Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

Development of new-type rapid analysis technology of polychlorinated biphenyls by using liquid chromatographic clean-up material (polyvinyl alcohol gel).

We developed a new-type rapid polychlorinated biphenyl (PCB) analysis technology on the basis of a liquid chromatographic clean-up system combined with a large-volume injection GC-LRMS. Among 18 kinds of materials such as polymer gels, normal-phase silica gels, reversed-phase silica gels, carbon material and ion-exchange material, polyvinyl alcohol (PVA) gel and poly (hydroxylmethacrylate) gel were found to give rather good separation performance for insulating oil. Especially, PVA gel was confirmed to be the most suitable for rapid PCB analysis because of its least required quantity of fraction liquid as well as the highest resolution. Then, we confirmed elution characteristics of all PCB isomers and removal efficiency of insulating oil on PVA gel under an optimized condition, and established high-performance clean-up system using a combination of octadecyl silica gel (ODS), porous graphite carbon (PGC) and PVA gel. In this system, we applied newly valve-switching method that could remove other impurities. In addition, it was demonstrated that the proposed clean-up system could become highly sensitive and rapid PCB analysis technology with 2-h analysis time, lower measurement limit of less than 0.05 mg/kg, and a variation coefficient of less than 5%, by coupling with a large-volume injection type GC-LRMS. Thus, we can conclude that this rapid PCB analysis technology has not only good correlativity (R2>0.999) with standard analysis method but also high durability and can be fully applied to actual PCB-treatment plants.