Binding assay of human Dectin-1 variants to DNA/ß-glucan complex for active-targeting delivery of antisense DNA.

The lectin Dectin-1 is a good target for ß-glucan-mediated drug delivery. Although many murine studies of Dectin-1 have been performed, its human analog has not been studied well in terms of being a drug delivery target. We thus analyzed human Dectin-1 cDNA obtained from chronic myelogenous leukemia-derived cells, CML-1, and confirmed the findings of previous studies that there are many isoforms of human Dectin-1 due to exon skipping, although murine Dectin-1 has only two forms. When we transfected the Dectin-1 gene into a non-Dectin-1-expressing cell line and examined cellular uptake of the antisense DNA/ß-glucan complex, we confirmed that expression of the target gene was effectively suppressed through ß-glucan/Dectin-1-mediated uptake. The present results suggest that the ß-glucan complex would be an effective tool to deliver antisense oligonucleotide (AS-ODN) to Dectin-1-expressing cells not only for mice but also for humans.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Optimal sequence of antisense DNA to silence YB-1 in lung cancer by use of a novel polysaccharide drug delivery system.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a ß-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid.

Extracellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa, an extremophile which naturally grows in water-soluble mineral cutting oil (pH 10) used as metalworking fluid (MWF) for cooling and lubrication in industrial metalworking processes. The molecular mass of the purified lipase was estimated by SDS-PAGE to be 54 kDa. The optimum pH and temperature were 11 and 70 degrees C, respectively. The enzyme is stabile over a broad pH range (pH 4-11.5). The lipase preferably acted on triacylglycerols with medium-chain fatty acids. The lipase was inhibited strongly by Zn(2+), Hg(2+), Cu(2+) and slightly by Ca(2+) and Mg(2+). Non-ionic detergents and sodiumdeoxycholate enhanced lipase activity. Alkaline lipase from P. aeruginosa, capable of growing in a water-restricted medium has excellent properties and good potential for biotechnological applications in the metal industry. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool in a water-restricted medium with a variety of applications including organosynthetic reactions and the control and prevention of MWF putrification in the metal industry.

Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil.

The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P. aeruginosa san-ai protease functions optimally at pH 9.0 and 60 degrees C. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P. aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.