Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.

No abstract

To investigate the structural characteristics and activation mechanism of the precursor caspase, genes encoding the inactive pro-form and the active mature form of caspase 6 were expressed in Escherichia coli and the proteins of both forms were purified to homogeneity. The structure of each protein was characterized by chemical cross-linking, size-exclusion chromatography, CD and fluorescence spectroscopies. The pro-form caspase 6 exhibits a dimeric structure and its overall secondary structure was found to be similar to that of the mature caspase 6. Upon the maturation of procaspase 6, the maximum fluorescence wavelength lambda(max) was red-shifted from 330 to 337 nm and the fluorescence intensity of lambda(max) was increased. This fluorescence spectral change indicates that the environment of a tryptophan residue in the substrate-binding site can be changed to a more polar one when the procaspase 6 is processed. Taken together, our results strongly demonstrate that precursor caspase 6 exists as a dimer and its overall structure is similar to that of the active caspase 6. Our results also suggest that the local conformational change at the substrate-binding site, with no drastic change in the overall structure, seems to enable precursor caspase 6 to become the active mature enzyme.

Aster yomena (A. yomena) extract has anti-inflammatory, antioxidant, anti-asthma, and anti-atopic effects. However, the commercial use of A. yomena extract requires a long processing time with specific processing steps (including heat treatment and ethanol precipitation), and there are various environmental problems. We aimed to build a system to produce A. yomena extract by culturing the callus in a bioreactor that can allow rapid process scale-up to test the effect of extract (AYC-CS-E) isolated from culture supernatant of A. yomena callus on photoaging of human keratinocytes (HaCaT) caused by ultraviolet B (UVB) exposure. Through screening analysis based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), 17 major metabolites were tentatively identified from AYC-CS-E for the first time. The suppression of cell proliferation caused by UVB was effectively alleviated in UVB-irradiated HaCaT cells treated with AYC-CS-E. Treatment with AYC-CS-E strongly induced the formation of type I procollagen and the inhibition of elastase in UVB-irradiated HaCaT cells and significantly reduced the expression of matrix metalloproteinase (MMP)-1. In addition, treatment of UVB-irradiated HaCaT cells with AYC-CS-E effectively improved various factors associated with an inflammatory reaction, skin damage recovery, skin moisture retention, and hyper-keratinization caused by photoaging, such as reactive oxygen species (ROS), pro-inflammatory cytokines, transforming growth factor beta (TGF-β), MMP-3, MMP-9, filaggrin, hyaluronic acid synthase 2 (HAS-2), keratin 1 (KRT-1), nuclear factor-kappa B (NF-κB), and nuclear factor erythroid 2-related factor 2 (Nrf2) at the gene and protein levels. These results suggest that AYC-CS-E can be used as a cosmetic ingredient for various skin diseases caused by photoaging, and the current callus culture system can be used commercially to supply cosmetic ingredients.

Plant tissue culture holds immense potential for the production of secondary metabolites with various physiological functions. We recently established a plant tissue culture system capable of producing secondary metabolites from Aster yomena . This study aimed to uncover the mechanisms underlying the potential therapeutic effects of Aster yomena callus pellet extract (AYC-P-E) on photoaging-induced skin pigmentation. Excessive melanogenesis was induced in B16F10 melanoma cells using α-melanocyte stimulating hormone (α-MSH). The effects of AYC-P-E treatment on melanin biosynthesis inducers and melanin synthesis inhibition were assessed. Based on the results, a clinical study was conducted in subjects with skin pigmentation. AYC-P-E inhibited melanogenesis in α-MSH-treated B16F10 cells, accompanied by decreased mRNA and protein expression of melanin biosynthesis inducers, including cyclic AMP response element-binding protein (CREB), tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), and TRP-2. This anti-melanogenic effect was mediated by mitogen-activated protein kinase (MEK) / extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) phosphorylation. Treatment of subjects with skin pigmentation with AYC-P-E-containing cream formulations resulted in 3.33%, 7.06%, and 8.68% improvement in the melanin levels at 2, 4, and 8 weeks, respectively. Our findings suggest that AYC-P-E inhibits excessive melanogenesis by activating MEK/ERK and AKT signaling, potentiating its cosmetic applications in hyperpigmentation treatment.