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  • 1
    ISSN: 1573-904X
    Keywords: oligonucleotide ; protamine sulfate ; artificial viral envelope ; confocal laser scanning microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS. Methods. Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligo- nucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE™-3 = artificial viral envelope). Results. PS-ON complexes and AVE™-3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs. Conclusions. AVE™ associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1238
    Keywords: Key words Intensive care ; Critical care ; Acute respiratory distress syndrome ; Persistent pulmonary hypertension of the newborn ; Feedback inhibition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Objective: To examine the kinetics of successful nitric oxide (NO) withdrawal in vivo and in vitro.¶Design and setting: Prospective study in a university pediatric intensive care ward and research laboratory.¶Patients and materials: Nineteen patients with acute respiratory distress syndrome (ARDS) or persistent pulmonary hypertension of the newborn (PPHN). Primary porcine pulmonary artery cells in vitro.¶Interventions: NO inhalation and withdrawal in patients; exposure to NO donor sodium nitroprusside (SNP) and gaseous NO in vitro.¶Measurements and results: In patients: a slight, but significant, increase of oxygenation index (OI) from 4.57 ± 0.24 cmH2O/torr (mean ± SEM) to 4.90 ± 0.26 cmH2O/torr after withdrawal of NO (p 〈 0.001). Recovery of OI (4.43 ± 0.23 cmH2O/torr) 30 min after weaning, a significant drop after 4 h (3.72 ± 0.17 cmH2O/torr; p 〈 0.001), values restored after 12 h.¶In vitro: NO synthase (NOS) activity was significantly lower in SNP-incubated cells (20.0 ± 4.0 μm/min) than in control cells (37.6 ± 7.0 μm/min; p 〈 0.05). Thirty minutes after SNP withdrawal there was NOS activity of 35.8 ± 10.0 μm/min with a significant increase by 4 h (p 〈 0.05). No alteration of endothelial NOS (ENOS) mRNA expression by NO (Northern Blot).¶Conclusion: In patients there is a slight, but significant, reversible increase of OI after successful weaning from NO. In vitro, NO leads to a reversible decrease of ENOS activity on a post mRNA level, resembling clinical observations.
    Type of Medium: Electronic Resource
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