Ha Chin Sung

Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum.

Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 °C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH·NADH binary complex, and Aa MDH·NAD·oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-Å resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH fromThermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55 °C, whereas Tf MDH is fully active at 90 °C for 1 h. Aa MDH shows 2–3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4–10 °C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.

Optimization of Lipid Composition in Cationic Emulsion as In Vitro and In Vivo Transfection Agents

Purpose. To enhance in vitro and in vivo transfection activity by optimizing lipid composition of cationic lipid emulsions.Methods. Various emulsion formulations having different cationic lipids as emulsifiers, and additional helper lipids as co-emulsifiers, were prepared. The stability of the emulsion and its complex with DNA was investigated by measuring the particle size change in phosphate buffer saline (PBS) over a period of 20 days. The activity of the emulsions in transfecting pCMV-beta into COS-1 cells in the presence or absence of 80% serum was evaluated. We also evaluated in vivo transfection activity using intravenously administered pCMV-Luc+ as a reporter gene.Results. Among the cationic emulsifiers, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) formed the most stable and efficient emulsion gene carrier. Addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increased in vitro transfection activity, but slightly compromised the stability of the emulsion. The loss was compensated for by including small amounts of Tween 80 in the emulsion. The in vitro and in vivo transfection activities were also increased by adding Tween 80. Even though in vitro transfection activity of liposomes was high in the absence of serum, the transfection activity of emulsions was far greater than that of liposomes in the presence of serum and for in vivo applications.Conclusions. By including DOPE as an endosomolytic agent and Tween 80 as a stabilization agent, the cationic emulsion becomes a more potent gene carrier for in vitro and in vivo applications, especially in the presence of serum.

The effects of serum on the stability and the transfection activity of the cationic lipid emulsion with various oils.

The emulsions with various oils such as linseed oil, soybean oil and squalene were prepared to obtain the relationship between the stability and the transfection activity of the emulsions. 1,2-Dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) was used as a single cationic lipid emulsifier. The droplet sizes and size distributions of DOTAP emulsions were dependent on oils which had different interfacial tensions. The droplet sizes followed the order of squalene emulsion<soybean oil emulsion<linseed oil emulsion. The squalene emulsion was the most stable carrier since it kept its integrity in serum and PBS solution. For in vitro gene transfer, the transfection activities of the lipid carriers in the presence of serum followed the order of squalene emulsion>soybean oil emulsion>linseed oil emulsion>DOTAP liposome. The squalene emulsion showed the least cytotoxicity with or without serum. For in vivo gene transfer, the squalene emulsion also had the most potent transfection activity in the mouse after intravenous administration. Squalene as the oil component can enhance the stability of cationic emulsion more effectively that could be useful for the transfer of genes in vitro and in vivo.

The role of non-ionic surfactants on cationic lipid mediated gene transfer

Cationic lipid carriers were made of 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP), squalene and different amounts of non-ionic surfactants. Various non-ionic surfactants were selected to elucidate the role of Tween 80 in the cationic lipid mediated gene delivery. They had a similar structure to Tween 80 such as various poly(ethyleneglycol) (PEG) chain lengths and acyl chain with different headgroups. For comparison, lipid carriers were also prepared with 1,2-dioleoyl-sn-glycero-3-trimethylammoniumpropane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Addition of non-ionic surfactants decreased the emulsion-DNA interaction and affected the transfection activity depending on the chain length and the content of PEG in the surfactant. Among the surfactants, Tween 80 yielded the best transgene expression without showing toxicity in COS-1 cells. The delivery mechanism of the complex was investigated by measuring the effects of endocytosis inhibitors (chloroquine and wortmannin). The emulsion-DNA complex seems to be taken up by the cells via endocytosis.

Airway gene transfer using cationic emulsion as a mucosal gene carrier

Delivery of genes to airway mucosa would be a very valuable method for gene therapy and vaccination. However, there have been few reports on suitable gene delivery systems for administration. In this study, we use a cationic emulsion system, which is physically stable and facilitates the transfer of genes in the presence of up to 90% serum, as a mucosal gene carrier.