Interaction of the tetracyclines with double-stranded RNAs of random base sequence: new perspectives on the target and mechanism of action

The 16S rRNA binding mechanism proposed for the antibacterial action of the tetracyclines does not explain their mechanism of action against non-bacterial pathogens. In addition, several contradictory base pairs have been proposed as their binding sites on the 16S rRNA. This study investigated the binding of minocycline and doxycycline to short double-stranded RNAs (dsRNAs) of random base sequences. These tetracyclines caused a dose-dependent decrease in the fluorescence intensities of 6-carboxyfluorescein (FAM)-labelled dsRNA and ethidium bromide (EtBr)-stained dsRNA, indicating that both drugs bind to dsRNA of random base sequence in a manner that is competitive with the binding of EtBr and other nucleic acid ligands often used as stains. This effect was observable in the presence of Mg2+. The binding of the tetracyclines to dsRNA changed features of the fluorescence emission spectra of the drugs and the CD spectra of the RNA, and inhibited RNase III cleavage of the dsRNA. These results indicate that the double-stranded structures of RNAs may have a more important role in their interaction with the tetracyclines than the specific base pairs, which had hitherto been the subject of much investigation. Given the diverse functions of cellular RNAs, the binding of the tetracyclines to their double-stranded helixes may alter the normal processing and functioning of the various biological processes they regulate. This could help to explain the wide range of action of the tetracyclines against various pathogens and disease condition

CD8+T cell-mediated enhancement of tumour necrosis factor-alpha (TNF-α) production and HIV-1 LTR-driven gene expression in human monocytic cells is pertussis toxin-sensitive

HIV replication and LTR-mediated gene expression can be modulated by CD8+T cells in a cell type-dependent manner. We have previously shown that supernatants of activated CD8+ T cells of HIV-infected individuals greatly enhanced p24 levels in human macrophages infected with NSI or SI primary isolates of HIV-1. Here we have examined the effect of culture with CD8+T cell supernatants on HIV-1 LTR-mediated gene expression in monocytic cells. CD8+T cell supernatants enhanced LTR-mediated gene expression in U38 cells activated with Tat in the absence or presence of phorbol myristate acetate (PMA) and ionomycin or TNF-α. Further, enhancement of LTR-mediated gene expression and virus replication in U38 cells and U1 cells, respectively, was pertussis toxin-sensitive. The enhancement of gene expression and virus replication was associated with increased levels of TNF-α and was significantly abrogated by antibody to TNF-α. In contrast, the suppression of LTR-mediated gene expression by CD8+T cell supernatants in Jurkat T cells was not pertussis toxin-sensitive and TNF-α levels were not affected. These results demonstrate that factors produced by CD8+T cells utilize different cellular pathways to mediate their effects on HIV transcription and replication in different cell types