[Abstract unavailable

The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer approximately hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerve terminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsin on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.

Diffusion-weighted single voxel experiments conducted at b-values up to 1 x 10(4) smm-2 yielded biexponential signal attenuation curves for both normal and ischemic brain. The relative fractions of the rapidly and slowly decaying components (f1, f2) are f1 = 0.80 +/- 0.02, f2 = 0.17 +/- 0.02 in healthy adult rat brain and f1 = 0.90 +/- 0.02, f2 = 0.11 +/- 0.01 in normal neonatal rat brain, whereas the corresponding values for the postmortem situation are f1 = 0.69 +/- 0.02, f2 = 0.33 +/- 0.02. It is demonstrated that the changes in f1 and f2 occur simultaneously to those in the extracellular and intracellular space fractions (fex, f(in)) during: (i) cell swelling after total circulatory arrest, and (ii) the recovery from N-methyl-D-aspartate induced excitotoxic brain edema evoked by MK-801, as measured by changes in the electrical impedance. Possible reasons for the discrepancy between the estimated magnitude components and the physiological values are presented and evaluated. Implications of the biexponential signal attenuation curves for diffusion-weighted imaging experiments are discussed.

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.

Immune cell-specific expression is one indication of the importance of a gene's role in the immune response. We have compiled a compendium of microarray expression data for virtually all human genes from six key immune cell types and their activated and differentiated states. Immune Response In Silico (IRIS) is a collection of genes that have been selected for specific expression in immune cells. The expression pattern of IRIS genes recapitulates the phylogeny of immune cells in terms of the lineages of their differentiation. Gene Ontology assignments for IRIS genes reveal significant involvement in inflammation and immunity. Genes encoding CD antigens, cytokines, integrins and many other gene families playing key roles in the immune response are highly represented. IRIS also includes proteins of unknown function and expressed sequence tags that may not represent genes. The predicted cellular localization of IRIS proteins is evenly distributed between cell surface and intracellular compartments, indicating that immune specificity is important at many points in the signaling pathways of the immune response. IRIS provides a resource for further investigation into the function of the immune system and immune diseases.

Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

No abstract

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 A) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure-function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.