binding protein [D-Lys3]-GHRP-6, [D-Lys3]-growth hormone releasing peptide-6 EIA, enzyme immunoassay ERK, extracellular signal–related kinase Ex-4, exendin-4 Ex-9, exendin-(9-39) FBS, fetal bovine serum GLP-1, glucagon-like peptide-1 GLP-1R, glucagon-like peptide-1 receptor GPR39, G-protein–coupled receptor 39 GRLN-R, ghrelin receptor IBMX, 3-isobutyl-1-methylxanthine IL-1β, interleukin-1β IFN-γ, interferon-γ IRS-2, insulin receptor substrate-2 KRBH, Krebs-Ringer bicarbonate HEPES buffer MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide PDX-1, pancreatic and duodenal homeobox-1 PI 3-kinase, phosphatidylinositol 3-kinase PKA, protein kinase A RIA, radioimmunoassay TNF-α, tumor necrosis factor-α UAG, unacylated ghrelin TUNEL, TdT-mediated dUTP nick-end labeling Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 9 January 2008. DOI: 10.2337/db07-1104. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted December 13, 2007. Received August 15, 2007. DIABETES