Ik Jung Kim

Synchrotron Small-Angle X-ray Scattering Studies of the Structure of Porcine Pepsin under Various pH Conditions

Structural characteristics of various conformational states of porcine pepsin in solution under different pH conditions were investigated in terms of size and shape by small-angle X-ray scattering (SAXS). Low-resolution structural models of porcine pepsin were reconstructed from SAXS data, which were made inside the search volume of maximum dimension (Dmax), calculated from the pair distance distribution function p(r). The reconstructed structural models were obtained without imposing any restrictions on the symmetry or anisometry of the pepsin molecule. Under conditions emulating those for physiological activity of the enzyme, the reconstructed structural models exhibited a more extended C-terminal domain compared to the crystal structure. The differences between the solution and crystal structures of pepsin can be explained by inherent conformations of the flexible subdomain in the C-terminal domain under the solution pH conditions. Under mild acidic conditions where the enzyme is inactive, the reconstructed structural models revealed a compact globular conformation similar in overall shape to the crystal structure. These results indicate that the changes in fluorescence and circular dichroism curves observed under acidic conditions could also arise from the inherent conformation of the flexible subdomain, which has a tendency to roll into a sphere in the overall structure, but without affecting the stability of internal structure. Furthermore, the conformational changes in the subdomain might explain the inactivity of pepsin under mildly acidic conditions. Finally, compared to neutral denaturing conditions, pepsin under alkaline denaturing conditions had a larger expanded vertical conformation in the reconstructed model, as a consequence of alkaline denaturation of the N-terminal domain and a fully extended conformation of the C-terminal domain. The structural evidence presented here may have important implications for understanding the relationship between the structure of porcine pepsin and enzymatic function.

Biological Affinity and Biodegradability of Poly(propylene carbonate) Prepared from Copolymerization of Carbon Dioxide with Propylene Oxide

In this study we investigated bacterial and cell adhesion to poly(propylene carbonate) (PPC) films, that had been synthesized by the copolymerization of carbon dioxide (a global warming chemical) with propylene oxide. We also assessed the biocompatibility and biodegradability of the filmsin vivo, and their oxidative degradation in vitro. The bacteria adhered to the smooth, hydrophobic PPC surface after 4 h incubation.Pseudomonas aeruginosa andEnterococcus faecalis had the highest levels of adhesion,Escherichia coli andStaphylococcus aureus had the lowest levels, andStaphylococcus epidermidis was intermediate. In contrast, there was no adhesion of human cells (cell line HEp-2) to the PPC films, due to the hydrophobicity and dimensional instability of the surface. On the other hand, the PPC films exhibited good biocompatibility in the mouse subcutaneous environment. Moreover, contrary to expectation the PPC films degraded in the mouse subcutaneous environment. This is the first experimental confirmation that PPC can undergo surface erosion biodegradationin vivo. The observed biodegradability of PPC may have resulted from enzymatic hydrolysis and oxidative degradation processes. In contrast, the PPC films showed resistance to oxidative degradationin vitro. Overall, PPC revealed high affinity to bioorganisms and also good biodegradability.

The biocompatability of mesoporous inorganic-organic hybrid resin films with ionic and hydrophilic characteristics.

New mesoporous silicate-titania resin systems hybridized with 4,5-dihydroxy-m-benzenedisulfonic acid and poly(ethylene glycol)-dimethacrylate component were developed. These inorganic-organic hybrid resins were found to reveal highly controlled ionic and hydrophilic surface with excellent durability and adhesion onto various substrates. The resin films revealed high resistance to nonspecific adsorption of fibrinogen and to adherence by several bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Furthermore, excellent biocompatibility of the developed resins was proved by both HEp-2 cell adhesion in vitro and subcutaneous implantation in mice. The inorganic-organic hybrid resins are strongly promising for biomedical applications including biomedical devices and biosensors.

Well-defined DNA-mimic brush polymers bearing adenine moieties: synthesis, layer-by-layer self-assembly, and biocompatibility.

Two new DNA-mimicking brush polymers were synthesized: poly[oxy(11-(3-(9-adeninyl)propionato)-undecanyl-1-thiomethyl)ethylene] (PECH-AP) and poly[oxy(11-(5-(9-adenylethyloxy)-4-oxopentanoato)undecanyl-1-thiomethyl)ethylene] (PECH-AS). These polymers were found to be thermally stable up to 220 °C and could be applied easily by conventional coating processes to produce good quality films. Interestingly, both brush polymers formed molecular multibilayer structures to provide an adenine-rich surface. Despite the structural similarities, PECH-AS surprisingly exhibited higher hydrophilicity and better water sorption properties than PECH-AP. These differences were attributed to the chemical structures in the bristles of the polymers. The adenine-rich surfaces of the polymer films demonstrated selective protein adsorption, suppressed bacterial adherence, facilitated HEp-2 cell adhesion, and exhibited good biocompatibility in mice. However, the high hydrophilicity and good water sorption characteristics of the PECH-AS film suggest that this brush polymer is better suited to applications requiring good biocompatibility and reduced chance of bacterial infection compared with the PECH-AP film.

