No abstract

Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate. In higher eukaryotes, the majority of genes produce nascent mRNA transcripts that contain several introns. Export of incompletely spliced RNAs with retained introns from these genes would potentially result in translation of aberrant proteins that could have deleterious effects on the cells. However, cells appear to have developed checkpoints to ensure that only fully spliced mRNAs are exported and expressed (Chang and Sharp 1989;Legrain and Rosbash 1989).Retroviruses use special mechanisms that allow unspliced and incompletely spliced (that is, intron-containing) viral transcripts to exit the nucleus and escape cellular proofreading mechanisms (Hammarskjöld 1997(Hammarskjöld , 2001). Replication of all of these viruses requires the cytoplasmic expression of intron-containing forms of the initial, genome-length viral RNA transcript, because the unspliced RNA serves both as the mRNA for the viral Gag and GagPol proteins and as the RNA that is packaged into viral particles (Berkowitz et al. 1996). In complex retroviruses such as HIV-1, export of unspliced and incompletely spliced RNA relies on the interaction of the viral Rev protein with a structured RNA sequence present in these RNAs, the Rev responsive element (RRE; Hadzopoulou-Cladaras et al. 1989; Hammarskjold et al. 1989;Malim et al. 1989; Pollard and Malim 1998).The resulting mRNP complex is exported by virtue of the nuclear export signal (NES) in Rev that interacts with the cellular export receptor Crm1 (Fornerod et al. 1997;Neville et al. 1997).Simple retroviruses do not encode an Rev-like transacting protein, and export of their unspliced RNA relies on the interaction of cis-acting RNA elements directly with cellular factors. The first element of this kind was identified in the simian type D retrovirus Mason Pfizer Monkey Virus (MPMV) and was given the name CTE (constitutive transport element; Bray et al. 1994; Ernst et al. 1997a,b; Hammarskjold 2001), because its interaction with cellular factors results in constitut...

Cells normally restrict the nuclear export and expression of intron-containing mRNA. In many cell lines, this restriction can be overcome by inclusion of cis-acting elements, such as the Mason-Pfizer monkey virus constitutive transport element (CTE), in the RNA. In contrast, we observed that CTE-mediated expression from human immunodeficiency virus Gag-Pol reporters was very inefficient in 293 and 293T cells. However, addition of Sam68 led to a dramatic increase in the amount of Gag-Pol proteins produced in these cells. Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used. Additionally, the effect of Sam68 was inhibited in a dose-dependent manner by coexpression of an activated form of the nuclear kinase Sik/BRK that hyperphosphorylated Sam68. RNA analysis showed that cytoplasmic Gag-Pol-CTE RNA levels were only slightly enhanced by the addition of Sam68, compared to a 60-to 70-fold increase in the levels of Gag-Pol protein expression. Thus, in this system, Sam68 functioned to enhance the cytoplasmic utilization of RNA containing the CTE. These results suggest that Sam68 may interact with specific RNAs in the nucleus to provide a "mark" that affects their cytoplasmic fate. They also provide further evidence of links between signal transduction and RNA utilization.

The aim of this study was to explore the feasibility of fused deposition modeling (FDM) 3D printing to prepare intragastric floating sustained release (FSR) tablets. Domperidone (DOM), an insoluble weak base, was chosen as a model drug to investigate the potential of FSR in increasing its oral bioavailability and reducing its administration frequency. DOM was successfully loaded into hydroxypropyl cellulose (HPC) filaments using hot melt extrusion (HME). The filaments were then printed into hollow structured tablets through changing the shell numbers and the infill percentages. Physical characterization results indicated that the majority of DOM gradually turned into the amorphous form during the fabrication process. The optimized formulation (contain 10% DOM, with 2 shells and 0% infill) exhibited the sustained release characteristic and was able to float for about 10 h in vitro. Radiographic images showed that the BaSO4-labeled tablets were retained in the stomach of rabbits for more than 8 h. Furthermore, pharmacokinetic studies showed the relative bioavailability of the FSR tablets compared with reference commercial tablets was 222.49 ± 62.85%. All the results showed that FDM based 3D printing might be a promising way to fabricate hollow tablets for the purpose of intragastric floating drug delivery.

We have studied the mechanism of steroid-induced osteonecrosis by examining the effect of dexamethasone on a multipotential cell line, D1, which is derived from bone marrow and is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, were determined. Osteocalcin promoter activity was measured by co-transfecting the cells with the phOC-luc and pSV beta-Gal plasmids. Dexamethasone increased PPARgamma2 gene expression 2-fold, while Cbfa1/Runx2 gene expression and osteocalcin promoter activity decreased by 50-60%, and VEGF protein, measured by ELISA, decreased by 55%. These changes indicate enhanced adipogenesis and decreased osteogenesis by mesenchymal cells in vitro, together with a decrease in VEGF, a potent angiogeneic factor, suggesting that dexamethasone may shunt uncommitted osteoprogenitor cells in marrow from osteoblastic differentiation into the adipocytic pathway, leading to diminished vascularization and eventual osteonecrosis.