Insights into the role of Nup62 and Nup93 in assembling cytoplasmic ring and central transport channel of the nuclear pore complex.

The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups) to form a versatile transport channel between the nucleus and the cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport, Nup93 for proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes such as the central transport channel (CTC), the cytoplasmic ring (CR), and the inner ring (IR). However, how they play their roles in NPC assembly and transport activity is not clear. Here we delineated the interacting regions and conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behavior and a distinct "4"-shaped architecture resembling the CTC complex. Our in vitro reconstitution data demonstrate that the Nup62 coiled-coil domain is critical to form both Nup62322-525 •Nup88517-742 and Nup62322-525•Nup88517-742•Nup214693-926 heterotrimers and both can bind to Nup931-150. We therefore propose that Nup93 acts as a "sensor" to bind to Nup62 shared heterotrimers including the Nup62•Nup54 heterotrimer of the CTC, which was not shown previously to be an interacting partner. Altogether, our biochemical study suggests that Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers and Nup93 binds these diverse heterotrimers nonselectively.

Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower (p<0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine- and tail skin-derived HMC blastocysts and in vitro-fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order (p<0.05) urine≥tail skin>ear skin-derived cells, whereas in blastocysts, it was higher (p<0.05) in urine- and tail skin-derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CASPASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower (p<0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n=10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.