Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.

Objectives : The present study was designed to investigate effects of Ssanghwa-tang (Shuānghuā-gēng) on oxidation/reduction reaction-related and aging-related enzymes in vitro.Methods : We performed MTT assay, collagenase inhibition assay, elastase inhibition assay, tyrosinase inhibition assay, DPPH free radical scavenging assay, SOD-like activity and xanthine oxidase (XO) inhibition assay. Results : The 50% ethanol (EtOH) extract of Ssanghwa-tang (SHT) showed 55% inhibition of collagenase activity, and 42% inhibition of elastase activity at 1 mg/ml concentration. Also it's treatment showed 18% inhibition of tyrosinase activity, to relate whitening effect, at the same dose of 50% ethanol extract of SHT. Antioxidant activities were determined by DPPH radical scavenging, XO inhibiting activity and SOD-like activity. These scavenging, XO-inhibiting and SOD-like activities were measured in 80%, 75%, and 28% inhibitions, respectively, at a 1 mg/ml treated dose, compared to those of control. The inhibitory effects of 50% EtOH extract on aging and oxidation-related enzyme activities were higher than those of water extract and 95% EtOH extract. Conclusions : Taken together, our findings suggest that the SHT has potential and applicable benefits for development of cosmetics to have anti-aging (anti-wrinkle and whitening) and anti-oxidation functions.

Abstract. In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.