Non-imidazole heterocyclic histamine H3 receptor antagonists

Continued exploration of the SAR around the lead imidazopyridine histamine H(3) antagonist 1 has led to the discovery of several related series of heterocyclic histamine H(3) antagonists. The synthesis and SAR of indolizine, indole and pyrazolopyridine based compounds are now described.     

Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) > Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.     

Expression in Escherichia coli, folding in vitro, and characterization of the carbohydrate recognition domain of the natural killer cell receptor NKR-P1A

NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity. Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro. The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry. These are characteristic of a long form CRD. The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the (15)N-labeled CRD are those of a folded protein. Chemical shifts of H(alpha) and NH protons indicate a considerable amount of beta-strand structure. Successful folding in the absence of Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein.     

ATP-dependent substrate occlusion by the human erythrocyte sugar transporter

Human erythrocyte sugar transport presents a functional complexity that is not explained by existing models for carrier-mediated transport. It has been suggested that net sugar uptake is the sum of three serial processes: sugar translocation, sugar interaction with an intracellular binding complex, and the release from this complex into bulk cytosol. The present study was carried out to identify the erythrocyte sugar binding complex, to determine whether sugar binding occurs inside or outside the cell, and to determine whether this binding complex is affected by cytosolic ATP or transporter quaternary structure. Sugar binding assays using cells and membrane protein fractions indicate that sugar binding to erythrocytes is quantitatively accounted for by sugar binding to the hexose transport protein, GluT1. Kinetic analysis of net sugar fluxes indicates that GluT1 sugar binding sites are cytoplasmic. Intracellular ATP increases GluT1 sugar binding capacity from 1 to 2 mol of 3-O-methylglucose/mol GluT1 and inhibits the release of bound sugar into cytosol. Reductant-mediated, tetrameric GluT1 dissociation into dimeric GluT1 is associated with the loss of ATP and 3-O-methylglucose binding. We propose that sugar uptake involves GluT1-mediated, extracellular sugar translocation into an ATP-dependent cage formed by GluT1 cytoplasmic domains. Caged or occluded sugar has three possible fates: (1) transport out of the cell (substrate cycling); (2) interaction with sugar binding sites within the cage, or (3) release into bulk cytosol. We show how this hypothesis can account for the complexity of erythrocyte sugar transport and its regulation by cytoplasmic ATP.     

Novel substituted pyrrolidines are high affinity histamine H3 receptor antagonists

Pre-clinical characterization of novel substituted pyrrolidines that are high affinity histamine H₃ receptor antagonists is described. These compounds efficiently penetrate the CNS and occupy the histamine H₃ receptor in rat brain following oral administration. One compound, (2S,4R)-1-[2-(4-cyclobutyl-[1,4]diazepane-1-carbonyl)-4-(3-fluoro-phenoxy)-pyrrolidin-1-yl]-ethanone, was extensively profiled and shows promise as a potential clinical candidate.     

Seagrasses of south–west Australia: A conceptual synthesis of the world's most diverse and extensive seagrass meadows

South–west Australia contains extensive seagrass meadows along 2,500 km of coastline from the shallow subtidal to 50+ m water depths, and in many of the 51 bar-built estuaries along the coast. There are geomorphological differences between the south and west coasts that result in different patterns of swell exposure influencing the processes that structure seagrass habitats. In this paper, ‘sheltered’, ‘exposed’ and ‘estuarine’ seagrass habitat types are defined for south–west Australia to synthesize processes influencing seagrass communities. Sheltered habitats in south–west Australia are characterized by high light, low to moderate water motion and sporadic disturbance from storms, making them ideal habitats for a diversity of seagrass assemblages. Exposed seagrass habitats are characterized by the presence of strong and consistent ocean swells (3–8 m), predominantly from the south or south–west and seagrasses exhibit a suite of adaptive traits to survive the effects of exposure to ocean swell and associated sand movement. These include morphological features such as heavy fiber reinforcement to strengthen the aboveground stems or leaves, deep vertical rhizomes and life history traits such as rapid growth and high seed set. Within estuarine habitats highly dynamic seagrass communities are the result of fluctuating annual cycles in temperature, light and salinity. Compared to global seagrass meadows, coastal south–west Australian seagrass habitats experience high light, low nutrients and high water movement. Despite these differences, similarities with other regions do exist and here we place the habitats of south–west Australia into a global context using comparative data. The wide array of morphology and life history traits displayed among seagrass species of south–west Australia are presented in a conceptual model including habitat type, physical stressors and seagrass responses. The combination of adaptations to the habitats and processes that define them make south–west Australia a region where there is an unusually high number of co-occurring seagrass species, the highest in the world for a temperate environment (19 species), and approaching the species diversity of many tropical environments. Linking aspects of seagrass habitat, physical aspects of the environment and seagrass life history provides a context for applying knowledge gained from seagrasses in south–west Australia to other coastal ecosystems throughout the world.     

Novel tetrahydroisoquinolines are histamine H3 antagonists and serotonin reuptake inhibitors

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.     

ATP-dependent sugar transport complexity in human erythrocytes

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar](intracellular) = [sugar](extracellular), where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of 相似文献   

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.