Inhibition of phagosome maturation by mycobacteria does not interfere with presentation of mycobacterial antigens by MHC molecules

Pathogenic mycobacteria escape host innate immune responses by surviving within phagosomes of host macrophages and blocking their delivery to lysosomes. Avoiding lysosomal delivery may also be involved in the capacity of living mycobacteria to modulate MHC class I- or II-dependent T cell responses, which may contribute to their pathogenicity in vivo. In this study, we show that the presentation of mycobacterial Ags is independent of the site of intracellular residence inside professional APCs. Infection of mouse macrophages or dendritic cells in vitro with mycobacterial mutants that are unable to escape lysosomal transfer resulted in an identical efficiency of Ag presentation compared with wild-type mycobacteria. Moreover, in vivo, such mutants induced CD4(+) Th1 or CD8(+) CTL responses in mice against various mycobacterial Ags that were comparable to those induced by their wild-type counterparts. These results suggest that the limiting factor for the generation of an adaptive immune response against mycobacteria is not the degree of lysosomal delivery. These findings are important in the rational design of improved vaccines to combat mycobacterial diseases.     

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.     

Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.     

Synthesis, structural analysis, and SAR studies of triazine derivatives as potent, selective Tie-2 inhibitors

A novel class of selective Tie-2 inhibitors was derived from a multi-kinase inhibitor 1. By reversing the amide connectivity and incorporating aminotriazine or aminopyridine hinge-binding moieties, excellent Tie-2 potency and KDR selectivity could be achieved with 3-substituted terminal aryl rings. X-ray co-crystal structure analysis aided inhibitor design. This series was evaluated on the basis of potency, selectivity, and rat pharmacokinetic parameters.     

Functional evaluation of domain-domain interactions and human protein interaction networks

MOTIVATION: Large amounts of protein and domain interaction data are being produced by experimental high-throughput techniques and computational approaches. To gain insight into the value of the provided data, we used our new similarity measure based on the Gene Ontology (GO) to evaluate the molecular functions and biological processes of interacting proteins or domains. The applied measure particularly addresses the frequent annotation of proteins or domains with multiple GO terms. RESULTS: Using our similarity measure, we compare predicted domain-domain and human protein-protein interactions with experimentally derived interactions. The results show that our similarity measure is of significant benefit in quality assessment and confidence ranking of domain and protein networks. We also derive useful confidence score thresholds for dividing domain interaction predictions into subsets of low and high confidence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus-induced interferon (IFN)-alpha. They exhibit a lineage-negative phenotype but all express BDCA-4, which is homologous to the neuronal receptor neuropilin-1. Specific staining with anti-BDCA-4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN-alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage-positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti-BDCA-4 mAb also resulted in at least two-fold lower or reduced virus-induced IFN-alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN-alpha release induced by non-viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA-4 on PDC as demonstrated for IFN-alpha response to virus.     

In vitro cell growth of marine archaeal-bacterial consortia during anaerobic oxidation of methane with sulfate

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.     

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

Background

Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.

Methods

Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10⁶ cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.

Results

We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.

Conclusion

Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.