Exendin-(9-39) corrects fasting hypoglycemia in SUR-1-/- mice by lowering cAMP in pancreatic beta-cells and inhibiting insulin secretion

Congenital hyperinsulinism is a disorder of pancreatic beta-cell function characterized by failure to suppress insulin secretion in the setting of hypoglycemia, resulting in brain damage or death if untreated. Loss-of-function mutations in the K(ATP) channel (composed of two subunits: Kir6.2 and SUR-1) are responsible for the most common and severe form of congenital hyperinsulinism. Most patients are unresponsive to available medical therapy and require palliative pancreatectomy. Similar to the human condition, the SUR-1(-/-) mouse is hypoglycemic when fasted and hyperglycemic when glucose-loaded. We have previously reported that the glucagon-like peptide-1 receptor antagonist exendin-(9-39) raises fasting blood glucose in normal mice. Here we examine the effect of exendin-(9-39) on fasting blood glucose in SUR-1(-/-) mice. Mice were randomized to receive exendin-(9-39) or vehicle. Fasting blood glucose levels in SUR-1(-/-) mice treated with exendin-(9-39) were significantly higher than in vehicle-treated mice and not different from wild-type littermates. Exendin-(9-39) did not further worsen glucose tolerance and had no effect on body weight and insulin sensitivity. Isolated islet perifusion studies demonstrated that exendin-(9-39) blocked amino acid-stimulated insulin secretion, which is abnormally increased in SUR-1(-/-) islets. Furthermore, cAMP content in SUR-1(-/-) islets was reduced by exendin-(9-39) both basally and when stimulated by amino acids, whereas cytosolic calcium levels were not affected. These findings suggest that cAMP plays a key role in K(ATP)-independent insulin secretion and that the GLP-1 receptor is constitutively active in SUR-1(-/-) beta-cells. Our findings indicate that exendin-(9-39) normalizes fasting hypoglycemia in SUR-1(-/-) mice via a direct effect on insulin secretion, thereby raising exendin-(9-39) as a potential therapeutic agent for K(ATP) hyperinsulinism.     

Characterization of a protein phosphatase 2A holoenzyme that dephosphorylates the clathrin adaptors AP-1 and AP-2

The AP-2 complex is a key factor in the formation of endocytic clathrin-coated vesicles (CCVs). AP-2 sorts and packages cargo membrane proteins into CCVs, binds the coat protein clathrin, and recruits numerous other factors to the site of vesicle formation. Structural information on the AP-2 complex and biochemical work have allowed understanding its function on the molecular level, and recent studies showed that cycles of phosphorylation are key steps in the regulation of AP-2 function. The complex is phosphorylated on both large subunits (alpha- and beta2-adaptins) as well as at a single threonine residue (Thr-156) of the medium subunit mu2. Phosphorylation of mu2 is necessary for efficient cargo recruitment, whereas the functional context of the large subunit phosphorylation is unknown. Here, we show that the subunit phosphorylation of AP-2 exhibits striking differences, with calculated half-lives of <1 min for mu2, approximately 25 min for beta2, and approximately 70 min for alpha. We were also able to purify a phosphatase that dephosphorylates the mu2 subunit. The enzyme is a member of the protein phosphatase 2A family and composed of a catalytic Cbeta subunit, a scaffolding Abeta subunit, and a regulatory Balpha subunit. RNA interference knock down of the latter subunit in HeLa cells resulted in increased levels of phosphorylated adaptors and altered endocytosis, showing that a specific PP2A holoenzyme is an important regulatory enzyme in CCV-mediated transport.     

Which in vitro models could be best used to study hepatocyte polarity?

The correct functioning of the liver is ensured by the setting and the maintenance of hepatocyte polarity. The complex polarity of the hepatocyte is characterized by the existence of several basolateral and apical poles per cell. Many in vitro models are available for studying hepatocyte polarity, but which are the more suitable? To answer this question, we aimed to identify criteria which determine the typical hepatocyte polarity. Therefore, we compiled a range of protein markers of membrane domains in rat hepatocytes and investigated their involvement in hepatocytic functions. Then, we focused on the relationship between hepatic functions and the cytoskeleton, Golgi apparatus and endoplasmic reticulum. Subsequently, we compared different cell lines expressing hepatocyte polarity. Finally, to demonstrate the usefulness of some of these lines, we presented new data on endoplasmic reticulum organization in relation to polarity.     

Chemotactic responses of IL-4-, IL-10-, and IFN-gamma-producing CD4+ T cells depend on tissue origin and microbial stimulus

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-gamma- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-gamma, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-gamma-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-gamma+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.     

Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C–C bond formation

The thiamin-diphosphate-dependent enzyme benzaldehyde lyase is a very import catalyst for chemoenzymatic synthesis catalyzing the formation and cleavage of (R)-hydroxy ketones. We have studied the stability of the recombinant enzyme and some enzyme variants with respect to pH, temperature, buffer salt, cofactors and organic cosolvents. Stability of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer. Reaction temperature should not exceed 37 °C. The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optimum of both the lyase and the ligase reaction. Potassium phosphate and Tris were identified as optimal reaction buffers and the addition of 20 vol% DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL. The initial broad product range of BAL-catalyzed reactions has been enlarged to include highly substituted hydroxybutyrophenones and aliphatic acyloins.     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

Heterologous expression of Arabidopsis UDP-glucosyltransferases in Saccharomyces cerevisiae for production of zearalenone-4-O-glucoside

Zearalenone, a secondary metabolite produced by several plant-pathogenic fungi of the genus Fusarium, has high estrogenic activity in vertebrates. We developed a Saccharomyces cerevisiae bioassay strain that we used to identify plant genes encoding UDP-glucosyltransferases that can convert zearalenone into zearalenone-4-O-glucoside (ZON-4-O-Glc). Attachment of the glucose moiety to zearalenone prevented the interaction of the mycotoxin with the human estrogen receptor. We found that two of six clustered, similar UGT73C genes of Arabidopsis thaliana encode glucosyltransferases that can inactivate zearalenone in the yeast bioassay. The formation of glucose conjugates seems to be an important plant mechanism for coping with zearalenone but may result in significant amounts of "masked" zearalenone in Fusarium-infected plant products. Due to the unavailability of an analytical standard, the ZON-4-O-Glc is not measured in routine analytical procedures, even though it can be converted back to active zearalenone in the digestive tracts of animals. Zearalenone added to yeast transformed with UGT73C6 was converted rapidly and efficiently to ZON-4-O-Glc, suggesting that the cloned UDP-glucosyltransferase could be used to produce reference glucosides of zearalenone and its derivatives.     

The design and synthesis of novel alpha-ketoamide-based p38 MAP kinase inhibitors

We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory conditions.     

Tyramine fragment binding to BACE-1

Fragment screening revealed that tyramine binds to the active site of the Alzheimer's disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.