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F C Szoka

Publications and source records attributed to F C Szoka.

11 recordsLinked to original sources
PubMed1992

Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices.

Antisense oligonucleotides (ASOs) are designed to bind to a specific mRNA and selectively suppress its translation. To facilitate selection of optimal ASO targets, we have developed three thermodynamic indices to evaluate putative structural complexes important in ASO action. These indices are: a secondary structure score (Sscore), which estimates the strength of local mRNA secondary structures at the ASO target site; a duplex score (Dscore), which estimates the delta Gformation for the ASO:mRNA target sequence duplex; and a competition score (Cscore), which is the difference between the Dscore and the Sscore. We also present two histograms to graphically display these indices from different regions of the mRNA. The indices are compared to the inhibition reported in five studies of ASO-mediated suppression of gene expression. The Dscore is the most consistent predictor of ASO efficacy in four of the five studies (r2 from 0.44 to 0.99), while the results of the fifth study could not be predicted by any thermodynamic or physical index. Thus the Dscores and their histogram may prove useful in selection of ASO targets.

Algorithms↗
PubMed1992

Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes.

We compare the transfection efficiency of plasmid DNA encoding either luciferase or beta-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-eth anolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.

Cell Line↗
PubMed1992

Amino acid side-chain contributions to free energy of transfer of tripeptides from water to octanol.

The location of amino acids in soluble or membrane proteins is related to the hydrophobicity of the side chains. Amino acid hydrophobicity values are based upon the thermodynamics of transfer from an aqueous to a nonaqueous environment. However, for certain hydrophilic residues uncertainty exists on the appropriate hydrophobicity values. We have measured the octanol-water partition coefficients (Po/w) of tripeptides of the sequence N-14C-acetyl-Ala-X-Ala-NH-tButyl (AcAlaXAlaNHtButyl), where the central residue X was either Gly, Ala, Phe, Trp, Pro, His, Asp, or Glu. The Po/w for the tripeptides agreed reasonably well with values calculated by the fragment method of D. J. Abraham and A. J. Leo (Proteins Struct. Func. Gen. 2, 130-152, 1987). The log Po/w of the uncharged form was 1.6, 2.7, and 2.5 greater than the log Po/w of the ionized form for the His, Asp, and Glu peptide, respectively. The new data on the pH dependence of the ionizable side chains, His, Asp, and Glu, should result in better prediction of the partition coefficient of peptides as a function of pH. The thermodynamic parameters were determined from the temperature dependence of partitioning. In the temperature range studied (2 to 65 degrees C) the transfer of tripeptides from water to octanol was entropy governed except for the ionized peptides. A heat capacity term was necessary to account for the transfer of tripeptides containing non polar residues. The heat capacity change for transfer from water into octanol was -45, -73, -81, and -88 cal/mol K for Ala, Phe, Trp, and Pro peptides, respectively. Peptides containing Gly, His (pH 7.2), and the uncharged forms of Asp, Glu, and His did not show a significant change in heat capacity. The side-chain contribution of the central residue X (delta Gx) to the free energy of transfer was obtained from the difference between the free energy of transfer of the peptide containing the central residue X and the Gly peptide; delta Gx = delta G(AcAlaXAlaNHtButyl) - delta G(AcAlaGlyAlaNHtButyl). The relative order of hydrophobicity of the side chains correlated well with previous studies. However, a significant difference was found for the absolute hydrophobicity between the present study and experimental data on N-acetyl amino acid amide derivatives (J. Fauchere and V. Pliska, Eur. J. Med. Chem. 18(4), 369-375, 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids, Branched-Chain↗
PubMed1992

Prolonged systemic delivery of peptide drugs by long-circulating liposomes: illustration with vasopressin in the Brattleboro rat.

The value of novel systemically long-circulating liposomes to prolong the duration of an antidiuretic hormone, arg8-vasopressin (VP), was investigated as a representative of low molecular weight peptides with rapid clearance. Cholesterol content was found to have a controlling effect on VP release in serum. Three types of liposomes were selected for urine production measurements in VP deficient Brattleboro rats. One contained phosphatidylserine (PS), which was rapidly cleared from the circulation. In the other two liposomes, the PS component was replaced by either phosphatidylglycerol or a novel phospholipid derivatized with polyethylene glycol (PEG); both showing prolonged circulation. Free VP (up to 8 micrograms/kg) gave reduced urine production for less than 24 hr. The PG formulation exhibited a dose-dependent prolonged duration of bioactivity of up to 4 days. Substitution of PEG-PE resulted in a 2-day delay followed by a prolonged duration of bioactivity for over 4 days. The duration of the prolonged bioactivity was not dose dependent but the amplitude was. This is attributed to VP release from liposomes which have distributed intact to another compartment without having been taken up by the RES. By balancing liposome circulation time, release rate, and dose, long-circulating liposomes can be applied to prolong the biological activity of a therapeutic peptide.

