Arthur O. Hawtrey

Effect of Polyethylene Glycol Conjugated to DNA-Transfecting Complexes Targeted at the Transferrin Receptor of HeLa Cells

AbstractModification of streptavidin (biotinylated transferrin)-biotinylated polylysine conjugates for DNA delivery by covalent addition of polyethylene glycol chains was investigated. It was found that addition of two to four monomethoxy-polyethylene glycol chains to bio20-polylysine100, a component of the transfecting conjugate, increased luciferase activity approximately four- to five-fold in a HeLa cell system. The increase occurred only in the presence of the lysosomotropic agent chloroquine and was inhibited by free transferrin. The latter result indicating that DNA uptake was via transferrin receptor-mediated endocytosis. Titration of cell cultures with transfecting complexes, with and without attached polyethylene glycol, showed luciferase activity to decrease at higher concentrations of the complexes. This was more marked with complexes containing polyethylene glycol. Complexes containing higher concentrations of attached polyethylene glycol inhibited luciferase expression strongly. Free polyethy...

Effect of Nicotinic Acid Conjugated to DNA-Transfecting Complexes Targeted at the Transferrin Receptor of HeLa Cells

A conjugate consisting of streptavidin (biotinylated transferrin)-biotinylated polylysine for DNA delivery to cells was modified by partial nicotinylation of the polylysine component of the conjugate and used for transfection studies. A conjugate of biotin10-nicotinyl60-polylysine250 containing 60 weakly basic nicotinyl (pyridine-3-carboxyl) residues was prepared. The design of the modified polylysine was directed to the possible binding of H+ ions in the endosome-lysosomal vesicles (pH 5-6) by the nicotinyl groups, thus circumventing the use of chloroquine. The results obtained, however, while showing a 5- to 6-fold increase in luciferase transfection activity still necessitated an absolute requirement for chloroquine. A further polylysine conjugate containing a larger number of nicotinyl residues, biotin10-nicotinyl120-polylysine250, also was prepared and studied. This macromolecule stimulated luciferase activity to a small extent and was also dependent on chloroquine. Smaller biotinylated polylysine100 conjugates containing nicotinyl groups were also prepared. These were biotin10-nicotinyl30-polylysine100, and biotin10-nicotinyl60-polylysine100, respectively. Both substances, however, gave opaque, hazy aqueous solutions with precipitates on standing and could not be used for further experimental work. The results indicate that the introduction of weakly basic nicotinyl (pyridine-3-carboxyl) groups onto polylysine250 give conjugates that are unable to replace the lysosomotrophic agent chloroquine in the HeLa cell sysem studied. A 5- to 6-fold increase in luciferase activity, however, was found with biotin10-nicotinyl60-polylysine250.A conjugate consisting of streptavidin (biotinylated transferrin)-biotinylated polylysine for DNA delivery to cells was modified by partial nicotinylation of the polylysine component of the conjugate and used for transfection studies. A conjugate of biotin10- nicotinyl60-polylysine250 containing 60 weakly basic nicotinyl (pyridine-3-carboxyl) residues was prepared. The design of the modified polylysine was directed to the possible binding of H+ ions in the endosome-lysosomal vesicles (pH 5-6) by the nicotinyl groups, thus circumventing the use of chloroquine. The results obtained, however, while showing a 5- to 6-fold increase in luciferase transfection activity still necessitated an absolute requirement for chloroquine. A further polylysine conjugate containing a larger number of nicotinyl residues, biotin10-nicotinyl120-polylysine250, also was prepared and studied. This macromolecule stimulated luciferase activity to a small extent and was also dependent on chloroquine. Smaller biotinylated polylysine100 conjugates containing nicotinyl groups were also prepared. These were biotin10-nicotinyl30-polylysine, and biotin10-nicotinyl60-polylysine100, respectively. Both substances, however, gave opaque, hazy aqueous solutions with precipitates on standing and could not be used for further experimental work. The results indicate that the introduction of weakly basic nicotinyl (pyridine-3-carboxyl) groups onto polylysine250 give conjugates that are unable to replace the lysosomotrophic agent chloroquine in the HeLa cell sysem studied. A 5- to 6-fold increase in luciferase activity, however, was found with biotin10-nicotinyl60- polylysine250.

The effect of a peptide-containing synthetic lung surfactant on gas exchange and lung mechanics in a rabbit model of surfactant depletion

Background Currently, a new generation of synthetic pulmonary surfactants is being developed that may eventually replace animal-derived surfactants used in the treatment of respiratory distress syndrome. Enlightened by this, we prepared a synthetic peptide-containing surfactant (Synsurf) consisting of phospholipids and poly-l-lysine electrostatically bonded to poly-l-glutamic acid. Our objective in this study was to investigate if bronchoalveolar lavage (BAL)-induced acute lung injury and surfactant deficiency with accompanying hypoxemia and increased alveolar and physiological dead space is restored to its prelavage condition by surfactant replacement with Synsurf, a generic prepared Exosurf, and a generic Exosurf containing Ca2+. Methods Twelve adult New Zealand white rabbits receiving conventional mechanical ventilation underwent repeated BAL to create acute lung injury and surfactant-deficient lung disease. Synthetic surfactants were then administered and their effects assessed at specified time points over 5 hours. The variables assessed before and after lavage and surfactant treatment included alveolar and physiological dead space, dead space/tidal volume ratio, arterial end-tidal carbon dioxide tension (PCO2) difference (mainstream capnography), arterial blood gas analysis, calculated shunt, and oxygen ratios. Results BAL led to acute lung injury characterized by an increasing arterial PCO2 and a simultaneous increase of alveolar and physiological dead space/tidal volume ratio with no intergroup differences. Arterial end-tidal PCO2 and dead space/tidal volume ratio correlated in the Synsurf, generic Exosurf and generic Exosurf containing Ca2+ groups. A significant and sustained improvement in systemic oxygenation occurred from time point 180 minutes onward in animals treated with Synsurf compared to the other two groups (P < 0.001). A statistically significant decrease in pulmonary shunt (P < 0.001) was found for the Synsurf-treated group of animals, as well as radiographic improvement in three out of four animals in that group. Conclusion In general, surfactant-replacement therapy in the animals did not fully restore the lung to its prelavage condition. However, our data show that the formulated surfactant Synsurf improves oxygenation by lowering pulmonary shunt.

Synthetic Templates and the RNA Polymerase of Influenza a Virus

Abstract Polyadenylic acid (poly A) and polyguanylic acid (poly G) have been modified to give polymers containing and Gpm5C termini. Polymers containing methylated (Gpmf C) termini are inactive as templates for the RNA-dependent RNA polymerase of Influenza A virus.

Synthesis of Guanylyl (3′→5′)-5-methylcytidine (Gpm5C), a Dinucleoside Monophosphate which is Unable to Function as a Primer in the Synthesis of RNA by the Influenza A Virus RNA Polymerase

Abstract Guanylyl (3′→5′)-5-methylcytidine (Gpm5C) has been synthesized enzymatically through the use of T1RNAse at high enzyme dilution. In contrast with GpC, the methylated dinucleoside monophosphate is shown to be inactive as a primer for RNA synthesis by the RNA-dependent RNA polymerase of Influenza A virus.