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.     

Genome-wide analysis of mRNAs targeted to yeast mitochondria

It is agreed that nuclear-encoded mitochondrial proteins are post-translationally targeted to mitochondria, even if, in some cases, a co-translational phase can assist the import of precursor proteins. We used yeast DNA microarrays to analyse the mRNA populations associated with free and mitochondrion-bound polysomes. As expected, many mRNAs, known to encode mitochondrial proteins, are localized to free cytoplasmic polysomes, but many are localized to mitochondrion-bound polysomes. Furthermore, the 3′-UTR of six randomly chosen mitochondrion-bound mRNAs contains sufficient information to target, in vivo, non-translatable RNA to the vicinity of mitochondria. Interestingly, genes producing mRNAs that are targeted to mitochondria are mainly of ancient bacterial origin, whereas those producing mRNAs that are translated in the cytoplasm are mainly of eukaryotic origin. These observations, which support the recent hypotheses concerning the dual origin of the mitochondrial proteome, provide new insights into the biogenesis of mitochondria.     

Total synthesis and evaluation of lamellarin alpha 20-Sulfate analogues

In order to explore the influence of sulfate groups on the bioactivity profiles of marine alkaloids of the lamellarin class, three such alkaloids, lamellarin alpha, lamellarin alpha 13,20-disulfate and lamellarin H, were synthesized and their activities against HIV-1 integrase and cancer cell lines were compared with those of lamellarin alpha 20-sulfate, which is a selective inhibitor of HIV-1 integrase. Lamellarin alpha does not inhibit HIV-1 integrase but shows moderate cytotoxicity with good cell line selectivity. Lamellarin alpha 13,20-disulfate is a moderate inhibitor of both HIV-1 integrase and cancer cell lines. Lamellarin H is a more potent inhibitor of HIV-1 integrase but lacked the specificity required to be medicinally useful.     

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.     

Human amyloid-beta causes changes in the levels of endothelial protein kinase C and its alpha isoform in vitro

Amyloid-beta (A(beta)) deposits and neurofibrillary pathology are characteristic features of Alzheimer's disease (AD). The association of A(beta) with cerebral vessels is an intriguing feature of AD. While there is considerable evidence of altered activities of the major isoforms of protein kinase C (PKC) in the vasculature and neurons of AD brains, little is known about the relationship between the Abeta toxicity and the altered PKC levels in cerebral endothelial cells.In this study, cultured brain endothelial cells exposed to A(beta)1-40 revealed a translocation of PKC from the membrane fraction to the cytosol. The content of the isoform PKC(alpha), involved in the regulation of amyloid precursor protein (APP) secretion, was decreased in the membrane-bound fraction of rat endothelial cells and increased in the cytosol after A(beta)1-40 treatment. These data suggest that the accumulation of A(beta) peptide in the cerebral vasculature may play a significant role in the down-regulation of PKC seen in the AD cerebral vasculature.     

Plk phosphorylation regulates the microtubule-stabilizing protein TCTP

The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.     

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.     

Miniaturized analytical assays in biotechnology

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques.In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.