Tubedown (Narg1, Tbdn), a member of the Nat1 family of proteins, associates with the acetyltransferase Ard1 and exerts an angiostatic function in adult retinal-blood-vessel homeostasis. The purpose of the present study was to gain a better understanding of the nature of the Tbdn protein complex and how it might exert a homeostatic influence on blood vessels. Immunoprecipitation of Tbdn from endothelial cells followed by gel electrophoresis and liquid-chromatography–tandem-mass-spectrometry identified the actin-cytoskeleton-binding protein cortactin as a co-immunopurifying species. Western blotting confirmed the association between Tbdn and cortactin. Immunofluorescence confocal microscopy revealed that Tbdn colocalizes with cortactin and F-actin in cytoplasmic regions and at the cortex of cultured endothelial cells. Because cortactin is known to regulate cellular permeability through its interaction with the actin cytoskeleton, a process that is crucial for endothelial cell homeostasis, the role of Tbdn on endothelial cell permeability was examined. Knockdown of Tbdn expression in endothelial cells led to the co-suppression of Ard1 protein expression and to a significant increase in cellular permeability measured by the transit of FITC-albumin across the cellular monolayer. Furthermore, the proliferative retinal neovascularization and thickening resulting from induction of Tbdn knockdown in endothelium in transgenic mice was associated with a significant increase in extravasation or leakage of albumin from abnormal retinal blood vessels in vivo. These results provide evidence that an association occurs between Tbdn and cortactin, and that Tbdn is involved in the regulation of retinal-endothelial-cell permeability to albumin. This work implicates a functional role for Tbdn in blood-vessel permeability dynamics that are crucial for vascular homeostasis.

A protein mass mapping approach using mass spectrometry (MS) combined with an experimentally derived protein mass database is presented for rapid and effective identification of bacterial species. A prototype mass database from the protein extracts of nine bacterial species has been created by off-line high-performance liquid chromatography (HPLC) matrix-assisted laser desorption/ionization (MALDI) MS, in which the microbiological parameter of bacterial growth time is considered. A numerical method using a statistical weight factor algorithm is devised for matching the protein masses of an unknown bacterial sample against the database. The sum of these weight factors produces a corresponding summed weight factor score for each bacterial species listed in the database, and the database species producing the highest score represents the identity of the respective unknown bacterium. The applicability and reliability of this protein mass mapping approach has been tested with seven bacterial species in a single-blind study by both direct MALDI MS and HPLC electrospray ionization MS methods, and identification results with 100% accuracy are obtained. Our studies have demonstrated that the protein mass database can be rapidly established and readily adopted with relatively less dependency on experimental factors. Furthermore, it is shown that a number of proteins can be detected using a protein sample amount equivalent to an extract of less than 1000 cells, demonstrating that this protein mass mapping approach can potentially be highly sensitive for rapid bacterial identification.

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