Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

We study the detectability of circular polarization in a stochastic gravitational wave background from various sources such as supermassive black hole binaries, cosmic strings, and inflation in the early universe with pulsar timing arrays. We calculate generalized overlap reduction functions for the circularly polarized stochastic gravitational wave background. We find that the circular polarization can not be detected for an isotropic background. However, there is a chance to observe the circular polarization for an anisotropic gravitational wave background. We also show how to separate polarized gravitational waves from unpolarized gravitational waves.

Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time-and concentration-dependent. Uridine uptake was decreased under Na ϩ -free conditions, and the system was presumed to be high affinity, indicating an Na ϩ -dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na ϩ -independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na ϩ indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis.

In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1) , IntroductionCytokines such as interleukin-3 (IL-3) and erythropoietin play an important role in the differentiation of hematopoietic stem cells. 1,2 However, the roles of small molecules such as vitamins, nucleic acids, and amino acids in cell differentiation have not been elucidated well, though it is likely that specific transporters are involved in importing such small hydrophilic molecules into the cells. The ABC transporter Bcrp1 (abcg2) is expressed mainly in hematopoietic stem cells, 3 and more recently, efflux transporter activities in murine hematopoietic stem cells (HSCs) were found to vary according to developmental and activation status. 4 Thiamine transporter Thtr-1 (slc19a2) gene knockout mice showed abnormalities of erythroid, myeloid, and megakaryocyte lineages in bone marrow when fed a thiamine-free diet. 5 The importance of CAT1 (cationic amino acid transporter)-mediated transport was recently underscored by the production of cat1 gene knockout mice, which exhibit anemia. 6 These reports indicate that substrates of transporters expressed on hematopoietic stem cells are physiologically essential in differentiation or proliferation of the cells, or both, and the expression levels and activities of these transporters are likely to be regulated stage and lineage specifically.Cationic amino acid transporter (CAT, slc7a), known as system y ϩ , transports cationic amino acids such as L-lysine, L-histidine, L-ornithine, and L-arginine. 7-9 System y ϩ is a facilitative process that is Na ϩ -independent and pH insensitive. CAT1 (slc7a1), whose mouse ortholog was first identified as a virus receptor, 10,11 is expressed ubiquitously, including fetal liver and bone marrow, though not adult liver. 12 It is known that fetal and embryonic liver plays a significant role in hematopoiesis. 13 Furthermore, CAT1 mRNA is induced in regenerating liver in a short-lived manner and has multiple sites for regulation of gene expression, indicating that system y ϩ is tightly regulated and essential for liver cells to enter mitosis. 14,15 These findings suggest that CAT1 could be involved in hematopoietic activity.One of the substrates of CAT1, L-arginine, is a precursor of nitric oxide (NO) and polyamines. NO can induce apoptosis in megakaryocytes and platelet formation. 16,17 L-ornithine is produced by arginase and further metabolized to polyamines, which regulate the cell cycle. 18 Recently, low concentrations of L-glutamine were reported to induce functional differentiation of U937 myelomonocytic cells. 19 These facts suggest that low-molecular-weight molecules such as amino acids may be involved in hematopoiesis, as well as large-molecular cytokines such as IL-3 and erythropoietin. In the present study, we investigated the effects of hCAT1 expression level and L-arginine on differentiation and proliferation of human cord blood cells and a cloned human erythroid model, K562 cells. Materials and methods Tissue cultur...

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Total synthesis of Hirsutellone B has been achieved by a convergent synthetic strategy. This synthesis features direct construction of the highly strained 13-membered macrocycle of Hirsutellone B utilizing the Ullmann-type reaction. To the best of our knowledge, this is the first application of macrocyclization utilizing an intramolecular Ullmann-type reaction between an aliphatic alcohol and aryl halide.

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