Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.     

Genomic correlates of hyperthermostability,an update

It has been shown (Cambillau, C., and Claverie, J. M. (2000) J. Biol. Chem. 275, 32383-32386) that a large difference between the proportions of charged versus polar (non-charged) amino acids (CvP-bias) was an adequate, if empirical, signature of the proteome of hyperthermophilic organisms (T(growth) >80 degrees C). Since that study, the number of available microbial genomes has more than doubled, raising the possibility that the simple CvP-bias rule might no longer hold. Taking advantage of the new sequence data, we re-analyzed the genomes of 9 fully sequenced thermophiles, 9 hyperthermophiles, and 53 mesothermophile microorganisms to identify the genomic correlates of hyperthermostability on a wider data set. Our new results confirm that the CvP-bias previously identified on a much smaller data set still holds. Moreover, we show that it is an optimal criterion, in the sense that it corresponds to the most discriminating factor between hyperthermophilic and mesothermophilic microorganisms in a principal component analysis. In parallel, we evaluated two other recently proposed correlates of hyperthermostability, the proteome average pI and the dinucleotide statistical index (Kawashima, T., Amano, N., Koike, H., Makino, S., Higuchi, S., Kawashima-Ohya, Y., Watanabe, K., Yamazaki, M., Kanehori, K., Kawamoto, T., Nunoshiba, T., Yamamoto, Y., Aramaki, H., Makino, K., and Suzuki, M. (2000) Proc. Natl. Acad. Sci. 97, 14257-14262). We show that the CvP-bias is the sole criterion that is able to clearly discriminate hyperthermophile from mesothermophile microorganisms on a global genomic basis.     

Synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins

Toyocamycin and some analogues have shown potent antitumor activities; however, none of them could be used clinically primarily owing to their cytotoxicity to normal human cells. In order to overcome the weakness of these nucleoside analogues, substitution of a variety of modified sugars for the ribofuranose was explored in our laboratories with expectation that certain sugar-modified toyocamycin analogues may be selectively cytotoxic to cancer cells. In this article, we report synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins, which were prepared via the condensations of 4-C- and 5-C-substituted ribofuranose derivatives 11, 12, 13, 20, 21, and 26 with the silylated form of 4-amino-6-bromo-5-cyanopyrrolo[2,3-]pyrimidine (27) and subsequent debromination and debenzoylation. When compared to the parent toyocamycin, all these analogues showed much lower cytotoxicity to human prostate cancer cells (HTB-81), mouse melanoma cancer cells (B16) as well as normal human fibroblasts. Compound 1e showed a significant cytotoxicity to the prostate cancer cells and a moderate selectivity. The results suggested that sugar modifications, especially those that may affect phosphorylation of nucleosides, could alter cytotoxicity profile significantly.     

trans-Sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction.     

Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.     

Proliferative generation of mammalian auditory hair cells in culture

Hair cell (HC) and supporting cell (SC) productions are completed during early embryonic development of the mammalian cochlea. This study shows that acutely dissociated cells from the newborn rat organ of Corti, developed into so-called otospheres consisting of 98% nestin (+) cells when plated on a non-adherent substratum in the presence of either epidermal growth factor (EGF) or fibroblast growth factor (FGF2). Within cultured otospheres, nestin (+) cells were shown to express EGF receptor (EGFR) and FGFR2 and rapidly give rise to newly formed myosin VIIA (+) HCs and p27(KIP1) (+) SCs. Myosin VIIA (+) HCs had incorporated bromodeoxyuridine (BrdU) demonstrating that they were generated by a mitotic process. Ultrastructural studies confirmed that HCs had differentiated within the otosphere, as defined by the presence of both cuticular plates and stereocilia. This work raises the hypothesis that nestin (+) cells might be a source of newly generated HCs and SCs in the injured postnatal organ of Corti.