Jong-Hwa Park

Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed Drosophila melanogaster S2 cells

A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4xa0weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50xa0kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300xa0mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of ∼8xa0Uxa0μg−1 for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ®SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.

Human colorectal cancer antigen GA733-2-Fc fused to endoplasmic reticulum retention motif KDEL enhances its immunotherapeutic effects

Objective: The aim of this is to compare the immunotherapeutic effects of human colorectal cancer antigen GA733-2 fused to the Fc fragment of antibody (GA733-2-Fc) and to Fc and endoplasmic reticulum (ER) retention motif KDEL (GA733-2-Fc-KDEL). Materials and Methods: Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were produced from infiltrated Nicotiana benthamiana leaves and purified by affinity chromatography. Glycan structures were determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The allergic and immunogenic responses of recombinant GA733-2-Fc and GA733-2-Fc-KDEL were estimated in an intraperitoneally immunized mouse. The tumor regression effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL was examined using a colorectal carcinoma CT-26 animal model. Results: Recombinant GA733-2-Fc contained plant-specific glycan structures including β(1,2)-xylose and α(1,3)-fucose whereas recombinant GA733-2-Fc-KDEL contained oligomannose type glycan structures. Mice immunized intraperitoneally with recombinant GA733-2-Fc and GA733-2-Fc-KDEL elicited strong GA733-2-Fc-specific immunoglobulin G (IgG) and IgA serum antibody responses. Recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE. Recombinant GA733-2-Fc-KDEL increased the production of interferon-γ. Intraperitoneal preimmunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL regressed tumor growth in a colorectal carcinoma CT-26 animal model. The tumor regression effect induced by recombinant GA733-2-Fc-KDEL was greater than that induced by recombinant GA733-2-Fc. The human and mouse colorectal carcinoma cell binding activities of recombinant GA733-2-Fc-KDEL-immunized sera were higher than those of recombinant GA733-2-Fc. Conclusions: Our results suggest that GA733-2-Fc conjugated to ER-retention motif KDEL is a more efficient antigen to prevent tumor growth induced by colorectal carcinoma and minimize an allergic response.

Developing a Dwarf Echinacea purpurea Line by Suppressing DWF3 Expression via RNA Interference

DWARF3 (DWF3) is a brassinosteroid (BR) biosynthesis-related enzyme that catalyzes the C-3 oxidation of early BR intermediates. For the first time, we isolated the EpDWF3 gene from Echinacea purpurea. EpDWF3 shares high homology with AtCYP90A1 and ZeCPD1, a steroid 23-alpha-hydroxylase involved in the BR-biosynthetic pathway. We generated transgenic Echinacea plants via transformation with an RNA interference (RNAi) construct that expresses hairpin double-stranded RNA for EpDWF3 to induce sequence-specific RNA silencing. The transgenic plants displayed dwarf phenotypes, including reduced plant height and small flower diameter. We confirmed the presence of the RNAi construct in the transgenic plants by genomic DNA PCR. The transcript level of EpDWF3 decreased by 48% in the transgenic plants compared to wild type. Our results indicate that the knock-down of BR biosynthesis-related genes by RNAi enables the production of valuable dwarf plants for commercial horticultural or agricultural applications.

SU-E-J-02: Analysis of Interfractional Setup Variation with Image Guided Radiation Therapy Using KV Cone Beam Computed Tomography

Purpose: The aim of this study was to analyze the daily setup variations for various anatomic sites with kV x‐ray volume images and to suggest optimal margin guidelines for these anatomic sites.Methods: Fifteen cases including three whole brains, three heads and necks (HNs), two lungs, three abdomens (two livers and one pancreas), and four pelvic cases were selected for this study. The images generated by kilovoltage cone beam computed tomography (kV CBCT) were overlapped with reference images by helical CT simulator and were analyzed and corrected with three shifts (anterior‐posterior, right‐left, and superior‐inferior directions) range before each interfractional radiation treatment delivery. Additionally, source to skin distance of patient surface was recorded and compared weekly. For precise image guided radiation therapy(IGRT), our image detectors were installed in the relatively less than 1mm.Results: In results, after correction of kv CBCT and reference images, values in patient surface for whole brain, HN, lung, abdomen, and pelvis were 1.5 mm, 2.4 mm, 5.0 mm 5.4 mm and 3.8 mm, respectively. The interfractional setup errors based on kV CBCT were analyzed and the suggested mean values for whole brain, HN, lung, abdomen, and pelvis in the vertical, lateral, and longitudinal directions were each 1.22 mm, 0.86 mm, and 1.18 mm, 1.57 mm, 1.52 mm, and 2.53 mm, 4.9 mm, 2.28 mm, and 3.65mm, 2.63 mm, 2.58 mm, and 2.34mm, 3.05 mm, 2.01 mm, and 2.44 mm Conclusions: Conventional radiation treatment depends on 2D and external surface marker is not good the setup verification rather than kV CBCTimage with 3D. Daily setup imaging could be decreasing the planning target volume (PTV). Ultimately, the dose delivered to the surrounding healthy tissue and critical organs should be minimized during the radiation treatment period. This work was supported by Nuclear Research & Development Program of the National Research Foundation of Korea (NRF) grant funded by the Korean goverment (MEST)