Biodegradable cationic nanogels with tunable size, swelling and pKa for drug delivery.

Cationic polymers have garnered significant interest for their utility in intracellular drug delivery and gene therapy. However, due to their associated toxicities, novel synthesis approaches must be explored to develop materials that are biocompatible. The novel library of nanoparticles synthesized in this study exhibit tunable hydrodynamic diameters, composition and pH-responsive properties as a function of synthesis parameters. In addition, differences in the responsiveness of these nanoparticles under different pH conditions affords greater control over intracellular drug release.

Porcine conceptus Oct-4 mRNA expression during peri-implantation development.

OCT-4 is a transcriptional regulator of pluripotent cells throughout embryonic and germ cell lineage development prior to cellular differentiation during murine, bovine and porcine peri-implantation development. In contrast to murine OCT-4 expression, bovine and porcine expression is detected in both the inner cell mass and trophoblast. Delayed down regulation of OCT-4 gene expression in farm species may be a consequence of the lengthened period of peri-implantation. Expression of OCT-4 mRNA has not been characterized during conceptus attachment to the uterine surface in the pig. The objective of the present study was to determine conceptus OCT-4 mRNA expression during porcine peri-implantation development from days 10-17 of gestation. Total RNA was extracted from multiple pools of conceptuses collected on days 10, 12, 13, 15 and 17 of pregnancy. Quantitative RT-PCR was utilized to assay conceptus OCT-4 mRNA synthesis. Day of conceptus development significantly affected (p < 0.001) OCT-4 mRNA expression. Conceptus expression of OCT-4 was greatest on days 10 and 12 of pregnancy being approximately 7.7- and 11.6-fold greater compared to expression on days 15 and 17, respectively. Results from the present study suggest that down regulation of OCT-4 may be critical in trophoblastic expansion and uterine epithelial attachment during establishment of pregnancy in the pig.

Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines.

To examine the importance of side chain packing to protein stability, each of the 11 leucines in staphylococcal nuclease was substituted with isoleucine and valine. The nine valines were substituted with leucine and isoleucine, while the five isoleucines, previously substituted with valine, were substituted with leucine and methionine. These substitutions conserve the hydrophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously substituted with valine, were substituted with isoleucine to test the importance of packing at sites normally not occupied by a hydrophobic residue. The stabilities of these 58 mutant proteins were measured by guanidine hydrochloride denaturation. To the best of our knowledge, this is the largest library of single packing mutants yet characterized. As expected, repacking stability effects are tied to the degree of side chain burial. The average energetic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit with a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic transfer energy of a methylene from octanol to water. These results appear to indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrophobic transfer effects.

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.

High apparent dielectric constants in the interior of a protein reflect water penetration.

A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein.

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.

The M32L substitution of staphylococcal nuclease: disagreement between theoretical prediction and experimental protein stability.

The M32L substitution mutation of staphylococcal nuclease was made to test the theoretical prediction by Yamaotsu, Moriguchi and Hirono that it would be approximately 1.6 kcal/mol more stable than the wild-type protein. Instead M32L and the closely related M32I mutant were 0.8 and 0.6 kcal/mol less stable than the wild-type protein, respectively. The theoretical treatment had successfully predicted the stability effects of other mutations in staphylococcal nuclease. The discrepancy found here may be due to a general problem of the theoretical treatment, such as inadequate molecular dynamics simulation time, or possibly due to more specific difficulty in assessing the strength of the sulfur-aromatic interaction that is present in the wild-type.

The Spencer Op-Site Applicator, a new technique for treating the burned hand.

A technique to apply Op-Site Incise Drape to the burned hand is described using the Spencer Op-Site Applicator. The technique allows for the use of a transparent, flexible, semi-permeable plastic glove in the management of the burned hand in all age-groups. This minimizes excoriation of normal tissue but allows full flexibility of the hand with good healing.