J. Neurochem. (2011) 118, 163–175. Abstract In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic‐sensors which can up‐regulate the functional expression of drug transporters, such as P‐glycoprotein (P‐gp). In the brain, an increase in P‐gp expression can further limit drug permeability across the blood–brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P‐gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P‐gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P‐gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand‐mediated P‐gp induction. Ligand‐induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down‐regulation of hPXR and hCAR proteins using small‐interfering RNA decreased P‐gp expression. Our findings provide first evidence for P‐gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P‐gp regulation at this site.

Our data suggest the testes are a complex pharmacological compartment that can restrict the distribution of certain antiretroviral drugs and potentially contribute to HIV-1 persistence.

Drug transporters such as P-glycoprotein and OATPs regulate intestinal permeability of atazanavir and may contribute to its poor oral bioavailability and drug-drug interactions with other protease inhibitors and TDF.

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.In eukaryotic cells, the replicated DNA (sister chromatid) remains connected to each other from the end of the S phase until the onset of anaphase. This sister chromatid cohesion ensures that the two daughter cells inherit an identical set of genetic information. It is well established that the sister chromatid cohesion is accomplished by a phylogenetically conserved protein complex called cohesin (reviewed in Ref. 1). Cohesin was identified genetically for the first time in budding yeast and contains at least four components: Scc1/Mcd1, Scc3, Smc1, and Smc3 (2-4). Subsequent studies revealed that similar complexes are responsible for cohesion in diverse eukaryotic organisms including fission yeast, Xenopus, chicken, and human (5-8). However, the exact behaviors of cohesin differ among species. Specifically, Scc1 dissociates from chromosomes in a single step of the metaphase-to-anaphase transition in budding yeast, mediated by its proteolysis by a protease called Esp1 or separase (9). In contrast, the dissociation occurs in two steps in vertebrates, from the arm regions in prophase and from the centromeric region in the metaphase-to-anaphase transition (5, 10, 11). The bulk Scc1 dissociation in prophase occurs independently of separase, and is regulated by Polo-like kinase in Xenopus (12), whereas the Scc1 dissociation from kinetochores at the metaphase-to-anaphase transition in human cells is achieved by the proteolysis of Scc1 by Esp1 (13).Scc1 also plays an important role in kinetochore function. In budding yeast, it was reported that kinetochore-microtubule association can be established, but sister kinetochores frequently associate with monopoles (14). It was therefore concluded that Scc1 is important for the bipolar attachment of the sister kinetochores. A similar observation was reported in Scc1-depleted chicken DT40 cells, albeit the number of sister chromatids showing monopo...

Sister chromatids duplicated in S phase are connected with each other during G 2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.Accurate chromosome segregation during mitosis into two daughter cells is one of the requirements for stable genome maintenance. Until recently, the molecular mechanisms regulating chromatid cohesion had not been well understood. However, recent studies, mostly conducted in budding yeast, have outlined the process generally (reviewed in Ref. 1). Concurrent with DNA replication, the sister chromatids become connected along their entire length. Several distinct groups of proteins are involved in establishing and maintaining chromatid cohesion. The most intensively investigated protein complex, budding yeast cohesin, consists of four subunit proteins, Scc1p/ Mcd1p, Scc3p, Smc1p, and Smc3p (2-4), and serves as a physical glue between sister chromatids. Cohesin is phylogenetically conserved. Rad21p in fission yeast and human Rad21 (hRad21) are homologues of Scc1p/Mcd1p (5, 6). Smc1p and Smc3p are found in budding yeast, Xenopus, and mammals (7-9). They are members of the SMC (structural maintenance of chromosome) protein family that is characterized by the presence of coiled-coil domains and ATPase domains (reviewed in Ref. 10). Very recently, two Scc3p homologues, SA1 and SA2, have been found in Xenopus and human cells (11). Interestingly, in Xenopus, two distinct classes of cohesin, termed xcohesin SA1 and x-cohesin SA2 , are present. These two complexes share Xenopus (X)SCC1, XSCC2, and XRAD21 and differ via containing either XSA1 or XSA2 (11). Immunodepletion of Xenopus cohesin from egg extracts led to a failure of chromatid cohesion (8). Therefore, the cohesin complex is likely to be conserved in all eukaryotes, including humans.H...