AT2 receptor interacting protein 1 (ATIP1) mediates COX-2 induction by an AT2 receptor agonist in endothelial cells.

Angiotensin II (Ang II) type 2 receptor (AT2R) is one of the major components of the renin-angiotensin-aldosterone system. Nevertheless, the physiological role is not well defined compared to the understanding of the Ang II type 1 receptor (AT1R), which is a well characterized G-protein coupled receptor in the cardiovascular system. While the AT2R signaling pathway remains unclear, AT2 receptor interacting protein 1 (ATIP1) has been identified as a candidate molecule for interacting with the C-terminal region of AT2R. In this study, we investigated the ATIP1 dependent AT2R inducible genes in human umbilical vein endothelial cells (HUVECs). CGP42112A, an AT2R specific agonist, resulted in an upregulation of inflammatory genes in HUVECs, which were inhibited by knocking down ATIP1 with siRNA (siATIP1). Among them, we confirmed by quantitative PCR that the induction of COX-2 mRNA expression was significantly downregulated by siATIP1. COX-2 was also upregulated by Ang II stimulation. This upregulation was suppressed by treatment with the AT2R specific antagonist PD123319, which was not replicated by the AT1R antagonist telmisartan. These findings suggest that ATIP1 plays an important role in AT2R dependent inflammatory responses. This may provide a new approach to the development of cardio-protective drugs.

Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II.

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ', PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal-type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.