Differential role of PKC-induced c-Jun in HTLV-1 LTR activation by 12-O-tetradecanoylphorbol-13-acetate in different human T-cell lines

We have previously shown that TPA activates HTLV-1 LTR in Jurkat T-cells by inducing the binding of Sp1-p53 complex to the Sp1 site residing within the Ets responsive region 1 (ERR-1) of the LTR and that this activation is inhibited by PKCalpha and PKCepsilon. However, in H9 T-cells TPA has been noted to activate the LTR in two consecutive stages. The first stage is activation is mediated by PKCetta and requires the three 21 bp TRE repeats. The second activation mode resembles that of Jurkat cells, except that it is inhibited by PKCdelta. The present study revealed that the first LTR activation in H9 cells resulted from PKCetta-induced elevation of non-phosphorylated c-Jun which bound to the AP-1 site residing within each TRE. In contrast, this TRE-dependent activation did not occur in Jurkat cells, since there was no elevation of non-phosphorylated c-Jun in these cells. However, we found that PKCalpha and PKCepsilon, in Jurkat cells, and PKCetta and PKCdelta, in H9 cells, increased the level of phosphorylated c-Jun that interacted with the Sp1-p53 complex. This interaction prevented the Sp1-p53 binding to ERR-1 and blocked, thereby, the ERR-1-mediated LTR activation. Therefore, this PKC-inhibited LTR activation started in both cell types after depletion of the relevant PKCs by their downregulation. In view of these variable activating mechanisms we assume that there might be additional undiscovered yet modes of HTLV-1 LTR activation which vary in different cell types. Moreover, in line with this presumption we speculate that in HTLV-1 carriers the LTR of the latent provirus may also be reactivated by different mechanisms that vary between its different host T-lymphocyte subclones. Since this reactivation may initiate the ATL process, understanding of these mechanisms is essential for establishing strategies to block the possibility of reactivating the latent virus as preventive means for ATL development in carriers.     

Development,Optimization, and Evaluation of Carvedilol-Loaded Solid Lipid Nanoparticles for Intranasal Drug Delivery

Carvedilol, a beta-adrenergic blocker, suffers from poor systemic availability (25%) due to first-pass metabolism. The aim of this work was to improve carvedilol bioavailability through developing carvedilol-loaded solid lipid nanoparticles (SLNs) for nasal administration. SLNs were prepared by emulsion/solvent evaporation method. A 2³ factorial design was employed with lipid type (Compritol or Precirol), surfactant (1 or 2% w/v poloxamer 188), and co-surfactant (0.25 or 0.5% w/v lecithin) concentrations as independent variables, while entrapment efficiency (EE%), particle size, and amount of carvedilol permeated/unit area in 24 h (Q ₂₄) were the dependent variables. Regression analysis was performed to identify the optimum formulation conditions. The in vivo behavior was evaluated in rabbits comparing the bioavailability of carvedilol after intravenous, nasal, and oral administration. The results revealed high drug EE% ranging from 68 to 87.62%. Carvedilol-loaded SLNs showed a spherical shape with an enriched core drug loading pattern having a particle size in the range of 66 to 352 nm. The developed SLNs exhibited significant high amounts of carvedilol permeated through the nasal mucosa as confirmed by confocal laser scanning microscopy. The in vivo pharmacokinetic study revealed that the absolute bioavailability of the optimized intranasal SLNs (50.63%) was significantly higher than oral carvedilol formulation (24.11%). Hence, we conclude that our developed SLNs represent a promising carrier for the nasal delivery of carvedilol.     

Accumulation of untranslated lactose-specific messenger ribonucleic acid during catabolite repression in Escherichia coli

When Escherichia coli K-12 Hfr.H was induced to synthesize beta-galactosidase in the presence of glucose, an untranslated lactose-specific mRNA (lac-mRNA), protected from decay, was found to accumulate progressively within the cells. The lac-mRNA accumulation was unaffected by the carbon source on which the cells had been grown before the induction. The amount of the lac-mRNA available for translation was affected by catabolite repression and 3':5'-cyclic AMP, but it remained unclear whether this was a direct effect on the formation of the lac-mRNA or a consequence of the effect on its translation.     

Topoisomerase-II activity in human leukemic and lymphoblastoid cells

Topoisomerase-II activity was analyzed in various human leukemic and lymphoblastoid cell-lines with comparison to normal human peripheral blood lymphocytes. All of the examined tumor cells contained this enzyme in both the nuclear and cytoplasmic fractions, whereas no appreciable activity of the enzyme was detected in either fraction of the resting normal lymphocytes. Using pBR322 plasmid as a substrate, undialyzed extracts of the tumor cells exhibited the typical ATP-dependent relaxation of supercoiled circles and formation of linear and catenated structures, as well as the ATP-independent knotting activity. On the other hand, dialyzed extracts exerted only the ATP-dependent supercoil relaxation. Novobiocin inhibited the linearization and catenation but not the supercoil relaxing or knotting activities. This study provides indications for an excessive level of a structurally abnormal topoisomerase-II in these tumor cell-lines.