Se Won Yie

Human cord blood-derived endothelial progenitor cells and their conditioned media exhibit therapeutic equivalence for diabetic wound healing.

Transplantation of human cord blood-derived endothelial progenitor cells (EPCs) is reported to contribute to neovascularization in various ischemic diseases. However, the possible beneficial role and underlying mechanisms in diabetes-impaired wound healing have been less well characterized. In this study, EPC transplantation stimulated keratinocyte and fibroblast proliferation substantially as early as 3 days after injury, leading to significantly accelerated wound closure in streptozotocin-induced diabetic nude mice, compared to PBS control. RT-PCR analysis showed that EPCs secreted various wound healing-related growth factors. Among them, keratinocyte growth factor and platelet-derived growth factor were highly expressed in the EPCs and were present at substantial levels in the EPC-injected dermal tissue. Using EPC-conditioned medium (CM), we found that paracrine factors from EPCs directly exerted mitogenic and chemotactic effects on keratinocytes and fibroblasts. Moreover, injection of EPC-CM alone into the same diabetic wound mice promoted wound healing and increased neovascularization to a similar extent as achieved with EPC transplantation. These results indicate that the beneficial effect of EPC transplantation on diabetic wounds was mainly achieved by their direct paracrine action on keratinocytes, fibroblasts, and endothelial cells, rather than through their physical engraftment into host tissues (vasculogenesis). In addition, EPC-CM was shown to be therapeutically equivalent to EPCs, at least for the treatment of diabetic dermal wounds, suggesting that conditioned medium may serve as a novel therapeutic option that is free from allograft-associated immune rejection concern.

Enhanced dermal wound neovascularization by targeted delivery of endothelial progenitor cells using an RGD-g-PLLA scaffold

Endothelial progenitor cells (EPCs), endothelial precursors that promote neovascularization in ischemic tissues, have shown the limited vascular regeneration efficacy due to their poor homing into injured sites and low survival, so that a variety of biosynthetic scaffolds have been employed as cell delivery vehicles to overcome the current cell transplantation methods. However, few paralleled studies that directly compare the efficacy of EPCs seeded within synthetic scaffolds to that of EPCs delivered by the conventional transplantation techniques used for EPC therapies have been performed. To address these issues, RGD-g-PLLA biosynthetic scaffold was developed for the targeted EPC delivery and was found to successfully support the in vitro growth and endothelial functions of EPCs. This scaffold also appeared to be good as in vivo targeted delivery carriers of EPCs as it promoted vascular regeneration in a murine dermal wound models. Furthermore, direct comparison with the intradermal EPC injection revealed that the targeted delivery of EPCs by using the RGD-g-PLLA scaffold was superior to their conventional local injection method in terms of the localization and survival/retention of the transplanted EPCs, and their vascular repairing potential. These results suggest that the development of an effective stem cell delivery system may help to maximize the tissue-repairing efficacy with a limited number of stem cells, thereby resolving the limited clinical success of current stem cell therapies that have utilized simple cell injections or infusions.

Identification of an OsPR10a promoter region responsive to salicylic acid

Orysa sativa pathogenesis-related protein 10a (OsPR10a) was induced by pathogens, salicylic acid (SA), jasmonic acid (JA), ethephon, abscisic acid (ABA), and NaCl. We tried to analyze the OsPR10a promoter to investigate the transcriptional regulation of OsPR10a by SA. We demonstrated the inducibility of OsPR10a promoter by SA using transgenic Arabidopsis carrying OsPR10a:GFP as well as by transient expression assays in rice. To further identify the promoter region responsible for its induction by SA, four different deletions of the OsPR10a promoter were made, and their activities were measured by transient assays. The construct containing 687-bp OsPR10a promoter from its start codon exhibited a six-fold increase of induction compared to the control in response to SA. Mutation in the W-box like element 1 (WLE 1) between 687 and 637-bp from TGACA to TGAAA completely abolished induction of the OsPR10a promoter by SA, indicating that the WLE 1 between −687 and −637 of OsPR10a promoter is important in SA-mediated OsPR10a expression. We show for the first time that the W-box like element plays a role in SA mediated PR gene expression.

Heterologous expression of OsWRKY6 gene in Arabidopsis activates the expression of defense related genes and enhances resistance to pathogens.

The WRKY proteins are a major family of plant transcription factors implicated in the regulation of plant defense mechanisms against pathogens. OsWRKY6 was isolated based on expression profiling data carried out with samples infected by Xanthomonas oryzae pv. oryzae (Xoo). OsWRKY6 encodes a DNA binding protein that contains one WRKY domain, a nuclear localization signal and C(2)H(2)-type zinc finger motif. OsWRKY6 is a member of the group II family of WRKY proteins. Based on the result of yeast one hybrid assay this OsWRKY6 protein binds to the typical W box ((T)TGACC/T). OsWRKY6 functions as a transcriptional activator in yeast. OsWRKY6 enhanced the expression of the reporter gene downstream of OsPR1 promoter, indicating that OsWRKY6 is a transcriptional activator in rice as well. Heterologous expression of OsWRKY6 enhanced disease resistance to pathogen. Defense-related genes were constitutively expressed in Arabidopsis transgenic lines overexpressing OsWRKY6. All together, OsWRKY6 functions as a positive transcriptional regulator of the plant defense response.

