• Journal of Life Science
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• v.21 no.2
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• pp.227-234
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• 2011

The hrp gene cluster in the plant pathogen Pseudomonas syringae is a key determinant of pathogenicity. Recent studies have demonstrated that specific host cell induction of the Ralstonia solanacearum hrp gene cluster is controlled by the PrhA (plant regulator of hrp) receptor. To characterize the role that P. syringae PrhA plays in the virulence of plant cells, a prhA homolog was isolated from P. syringae pv. tabaci and a $\Delta$prhA mutant was constructed by allelic exchange. The $\Delta$prhA mutant had reduced virulence in the host plant, and co-culture of P. syringae pv. tabaci and plant cell suspensions induced a much higher level of hrpA gene transcription than culture in hrp-inducing minimal medium. These results indicate that PrhA of P. syringae is a putative pathogen-plant cell contact sensor, therefore, we used a hrpA-gfp reporter fusion to monitor the in situ expression of PrhA. The results of this study demonstrated that PrhA induces hrp gene expression in P. syringae pv. tabaci in the presence of plant cells.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.144-151
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• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.277-286
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• 2001

Improvement and possible commercialization of a home-made electroporation apparatus(home-made) were further tried to establish a simple and effective introduction of foreign gene into sperm followed by in vitro fertilization. Expressions of introduced pJJ9 and pNT plasmids were shown in all fertilized eggs with electroporated spermatozoa. In particular, with this gene transfer system all the fry showed a consistently transient expression in the syncytium of the yolk sac. This fact is important since some required, minute quantity of human proteins can be produced from the established transient expression on the yolk sac of all fry derived from in vitro fertilization with electroporated spermatozoa. To explore tissue-specific expression in fish, which we will use a similar system later, we targeted the nerve tissue to see whether tissue-specific promoter is working in fish properly. pNT plasmid containing a nerve cell-specific tubulin promoter gene demonstrated consistently exact targeted expressions among the developing nerve cells in later stages of embryos and hatched fry. Finally, liver-specific genes are now being cloned by using already selected primers for useful human protein gene fusion.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.483-493
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• 2010

Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.

• Genes and Genomics
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• v.40 no.12
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• pp.1259-1267
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• 2018

Litchi (Litchi chinensis Sonn.) is an important subtropical fruit crop with high commercial value due to its high nutritional values and favorable tastes. However, irregular bearing attributed to unstable flowering is a major ongoing problem for litchi producers. Previous studies indicate that low-temperature is a key factor in litchi floral induction. In order to reveal the genetic and molecular mechanisms underlying the reproductive process in litchi, we had analyzed the transcriptome of buds before and after low-temperature induction using RNA-seq technology. A key flower bud differentiation associated gene, a homologue of FLORICAULA/LEAFY, was identified and named LcLFY (GenBank Accession No. KF008435). The cDNA sequence of LcLFY encodes a putative protein of 388 amino acids. To gain insight into the role of LcLFY, the temporal expression level of this gene was measured by real-time RT-PCR. LcLFY was highly expressed in flower buds and its expression correlated with the floral developmental stage. Heterologous expression of LcLFY in transgenic tobacco plants induced precocious flowering. Meantime, we investigated the sub-cellular localization of LcLFY. The LcLFY-Green fluorescent protein (GFP) fusion protein was found in the nucleus. The results suggest that LcLFY plays a pivotal role as a transcription factor in controlling the transition to flowering and in the development of floral organs in litchi.

• Biomedical Science Letters
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• v.13 no.3
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• pp.207-212
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• 2007

