• Journal of Microbiology and Biotechnology
• /
• v.12 no.1
• /
• pp.65-70
• /
• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• Polymer(Korea)
• /
• v.35 no.5
• /
• pp.378-384
• /
• 2011

Silk fibroin is a biocompatible and slowly biodegradable natural polymer. This natural polymer has excellent mechanical properties, non-toxicity, and non-immunogenic properties and has been demonstrated to support tissue regeneration. Also, gelatin is a natural material derived from collagen by hydrolysis and has an almost identical composition as that of collagen. Silk fibroin/gelatin scaffolds have been fabricated by using the freeze-drying method. To establish the scaffold manufacturing condition for silk fibroin and gelatin, we made scaffolds with various compositions of gelatin, glutaldehyde and silk fibroin. The silk fibroin/gelatin scaffolds were characterized using SEM, DSC, and water absorption ability tests. The cellular proliferation was evaluated by WST assay. These results suggested that a scaffold containing 8% of gelatin, 1% of glutaldehyde and 0.3 g of silk fibroin provided suitable characterstics for cell adhesion and proliferation. In conclusion, the silk fibroin/gelatin scaffold may serve as a potential cell delivery vehicle and a structural basis for tissue engineering.

• Anatomy and Cell Biology
• /
• v.56 no.3
• /
• pp.367-373
• /
• 2023

One of the main parameters in the analysis of skeletal remains in forensic anthropological cases is the estimation of age. This study aimed to investigate the correlation between age and the fusion status of the sternal junction. This cross-sectional study was carried out on 184 sterna from 94 females and 90 males obtained from known-age cadavers in the Thai population. By direct observation, the fusion stage of the manubrio-sternal and sterno-xiphoidal junctions was studied and divided into unfused and fused joints. The results showed that a large proportion of the sterna remain unfused throughout adulthood, with fusion observed in both young and old cadavers. Insignificant differences in the rate of fusion, the sexes and ages were observed. None of the sterna under 30 years of age in females and 32 years of age in males showed fusion of the manubrio-sternal and sterno-xiphoidal junctions. Based on the variability of the sternal fusions observed in this study, we highlighted a very limited role of the sternum alone in the estimation of age in the Thai population.

• Polymer(Korea)
• /
• v.33 no.1
• /
• pp.26-32
• /
• 2009

The aim of this research was to prepare microparticulate systems based on poly (lactide-co-glycolide)(PLGA) for the local release of ipriflavone in order to reduce bone loss. We developed the IP loaded PLGA microspheres using relatively simple oil-in-water(O/W) solvent evaporation method. HPLC was used to perform the in vitro release test of IP and morphology of cell attached on the micro-spheres was investigated using SEM. Cytotoxicity was assayed by cell counting kit-8 (CCK-8) test. Osteogenic differential cells were analyzed by ALP activity. Through RT-PCR analysis, we observed osteocalcin, ALP, and Type I collagen mRNA expression. The release of IP in vitro was more prolonged over 42 days and IP/PLGA microspheres showed the improvement on the cell proliferation, ALP activity and RT-PCR comparing with control (only PLGA). This initial research will be used to direct future work involved in developing this composite injectable bone tissue engineering system.

• Journal of the Korean Society of Tobacco Science
• /
• v.1 no.2
• /
• pp.138-149
• /
• 1979

For the preliminary study on tobacco cell fusion as one of new breeding techniques, the conditions that would be most effective in isolation, fusion, and culture of tobacco protoplasts were examined ; 1. The enzyme solution of 0.5% macerozyme and 2% cellulase( or meicellase) was the most economic and efficient in isolating protoplasts from tobacco leaves. 2. The proper incubation period of tobacco leaves in cell wall digesting solution was 4 hours. 3. As an osmotic stabilizer, sorbitol or mannitol solutions were employed. The concentration of 0.5~0.7 M of either hexitol gave satisfying results as the osmotic stabilizer. 4. The calcium concentration appeared to be an important factor in protoplast fusion. The adhesion of protoplasts was enhanced by enrichment of calcium ion in PEG solution. The highest frequency of protoplast fusion was obtained when tobacco protoplasts were incubated in PEG solution. containing 9mM CaCl2. 5. Cell divisions of the isolated protoplasts were continued and have generated colonies when they were grown on B-5 medium at 28$^{\circ}C$.

• Biomolecules & Therapeutics
• /
• v.30 no.4
• /
• pp.360-367
• /
• 2022

Tropomyosin receptor kinase A (TrkA) protein is a receptor tyrosine kinase encoded by the NTRK1 gene. TrkA signaling mediates the proliferation, differentiation, and survival of neurons and other cells following stimulation by its ligand, the nerve growth factor. Chromosomal rearrangements of the NTRK1 gene result in the generation of TrkA fusion protein, which is known to cause deregulation of TrkA signaling. Targeting TrkA activity represents a promising strategy for the treatment of cancers that harbor the TrkA fusion protein. In this study, we evaluated the TrkA-inhibitory activity of the benzoxazole compound KRC-108. KRC-108 inhibited TrkA activity in an in vitro kinase assay, and suppressed the growth of KM12C colon cancer cells harboring an NTRK1 gene fusion. KRC-108 treatment induced cell cycle arrest, apoptotic cell death, and autophagy. KRC-108 suppressed the phosphorylation of downstream signaling molecules of TrkA, including Akt, phospholipase Cγ, and ERK1/2. Furthermore, KRC-108 exhibited antitumor activity in vivo in a KM12C cell xenograft model. These results indicate that KRC-108 may be a promising therapeutic agent for Trk fusion-positive cancers.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.468-472
• /
• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• BMB Reports
• /
• v.44 no.8
• /
• pp.529-534
• /
• 2011

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.10
• /
• pp.1607-1615
• /
• 2007

We investigated the applicability of the TEM-l ${\beta}$-lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

• Genomics & Informatics
• /
• v.17 no.3
• /
• pp.26.1-26.12
• /
• 2019

Identification of fusion gene is of prominent importance in cancer research field because of their potential as carcinogenic drivers. RNA sequencing (RNA-Seq) data have been the most useful source for identification of fusion transcripts. Although a number of algorithms have been developed thus far, most programs produce too many false-positives, thus making experimental confirmation almost impossible. We still lack a reliable program that achieves high precision with reasonable recall rate. Here, we present FusionScan, a highly optimized tool for predicting fusion transcripts from RNA-Seq data. We specifically search for split reads composed of intact exons at the fusion boundaries. Using 269 known fusion cases as the reference, we have implemented various mapping and filtering strategies to remove false-positives without discarding genuine fusions. In the performance test using three cell line datasets with validated fusion cases (NCI-H660, K562, and MCF-7), FusionScan outperformed other existing programs by a considerable margin, achieving the precision and recall rates of 60% and 79%, respectively. Simulation test also demonstrated that FusionScan recovered most of true positives without producing an overwhelming number of false-positives regardless of sequencing depth and read length. The computation time was comparable to other leading tools. We also provide several curative means to help users investigate the details of fusion candidates easily. We believe that FusionScan would be a reliable, efficient and convenient program for detecting fusion transcripts that meet the requirements in the clinical and experimental community. FusionScan is freely available at http://fusionscan.ewha.ac.kr/.