Silver-Catalyzed Tandem Cycloisomerization/[5 + 2] Cycloaddition of 3-Cyclopropylideneprop-2-en-1-ones with Oxidopyrylium Ylides to Form Bibridged Benzocycloheptanones.

Herein, we report a mild, one-pot method for silver-catalyzed tandem cycloisomerization/[5 + 2] cycloaddition reactions between readily accessible cyclopropyl-tethered allenyl ketones and benzopyranone-derived oxidopyrylium ylides. The reactions proceed via a cyclobutene-fused furan intermediate generated in situ by a cycloisomerization/1,2-carbene transfer/ring-expansion cascade. This method, which features an unprecedented formal [5 + 2] cycloaddition, delivers good to excellent yields of structurally complex bibridged benzocycloheptanones bearing a strained cyclobutane ring and an O-bridged ring.

Mitochondrial dysfunction resulting from the down-regulation of bone morphogenetic protein 5 may cause microtia.

BACKGROUND: Bone morphogenetic protein 5 (BMP5) has been identified as one of the important risk factors for microtia; however, the link between them has yet to be clarified. In this study, we aimed to demonstrate the relationship of BMP5 with mitochondrial function and investigate the specific role of mitochondria in regulating microtia development. METHODS: BMP5 expression was measured in auricular cartilage tissues from patients with and without microtia. The effects of BMP5 knockdown on cellular function and mitochondrial function were also analyzed in vitro. Changes in genome-wide expression profiles were measured in BMP5-knockdown cells. Finally, the specific impact of BMP5 down-regulation on mitochondrial fat oxidation was analyzed in vitro. RESULTS: BMP5 expression was down-regulated in the auricular cartilage tissues of microtia patients. BMP5 down-regulation inhibited various cellular functions in vitro, including cell proliferation, mobility, and cytoactivity. The functional integrity of mitochondria was also damaged, accompanied by a decrease in mitochondrial membrane potential, reactive oxygen species (ROS) neutralization, and reduced adenosine triphosphate (ATP) production. Carnitine O-palmitoyltransferase 2 and diacylglycerol acyltransferase 2, two of the key regulators of mitochondrial lipid oxidation, were also found to be decreased by BMP5 down-regulation. CONCLUSIONS: Down-regulation of BMP5 affects glycerolipid metabolism and fatty acid degradation, leading to mitochondrial dysfunction, reduced ATP production, and changes in cell function, and ultimately resulting in microtia. This research provides supporting evidence for an important role of BMP5 down-regulation in affecting mitochondrial metabolism in cells, and sheds new light on the mechanisms underlying the pathogenesis of microtia.

The intracellular NPxY motif is critical in maintaining the function and expression of human organic anion transporting polypeptide 1B1.

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate sodium-independent transport of endogenous compounds such as bile salts, hormones and their conjugates as well as toxins and drugs. OATP1B1 is the major OATP specifically expressed at the basolateral membrane of human hepatocytes and many clinically important drugs have been shown to be substrates of the transporter. According to the computer-based hydropathy analysis, a large intracellular loop 3 (IL3) is situated between transmembrane domain 6 and 7 of OATPs, in which a conserved NPxY motif is found. In the current study, HEK293 cells expressing the HA-tagged OATP1B1 was utilized to investigate the role of the NPxY motif for the function and expression of the transporter. Alanine replacement of N335 or P336 retained substantial uptake function; while simultaneous mutation of these residues resulted in a double mutant that lost almost all the transport activity. On the other hand, Y338A showed >80% reduction for estrone-3-sulfate uptake. Plasma membrane protein analysis revealed that N335/P336A completely lost its cell surface protein expression; while that of Y338A is dramatically reduced. Further investigation with pharmacological inhibitors and immunocytochemistry demonstrated that N335/336A is detained in the Golgi apparatus and Y338A exhibited accelerated protein degradation rate compared to that of the wild-type. Conservative replacement of Y338 with phenylalanine fully recovered uptake and expression of the transporter. In summary, a new role was observed for the NPxY motif located in the IL3 of OATP1B1, which may affect processing and stability of the transporter.