Hyean-Woo Lee

Human zinc fingers as building blocks in the construction of artificial transcription factors.

We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Chitosan oligosaccharides, dp 2–8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp.

In order to investigate the prebiotic potential of chitosan oligosaccharide (COS), prepared by enzymatic hydrolysis of fully deacetylated chitosan polymer, the effect of COS on bacterial growth was studied. The degree of polymerization (dp) of COS was determined by MALDI-ToF mass spectrometry, and the COS was found to be composed of dimer (33.6%), trimer (16.9%), tetramer (15.8%), pentamer (12.4%), hexamer (8.3%), heptamer (7.1%), and octamer (5.9%). The minimum inhibitory concentrations (MIC) of chitosan polymer against lactic acid bacteria and bifidobacteria were below 0.31%. However, this only applied to two strains, the other bacteria tested grew on MRS broth containing 5% COS. The effects of COS on the growth of bifidobacteria and lactic acid bacteria were compared with those of fructo-oligosaccharide (FOS). FOS was found to have a growth stimulatory effect on only three strains: Bifidobacterium bifidium, B. infantis and Lactobacillus casei. However, COS stimulated the growth of most Lactobacillus sp. and B. bifidium KCTC 3440. The amount of the growth and the specific growth rate of B. bifidium increased with increasing COS concentration. The cultivation time required to obtain maximum growth was reduced to about 25% in MRS broth supplemented with 0.2-0.4% COS. These results demonstrate that COS has considerable bifidogenic potential. Both cell growth and specific growth rates of L. brevis in MRS broth supplemented with 0.1% COS increased by 25%. The present study shows that COS stimulates the growth of some enteric bacteria, and that COS has potential use as a prebiotic health-food.

Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation

Emodin (1,3,8‐trihydroxy‐6‐methylanthraquinone), an active component in the root and rhizome of Rheum palmatum, is a tyrosine kinase inhibitor with a number of biological activities, including antitumor effects. Here, we examine the effects of emodin on vascular endothelial growth factor (VEGF)‐A‐induced angiogenesis, both in vitro and in vivo. In vitro, emodin dose‐dependently inhibits proliferation, migration into the denuded area, invasion through a layer of Matrigel and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated with VEGF‐A. Emodin also inhibits basic fibroblast growth factor‐induced proliferation and migration of HUVECs and VEGF‐A‐induced tube formation of human dermal microvascular endothelial cells. Specifically, emodin induces the cell cycle arrest of HUVECs in the G0/G1 phase by suppressing cyclin D1 and E expression and retinoblastoma protein phosphorylation, and suppresses Matrigel invasion by inhibiting the basal secretion of matrix metalloproteinase‐2 and VEGF‐A‐stimulated urokinase plasminogen activator receptor expression. Additionally, emodin effectively inhibits phosphorylation of VEGF‐A receptor‐2 (KDR/Flk‐1) and downstream effector molecules, including focal adhesion kinase, extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase, Akt and endothelial nitric oxide synthase. In vivo, emodin strongly suppresses neovessel formation in the chorioallantoic membrane of chick and VEGF‐A‐induced angiogenesis of the Matrigel plug in mice. Our data collectively demonstrate that emodin effectively inhibits VEGF‐A‐induced angiogenesis in vitro and in vivo. Moreover, inhibition of phosphorylation of KDR/Flk‐1 and downstream effector molecules is a possible underlying mechanism of the anti‐angiogenic activity of emodin. Based on these data, we propose that an interaction of emodin with KDR/Flk‐1 may be involved in the inhibitory function of emodin toward VEGF‐A‐induced angiogenesis in vitro and responsible for its potent anti‐angiogenic in vivo.

Effects of silver and potassium ions on ion exchange in float glass

The variation of optical and mechanical properties in ion-exchanged glasses was investigated. Ion exchange was carried out in KNO3, NaNO3 melts and their mixed melts with AgNO3. The glasses used were soda-lime-silicate glasses produced by the float process. In order to analyse the effects of ion exchange, colour variation for optical properties, bending strength, surface microhardness and softening point for mechanical properties, and resistivity for electrical properties, were measured. In the optical properties, silver ions play an important role in colouring, ion exchange in the mixed melt of KNO3 and AgNO3, and additional heat treatment in air made it possible to obtain a range of colours from yellow to reddish-brown. Bending strength was increased by five times over the values of the parent glasses, surface microhardness was increased about 50 kg mm−2, and the softening point was increased linearly with the amount of ion exchange resulting from ion exchange in the KNO3 melt, because the potassium ion plays a great role in strengthening in the mechanical properties. On the other hand, in the mixed melt of KNO3 and AgNO3, bending strength and surface microhardness were increased slightly in contrast with the KNO3 melt, and the softening point decreased on increasing the amount of ion exchange.

F508 amino acid deletion mutation of CFTR gene in Korean lung cancer patients.

Mutations of the transmembrane conductance regulator (CFTR) gene in cystic fibrosis lead to dysfunction of the lung, pancreas, and sweat glands, etc. To investigate the possibility of the relationship between lung cancer and the mutations of CFTR gene, we determined amino acid sequences using reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In this study, the deletion mutation of 508th amino acid in one of nine lung caner patients was found confirming that CFTR gene mutation exists in a Korean lung cancer patient.

Apoptosis induced by nonspecific effects of siRNA in human umbilical vein endothelial cell

RNA interference (RNAi) is a powerful tool used to produce post-translational gene silencing in a sequence-dependent manner. However, sequence-independent activation of interferon systems induced by double-stranded RNA can interfere with the data interpretation of RNAi experiments. In this study, we observed cytokine activation and cytotoxicity caused by small interfering RNA (siRNA) in human umbilical vein endothelial cells (HUVECs). Transfection with different sequences of siRNAs (21-nucleotides) induced apoptosis in HUVECs within 24 hours, in a dose-dependent and sequence-independent manner. These effects could not be achieved by the application of transfection agents nor by siRNA only. In HUVECs, the expression of toll-like receptor 3 (TLR3), a receptor for double-stranded RNA, was significantly increased by the transfection with siRNAs regardless of their sequences. The expressions of interleukin 6, interleukin 8, and interferon-β were also up-regulated by siRNA transfection. The activation of RNA-dependent protein kinase (PKR) peaked within 30 min and slowly decreased 4 hours after transfection with siRNA, which was followed by the phosphorylation of eukaryotic initiation factor 2α(eIF2α). These studies suggest that transfection with double-stranded oligonucleotides in HUVECs shows sequence-independent apoptotic effects associated with the upregulation of TLR3 and cytokines, which should be considered when designing experiments using RNAi.