Hepatitis C virus core protein potentiates c-Jun N-terminal kinase activation through a signaling complex involving TRADD and TRAF2

The hepatitis C virus (HCV) core protein is a multifunctional viral nucleocapsid protein. Previously, it has been demonstrated that the HCV core protein interacts with the cytoplasmic domain of tumor necrosis factor receptor 1 (TNFR1). Since the TNFR1 is engaged in stimulation of transcriptional factor NF-kappaB and AP-1 through activation of IkappaB kinase and c-Jun N-terminal kinase (JNK, or stress-activated protein kinase), respectively, we have examined whether the interaction between core protein and TNFR1 can modulate JNK. In this study, we demonstrate that the HCV core protein synergistically activates TNFalpha-induced JNK at a core concentration dependent manner in human embryonic kidney (HEK) 293 cells. HCV core-mediated synergism of JNK activation was also detected in stable cells expressing HCV core protein. Furthermore, we demonstrate that HCV core protein does not compete with TNF receptor-associated death domain (TRADD) for its interaction with the death domain of TNFR1. Our in vivo data show that HCV core and TRADD form a ternary complex with TNFR1. These findings suggest that the HCV core protein modulates TNFR1 signaling and may, thus, play a role in chronic infection of HCV patients.

RT-PCR Detection of dsRNA Mycoviruses Infecting Pleurotus ostreatus and Agaricus blazei Murrill

The partial nucleotide sequences of the genomic dsRNA mycoviruses infecting Pleurotus ostreatus (isolates ASI2596, ASI2597, and Bupyungbokhoe) and Agaricus blazei Murrill were determined and compared with those of the other dsRNA mycoviruses. Partial nucleotide sequences of the purified dsRNA from ASI2596 and ASI2597 revealed RNA-dependent RNA polymerase sequences that are closely related to Oyster mushroom isometric virus 2, while nucleotide sequences and the deduced amino acid sequence from dsRNA mycovirus infecting Agaricus blazei did not show any significant homology to the other dsRNA mycoviruses. Specific primers were designed for RT-PCR detection of these dsRNA viruses and were found to specifically detect each dsRNA virus. Northern blot analysis confirmed the homogeneity of RT-PCR products to each purified dsRNA. Altogether, our results suggest that these virus-specific primer sets can be employed for the specific detection of each dsRNA mycovirus in infected mushrooms.

REORGANIZATION OF MYOSIN AND FOCAL ADHESION PROTEINS IN SWISS 3T3 FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR BETA

Certain types of cells show a dramatic change in cell morphology cultured in the presence of transforming growth factor beta (TGF‐β). To identify cellular components or factors leading to morphological changes, we investigated if any members of cytoskeletal proteins and cell‐adhesion molecules were redistributed in TGF‐β‐treated Swiss 3T3 fibroblasts by indirect immunofluorescence and Western‐blot analysis. Changes in cell morphology became apparent within 12h of the addition of TGF‐β and new RNA and protein synthesis was necessitated by the changes. While TGF‐β induced reorganization of microfilaments as reported in earlier studies, one of the actin isoforms, alpha actin of smooth muscle, was induced to form stress fibers in Swiss 3T3 cells. It was observed that myosin light chain was relocated from cell periphery to cytoplasmic filamentous structures by TGF‐β treatment, with an increased amount. In addition, the cell‐shape change was accompanied by an increase in the level of vinculin and tyrosine phosphorylation at focal adhesions. These results suggest that new protein synthesis is required for the cell‐shape change, and acto‐myosin filaments and focal adhesion proteins are involved in the alteration of cell morphology induced by TGF‐β in Swiss 3T3 fibroblasts.

The identification of a novel Pleurotus ostreatus dsRNA virus and determination of the distribution of viruses in mushroom spores

Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.

Characterization and RT-PCR Detection of dsRNA Mycoviruses from the Oyster Mushroom, Pleurotus ostreatus

The partial nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus isolates ASI2223 and Suhan were determined and compared with those of mycoviruses belonging to partitiviruses and totiviruses. Partial nucleotide sequences of the purified dsRNA from ASI2223 and Suhan showed RNA-dependent RNA polymerase sequences that are closely related to those of partitiviruses, including Fusarium poae virus 1, Fusarium solani virus, Rhizoctonia solani virus, Discula destructiva virus 2, and Oyster mushroom isometric virus 2. Specific primers were designed for RT-PCR detection of dsRNA viruses from the P. ostreatus isolate ASI2223 and Suhan. Two virus specific primer sets were found to specifically detect each virus among six sets of designed oligonucleotide primers. Collectively, these results suggest that dsRNA mycoviruses from P. ostreatus isolates ASI2223 and Suhan belong to the family Partitiviridae, although, they are not the same virus species. Our results also suggest that these virus-specific primer sets can be employed for the specific detection of each viral sequence in infected tissues.