Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure

The structure of the precursor form of catechol oxidase from sweet potatoes (Ipomoea batatas) has been modeled on the basis of the 3D structural data of mature catechol oxidase [Nat. Struct. Biol. 5 (1998) 1084] and of hemocyanin from giant octopus (Octopus dofleini) [J. Mol. Biol. 278 (1998) 855]. A C-terminal extension peptide is found in the cDNA sequence but not in the purified, mature form of catechol oxidase. Superimposition of the 3D structures of the native hemocyanin and catechol oxidase reveals a close relationship except for an additional C-terminal domain only found in the hemocyanin structure. As sequence alignment shows good homology this domain of the hemocyanin structure was used as a template to model the 3D structure of the C-terminal extension peptide of catechol oxidase. As hemocyanins show no or only weak catecholase activity due to this domain this indicates an inhibitory function of this extension peptide. Beside this possible shielding function for the precursor form, evidence for a function in copper-uptake also increases due to the location of three histidine residues in the model.     

The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases beta-cell mass in diabetic mice

NN2211 is a long-acting, metabolically stable glucagon-like peptide-1 (GLP-1) derivative designed for once daily administration in humans. NN2211 dose dependently reduced the glycemic levels in ob/ob mice, with antihyperglycemic activity still evident 24 h postdose. Apart from an initial reduction in food intake, there were no significant differences between NN2211 and vehicle treatment, and body weight was not affected. Histological examination revealed that beta-cell proliferation and mass were not increased significantly in ob/ob mice with NN2211, although there was a strong tendency for increased proliferation. In db/db mice, exendin-4 and NN2211 decreased blood glucose compared with vehicle, but NN2211 had a longer duration of action. Food intake was lowered only on day 1 with both compounds, and body weight was unaffected. beta-Cell proliferation rate and mass were significantly increased with NN2211, but with exendin-4, only the beta-cell proliferation rate was significantly increased. In conclusion, NN2211 reduced blood glucose after acute and chronic treatment in ob/ob and db/db mice and was associated with increased beta-cell mass and proliferation in db/db mice. NN2211 is currently in phase 2 clinical development.     

Aluminum impairs gap junctional intercellular communication between astroglial cells in vitro

The purpose of the present study was to investigate the effect of aluminum on gap junctional intercellular communication (GJIC) in cultured astrocytes. In the CNS the extracellular environment and metabolic status of neurons is dependent upon astrocytes, which are known to exhibit GJIC. This cell-to-cell communication provides a cytoplasmic continuity between adjacent cells, allowing exchange of diverse ions, second messengers, and metabolites. To study the effects of aluminum intoxication on GJIC in cultured glial cells, astroglial cell cultures obtained from fetal rat brains were exposed to aluminum lactate for 2-6 weeks. To demonstrate the metabolic coupling of neighboring cells, the technique of microinjection of the gap junction permeable substance neurobiotin was performed. Whereas in controls intensive GJIC was observed by dye transfer of neurobiotin from the microinjected cell into the adjacent astrocytes, aluminum treatment significantly impaired this cellular communication. As aluminum is known to affect cytoskeletal elements, additional investigations into the organization of intermediate filaments (glial fibrillary acid protein, GFAP) and microfilaments in control astrocytes and subsequent aluminum exposure were performed with the aid of fluorescence microscopy and rapid-freeze, deep-etch electron microscopy. Aluminum exposure led to an aggregation of GFAP-positive filaments near to the cell nucleus, accompanied by a destruction of the actin cytoskeleton, especially close to the cell membrane. Ultrastructurally these data could be verified as prominent areas without actin filaments contacting the cell membrane detectable in aluminum-treated astrocytes. Immunohistochemical staining of Cx43 revealed an impaired trafficking of this connexin into the cell prolongations following aluminum treatment, although electron-microscopic data revealed that gap junctions between adjacent astrocytes were still present after aluminum incubation for 24 days. In conclusion, in cultured astrocytes the morphological integrity of microfilaments and the intermediate filament network seem to be fundamental for the translocation of connexins from Golgi complex into the cellular prolongation to exhibit proper and extensive cellular communication through gap junctions.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.     

The NKG2D receptor and its ligands-recognition beyond the "missing self"?

NKG2D is a surface receptor that activates natural killer (NK) cells and delivers a co-stimulatory signal to CD8-positive T cells. The ligands of NKG2D are induced by cellular stress and are specifically expressed by some tumor cells. This sparked the idea of an alternative regulation of NK cells by expression of "induced self" ligands on target cells which can overcome the inhibition imparted by MHC class I-specific inhibitory receptors.