Animals↗
PubMed1991

Iontophoretic delivery of a series of tripeptides across the skin in vitro.

The iontophoresis of eight tripeptides, of the general structure alanine-X-alanine, has been measured across hairless mouse skin in vitro. The peptides were blocked (a) at the carboxyl terminus using the mixed anhydride reaction with t-butylamine and (b) at the amino terminus by acetylation with 14C-acetic anhydride. The nature of the central residue (X) was varied by selecting one of five neutral amino acids, two negatively chargeable moieties (aspartic and glutamic acids), and a positively chargeable species (histidine). Constant current iontophoresis at 0.36 mA/cm2, using Ag/AgCl electrodes, was performed for 24 hr in diffusion cells, which allowed both anode and cathode to be situated on the same (epidermal) side of a single piece of skin. Due to a combination of osmotic and electroosmotic forces, the anodal iontophoretic flux of neutral peptides was significantly greater than passive transport. Steady-state fluxes were not achieved, however, suggesting that time-dependent changes in the properties of the skin barrier may be occurring. Limited, further experiments confirmed that, on a 24-hr time scale, these changes were not fully reversible. The cathodal delivery of anionic permeants was well controlled at a steady and highly enhanced rate by the current flow. This behavior closely paralleled earlier work using simple negatively charged amino acids and N-acetylated amino acid derivatives. It appears that the normalized iontophoretic flux of these anionic species is independent of lipophilicity but may be inversely related to molecular weight. The positively charged peptide, Ac-Ala-His-Ala-NH(But), showed greater anodal iontophoretic enhancement when delivered from a donor solution at pH 4.0 than from a solution at pH 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Cutaneous↗
PubMed1990

Rapid nuclear accumulation of injected oligodeoxyribonucleotides.

The intracellular transport and fate of nucleic acids is poorly understood. To study this process, we injected fluorescent oligodeoxyribonucleotides (oligos) into the cytoplasm of CV-1 epithelial cells and primary human fibroblasts. Rapid nuclear accumulation was found with the phosphodiester (PD), phosphorothioate (PT), and methylphosphonate (MP) forms of a 28-mer oligo complimentary to the rev mRNA of the human immunodeficiency virus type 1. Migration of the oligos in the cytoplasm was slower than diffusion of a coinjected dextran, but the oligos freely diffused into the nucleus. Nuclear incorporation was temperature but not energy dependent. The intranuclear distribution of the oligos was influenced by the chemistry of internucleoside linkages. The PD oligos and, to a lesser extent, the PT oligos colocalized with small nuclear ribonucleoproteins (snRNPs), whereas the MP oligos colocalized with concentrated regions of genomic DNA. These data have important implications for our understanding of the transport and accumulation of exogenous nucleic acids in mammalian nuclei, and the assay described could potentially be used for testing the efficacy of oligos designed as therapeutic agents.

Base Sequence↗
PubMed1979

Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins.

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.

Animals↗
PubMed1979

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 micron membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 micron while maintaining an acceptable level of encapsulation of the aqueous phase.

Carbonates↗
PubMed1979

The use of aqueous space markers to determine the mechanism of interaction between phospholipid vesicles and cells.

A method has recently been introduced that quantitates the extent of phospholipid vesicle-cell interactions by following the amount of a vesicle-entrapped water-soluble fluorescent probe, carboxyfluorescein (CF) that becomes cell associated (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Hagins, W.A. (1977) Science 195, 489--492). We have characterized some of the properties of this probe in sonicated phospholipid vesicles. The CF undergoes a pH-dependent quenching as previously reported and both a pH- and temperature-dependent efflux from vesicles. Decreasing the pH from 7.4 to 5.0 results in almost a 100-fold increase in CF efflux from the vesicles. The simultaneous measurement of cell-associated tritiated lipid and CF fluorescence reveals a discrepancy between the two markers with the tritiated phospholipid becoming associated to 5--10-fold greater extent than the CF. In the presence of cells the leakage of CF from vesicles increases from 1.5- to 10-fold depending on the vesicle composition. This data suggests that interpretations of cell-vesicle interactions followed by the CF technique or other aqueous space markers should be done with caution. However, in experiments where the leakage of CF from vesicles can be controlled, the technique can provide useful information.

Animals↗