The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.

Endogenous expression and localization of myostatin and its relation to MHC distribution in C2C12 skeletal muscle cells

Myostatin is a negative regulator of skeletal muscle growth. We have previously reported that recombinant myostatin protein inhibits DNA and protein synthesis in C2C12 cells. Our objective was to assess if C2C12 cells express myostatin, determine its sub-cellular localization and the developmental stage of C2C12 cells in which myostatin mRNA and protein are expressed. To study the endogenous expression of myostatin, C2C12 myoblasts were allowed to progress to myotubes, and changes in the levels of endogenous myostatin mRNA expression were determined by RT-PCR. The myostatin protein and the two major myosin heavy chain (MHC) isoforms (MHC-I and -II) were determined by Western blot. Confirmation of the relative MHC expression patterns was obtained by a modified polyacrylamide gel electropheretic (PAGE) procedure. Imunofluorescence staining was employed to localize the site of myostatin expression and the relative distribution of the MHC isoforms. Co-expression of these proteins was studied using a dual staining approach. Expression of myostatin mRNA was found in myotubes but not in myoblasts. Myostatin protein was seen in most but not all, of the nuclei of polynucleated fibers expressing MHC-II, and myostatin was detected in the cytoplasm of myotube. The localization of myostatin protein in myotube nuclei was confirmed by Western blot of isolated nuclear and cytoplasmic fractions. Incubation of C2C12 myotubes with graded doses of dexamethasone dose-dependently increased the intensity of nuclear myostatin immunostaining and also resulted in the appearance of cytoplasmic expression. In conclusion, myostatin was expressed mostly in C2C12 myotubes nuclei expressing MHC-II. Its predominant

The endogenous granulocyte colony-stimulating factor response following autologous peripheral blood stem cell transplantation is inpaired in patients with myeloma

Granulocyte colony-stimulating factor (G-CSF) levels were studied in 23 patients (10 myeloma, 13 relapsed Hodgkin's disease, non-Hodgkin's lymphoma or germ cell tumours), post autologous peripheral blood stem cell transplantation (PBSCT). The two groups had similar previous chemotherapy and numbers of CD34+ cells transplanted. All patients received G-CSF by injection starting 8 d post transplantation. Twenty out of 23 patients showed raised endogenous levels of G-CSF before cytokine administration. Myeloma patients showed significantly lower levels of endogenous G-CSF than the other patients (0.767 versus 3.262 ng/ml, P <0.05). Further rises in G-CSF levels were seen following the administration of exogenous G-CSF which then fell, despite ongoing administration of G-CSF, as neutrophil recovery occurred.

Kir2.2 inward rectifier potassium channels are inhibited by an endogenous factor in Xenopus oocytes independently from the action of a mitochondrial uncoupler.

We previously showed inhibition of Kir2 inward rectifier K+ channels expressed in Xenopus oocytes by the mitochondrial agents carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide. Mutagenesis studies suggested that FCCP may act via phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. This mechanism could be reversible in intact cells but not in excised membrane patches which preclude PIP2 regeneration. This prediction was tested by investigating the reversibility of the inhibition of Kir2.2 by FCCP in intact cells and excised patches. We also investigated the effect of FCCP on Kir2.2 expressed in human embryonic kidney (HEK) cells. Kir2.2 current, expressed in Xenopus oocytes, increased in inside-out patches from FCCP-treated and untreated oocytes. The fraction of total current that increased was 0.79?±?0.05 in control and 0.89?±?0.03 in 10 µM FCCP-treated (P?>?.05). Following “run-up,” Kir2.2 current was re-inhibited by “cramming” inside-out patches into oocytes. Therefore, run-up reflected not reversal of inhibition by FCCP, but washout of an endogenous inhibitor. Kir2.2 current recovered in intact oocytes within 26.5 h of FCCP removal. Injection of oocytes with 0.1 U apyrase completely depleted ATP (P?<?.001) but did not inhibit Kir2.2 and inhibited Kir2.1 by 35% (P?<?.05). FCCP only partially reduced [ATP] (P?<?.001), despite inhibiting Kir2.2 by 75% (P?<?.01) but not Kir2.1. FCCP inhibited Kir2.2 expressed in HEK cells. The recovery of Kir2.2 from inhibition by FCCP requires intracellular components, but direct depletion of ATP does not reproduce the differential inhibitory effect of FCCP. Inhibition of Kir2.2 by FCCP is not unique to Xenopus oocytes. J. Cell. Physiol. 219: 8–13, 2009. © 2008 Wiley-Liss, Inc.

Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors.

Endogenous Two-Sided Markets with Repeated Transactions

We consider homogeneous two-sided markets, in which connected buyer-seller pairs bargain and trade repeatedly. In this infinite market game with exogenous matching probabilities and a common discount factor, we prove the existence of equilibria in stationary strategies. The equilibrium payoffs are given implicitly as a solution to a system of linear equations. Then, we endogenize the matching mechanism in a link formation stage that precedes the market game. When agents are sufficiently patient and link costs are low, we provide an algorithm to construct minimally connected networks that are pairwise stable with respect to the expected payoffs in the trading stage. The constructed networks are essentially efficient and consist of components with a constant buyer-seller ratio. The latter ratio increases (decreases) for a buyer (seller) that deletes one of her links in a pairwise stable component.

ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor

The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca2+ concentration ([Ca2+](i)) remains unknown. We measured [Ca2+](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary beta-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca2+](i) in the form of a peak followed by a plateau dependent on extracellular Ca2+. NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca2+](i). The ADPr-induced [Ca2+](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP3 receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPrinduced [Ca2+](i) increase. ADPr increased [Ca2+](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors. (c) 2010 Elsevier Ireland Ltd. All rights reserved.