The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.

Abstract-Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the ␣-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QT c intervals, reflecting reductions in the densities of repolarizing, voltage-gated K ϩ currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo. Key Words: lipids Ⅲ metabolism Ⅲ cardiomyopathy C ardiomyopathy has been observed in a variety of metabolic disorders. In inherited disorders of ␤-oxidation, accumulation of unmetabolized lipid in cardiac myocytes is associated with ventricular systolic dysfunction. 1 In obesity, increased myocardial oxygen consumption and decreased efficiency may contribute to diastolic and systolic dysfunction. 2,3 In diabetes mellitus, heart failure in the absence of valvular or congenital heart disease, alcoholism, hypertension, or significant epicardial coronary atherosclerosis is defined as diabetic cardiomyopathy and accounts for significant morbidity and mortality in people with type 1 and type 2 diabetes. 4 Echocardiographic and hemodynamic studies suggest left ventricular (LV) diastolic impairment represents an early preclinical manifestation of diabetic cardiomyopathy that may progress over an extended period of time to both diastolic and systolic dysfunction. 5,6 In these metabolic disorders, systemic metabolic perturbations lead to myocyte dysfunction and/or loss. Glucotoxicity, 7 ATP depletion, 8 and maladaptive changes in metabolic substrate utilization 9 are mechanisms proposed to contribute to cardiac dysfunction. It has also been hypothesized that mismatch between tissue free fatty acid (FFA) import and utilization leads to lipid accumulation and results in lipotoxicity. In diabetes, this imbalance results from high-serum F...

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Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.

An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.