The purpose of this preliminary study is to improve the efficiency of gene transfer of nonviral plasmid DNA by in vivo electroporation in ischemic hindlimb muscle, tibialis anterior. Hindlimb ischemic model was aseptically made by excision of left femoral artery. Each $50\;{\mu}g$ of pEGFP-C1 and pGL3-control in $100\;{\mu}l$ 0.9% NaCl was injected in tibialis anterior muscle. In vivo electroporation was applied on the same site with 10 mm-distance 2 needle array electrodes and ECM830. In 3 groups of normal rat with different electric field strength 0, 200 and 800 V/cm, the expression of pEGFP-C1 was comparatively evaluated. In 8 groups of normal rats, the expression of pGL3-control was evaluated in 0, 40, 50, 80, 100, 150, 200 and 300 V/cm of electric field strength. In 5 groups of ischemic models, the expression of pGL3-control was analyzed on 0, 4, 7, 10 and 14 days elapsed after making ischemic models. In 9 groups of ischemic rats, the expression of pGL3-control was analyzed in the electric field strength 0, 60, 70, 80, 100, 150, 200, 250 and 300 V/cm. GFP expressions in normal tibialis anterior were high in the extent and degree in order of electric field strength of 200, 800 and 0 V/cm. Luciferase value was highest in $50{\sim}100\;V/cm$ electric field strength. In the case of ischemic models, luciferase expression was significantly increasing in the order elapsed time after making the model. The degree of luciferase expression was higher in cases of application of in vivo electroporation than in that of non-application and was highest in $100{\sim}150\;V/cm$. In conclusion, in vivo electroporation is effective in transfer and expression of plasmid DNA in normal and ischemic tibialis anterior of rat.

• Nuclear Medicine and Molecular Imaging
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• v.42 no.5
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• pp.394-400
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• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

• IMMUNE NETWORK
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• v.6 no.1
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• pp.33-42
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• 2006

Background: Calcineurin plays a crucial role in T cell activation, cell growth, apoptosis, and angiogenesis, and its over-expression has been implicated in the pathogenesis of cardiomyopathy and stroke. However, the expression and function of calcineurin in the pathologic lesion of chronic inflammatory diseases, like rheumatoid synovium, remain to be defined. This study was aimed to determine the role of calcineurin in inflammatory arthritis and investigate the expression and function of calcineurin in the rheumatoid synovium and synoviocytes, the actual site of chronic inflammation. Methods: Immuno-histochemical staining using specific antibody to calcineurin was perfomed in the synovium of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients were isolated from RA and OA patients, and cultured with IL-1${\beta}$ and TNF-${\alpha}$ in the presence or absence of cyclosporin A, a calcineurin inhibitor. The calcineurin expression was assessed by phosphatase assay and Western blotting analysis. IL-6, -10, -17, matrix metalloproteinase (MMP)-1, -2, -3, and -9 released into the culture supernatants were measured by ELISA. After transfection with GFP-Cabin 1 gene into synoviocytes, the levels of IL-6 and MMPs were measured by ELISA. Results: Calcineurin was highly expressed in the lining layer of synovium and cultured synoviocytes of RA patients. The elevated calcineurin activity in the rheumatoid synoviocytes was triggered by proin flammatory cytokines such as IL-1${\beta}$ and TNF-${\alpha}$. In contrast, IL-10, an anti-inflammatory cytokine, failed to increase the calcineurin activity. The targeted inhibition of calcineurin by the over-expression of Cabin 1, a natural calcineurin antagonist, inhibited the production of IL-6 and MMP-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Conclusion: These data suggest that abnormal activation of calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis, and thus provide a potential target for controlling inflammatory arthritis.

• BMB Reports
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• v.55 no.2
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• pp.98-103
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• 2022

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.443-448
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• 2008

Multidomain proteins for the biochemical analysis of the scouring efficiency of cotton fabrics were constructed by the fusion of a reporter moiety in the N-terminal and the cellulose binding domain (CBD) in the C-terminal. Based on the specific binding of the CBD of Cellulomonas fimi exoglucanase (Cex) to crystalline cellulose (Avicel), the reporter protein is guided to the cellulose fibers that are increasingly exposed as the scouring process proceeds. Among the tested reporter proteins, a thermostable ${\beta}$-glycosidase (BglA) from Thermus caldophilus was found to be most appropriate, showing a higher applicability and stability than GFP, DsRed2, or a tetrameric ${\beta}$-glycosidase (GUS) from Escherichia coli, which were precipitated more seriously during the expression and purification steps. When cotton fabrics with different scouring levels were treated with the BglA-CBD and incubated with X-Gal as the chromogenic substrate, an indigo color became visible within 2 h, and the color depth changed according to the conditions and extent of the scouring.