The biocompatibility of self-assembled brush polymers bearing glycine derivatives

We have synthesized brush polymers with various glycine derivatives as the end groups of their long alkyl bristles. The polymers are thermally stable up to 170-210 degrees C and form good quality films through conventional spin- or dip-coating and subsequent drying. Interestingly, the thin films of these brush polymers exhibit different molecular multi-layer structures that arise through the efficient self-assembly of the bristles with glycine derivative end groups. These brush polymer films have hydrophilic surfaces and exhibit some water sorption. The extent of the water sorption by these films depends upon the nature of the glycine derivatives in the bristle end. These films not only repel fibrinogen molecules and platelets from their surfaces, but also have high resistance to bacterial adherence. Moreover, the films were found to provide conducive surface environments for the successful anchoring and growth of HEp-2 cells, and to exhibit excellent biocompatibility in mice. These brush polymers have potential uses in biomedical applications including medical devices, especially blood contacting devices such as catheters, stents, blood vessels, and biosensors, due to their enhanced biocompatibility and the reduced possibility of post-operative infection.

Inhibition of the EGF-induced activation of phospholipase C-γ1 by a single chain antibody fragment

Phospholipase C-γ1(PLC-γ1) is known to play an essential role in various cellular responses, such as proliferation and tumorigenesis, and PLC-γ1-specific inhibitors are commonly employed to investigate the mechanism of the PLC-γ1-mediated signaling pathway. In this study, we developed a single chain antibody fragment (scFv) as a blocker for PLC-γ1 mediated signaling. scFv, designated F7-scFv, specifically bound to PLC-γ1 with high affinity (Kd=1.9×10−8 M) in vitro. F7-scFv also bound to PLC-γ1 in vivo and altered the distribution pattern of PLC-γ1 from the cytoplasm to the intracellular aggregates, where F7-scFv was localized. Moreover, F7-scFv interrupted the EGF-induced translocation of PLC-γ1 from the cytosol to the membrane ruffle and attenuated EGF-induced inositol phosphates generation and intracellular calcium mobilization. These results indicate that F7-scFv blocks EGF-induced PLC-γ1 activation by causing sequestering of PLC-γ1 into intracellular aggregates, and may therefore be useful in studies of the PLC-γ1-mediated signaling pathway.

New self-assembled brush glycopolymers: Synthesis, structure and properties

A new series of chemically well-defined brush glycopolymers consisting of a polyoxyethylene backbone and bristles bearing glycosyl and methyl end groups was synthesized with various compositions. The glycopolymers were thermally stable up 200 °C and were soluble in a variety of common solvents. The brush polymer films formed multibilayer structures, the layers of which were stacked along the direction normal to the film plane so as to display a glycosyl group-rich surface or a methyl group-rich surface or their mixture, depending on the bristle end group composition. The multibilayer structures were stabilized by the self-assembly of the bristles via lateral packing. The glycosyl-rich surface played a critical role in enhancing the surface hydrophilicity and water sorption to a certain level; thus, the glycopolymer films easily formed a hydration layer to a certain depth on the film surface. The hydrophilic surfaces and hydration layer efficiently prevented protein adsorption onto the brush glycopolymers and suppressed bacterial adherence while promoting mammalian cell adhesion and displaying excellent biocompatibility in an in vivo mouse study.

Biocompatible characteristics of sulfobetaine-containing brush polymers

AbstractA series of well-defined brush polymers, poly(oxy(11-(3-sulfonylpropyltrimethyl-glycinyl)undecylesterthiomethyl) ethylene-co-oxy(n-dodecylthio-methyl)ethylene)s (PECH-DMAPSm, where m is the mol% of the DMAPS [sulfobetaine] end group) were synthesized. The thermal properties and phase transitions of these polymers were investigated. The polymers were thermally stable up to 185 °C and were found to form favorably into multibilayer structures, always providing hydrophilic, zwitterionic sulfobetaine end groups at the film surface. Because of the presence of these sulfobetaine groups at the surface, the polymer films promoted HEp-2 cell adhesion and revealed biocompatibility in mice but significantly suppressed protein adsorption. These results collectively indicate that the sulfobetaine-containing brush polymers are suitable for use in biomedical applications, including medical devices and biosensors that require biocompatibility.