• KSBB Journal
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• v.31 no.4
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• pp.277-283
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• 2016

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

• BMB Reports
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• v.39 no.5
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• pp.502-510
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• 2006

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family. Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of $\beta$-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.

• Analytical Science and Technology
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• v.30 no.6
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• pp.348-354
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• 2017

In this study, patterns of proteome expression were monitored and specifically expressed proteins in human milk were detected in collected human milk after 1 week, 3 weeks, and 6 weeks from delivery. A quantitative shotgun proteomic approach was used to identify human milk proteins and reveal their relative expression amounts. For each sample, two independent human milk samples from two mothers were pooled, and then three replicated shotgun proteomic analyses were carried out. Casein, which is a highly abundant protein in human milk, was removed, and then trypsin was treated to produce a digested peptide mixture. The peptides were loaded in the home-made reversed-phase C18 fused-silica capillary column, and then the eluted peptides were analyzed by using a linear ion-trap mass spectrometer. The relative quantitation of proteins was performed by the normalized spectral count method. For each sample, 81-109 non-redundant proteins were identified. The identified proteins consisted of glycoproteins, metabolic enzyme, and chaperon enzymes such as lactoferrin, carboxylic ester hydrolase, and clusterin. The comparative analysis for the 63 proteins, which were reproducibly identified in all three replications, revealed that 25 proteins were statically significant differentially expressed. Among the differentially expressed proteins, Ig lambda-7 chain C region and tenascin drastically decreased with the delivery time.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.83-89
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• 2001

Recent advances in the structural and molecular biology uncovered that a set of translation factors resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic ：ode. Nature must have evolved this 'art' of molecular mimicry between protein and ribonucleic acid using different protein architectures to fulfill the requirement of a ribosome 'machine'. Termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the 'decoding' site (A site). Translation termination requires two classes of polypeptide release factors (RFs)： a class-I factor, codon-specific RFs (RFI and RF2 in prokaryotes; eRFI in eukaryotes), and a class-IT factor, non-specific RFs (RF3 in prokaryotes; eRF3 in eukaryotes) that bind guanine nucleotides and stimulate class-I RF activity. The underlying mechanism for translation termination represents a long-standing coding problem of considerable interest since it entails protein-RNA recognition instead of the well-understood codon-anticodon pairing during the mRNA-tRNA interaction. Molecular mimicry between protein and nucleic acid is a novel concept in biology, proposed in 1995 from three crystallographic discoveries, one, on protein-RNA mimicry, and the other two, on protein-DNA mimicry. Nyborg, Clark and colleagues have first described this concept when they solved the crystal structure of elongation factor EF- Tu：GTP：aminoacyl-tRNA ternary complex and found its overall structural similarity with another elongation factor EF-G including the resemblance of part of EF-G to the anticodon stem of tRNA (Nissen et al. 1995). Protein mimicry of DNA has been shown in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex (Mol et al. 1995; Savva and Pear 1995) as well as in the NMR structure of transcription factor TBP-TA $F_{II}$ 230 complex (Liu et al. 1998). Consistent with this discovery, functional mimicry of a major autoantigenic epitope of the human insulin receptor by RNA has been suggested (Doudna et al. 1995) but its nature of mimic is. still largely unknown. The milestone of functional mimicry between protein and nucleic acid has been achieved by the discovery of 'peptide anticodon' that deciphers stop codons in mRNA (Ito et al. 2000). It is surprising that it took 4 decades since the discovery of the genetic code to figure out the basic mechanisms behind the deciphering of its 64 codons.

• Journal of Chest Surgery
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• v.48 no.3
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• pp.164-173
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• 2015

Background: Hypertrophied myocardium is especially vulnerable to ischemic injury. This study aimed to compare the early and late clinical outcomes of three different methods of myocardial protection in patients with aortic stenosis. Methods: This retrospective study included 225 consecutive patients (mean age, 65{\pm}10 years; 123 males) with severe aortic stenosis who underwent aortic valve replacement. Patients were excluded if they had coronary artery disease, an ejection fraction <50%, more than mild aortic regurgitation, or endocarditis. The patients were divided into three groups: group A, which was treated with antegrade and retrograde cold blood cardioplegia; group B, which was treated with antegrade crystalloid cardioplegia using histidine-tryptophan-ketoglutarate (HTK) solution; and group C, treated with retrograde cold blood cardioplegia. Results: Group A contained 70 patients (31.1%), group B contained 74 patients (32.9%), and group C contained 81 patients (36%). The three groups showed significant differences with regard to the proportion of patients with a New York Heart Association functional classification ${\geq}III$ (p=0.035), N-terminal pro-brain natriuretic peptide levels (p=0.042), ejection fraction (p=0.035), left ventricular dimensions (p<0.001), left ventricular mass index (p<0.001), and right ventricular systolic pressure (p <0.001). Differences in cardiopulmonary bypass time (p=0.532) and aortic cross-clamp time (p=0.48) among the three groups were not statistically significant. During postoperative recovery, no significant differences were found regarding the use of inotropes (p=0.328), mechanical support (n=0), arrhythmias (atrial fibrillation, p=0.347; non-sustained ventricular tachycardia, p=0.1), and ventilator support time (p=0.162). No operative mortality occurred. Similarly, no significant differences were found in long-term outcomes. Conclusion: Although the three groups showed some significant differences with regard to patient characteristics, both antegrade crystalloid cardioplegia with HTK solution and retrograde cold blood cardioplegia led to early and late clinical results similar to those achieved with combined antegrade and retrograde cold blood cardioplegia.

• Polymer(Korea)
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• v.30 no.5
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• pp.445-452
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• 2006

In this study, we investigated interaction of Schwann cells (SCs) with various cell-adhesive coated polymer surface. We used cell-adhesives that like a fibronectin (FN), fibrinogen(FG), laminin(LM), vitronectin (VN), poly-D-Iysine (PDL), and poly-L-Iysine (PLL) to coat PLGA film surface and evaluated the surface property of coated or not PLGA films by measurement of water contact angle and ESCA. SCs were cultured on coated or non-coated PLGA film surface, and then examined the cell adhesion and proliferation by cell count and SEM observation. Cell count results revealed initial cell adhesion related to protein adsorption on PLGA surface. In addition, serum content in media related to cell proliferation rate. In this result, we recognized that adhesion and proliferation of SCs were affected by specific cell-adhesives. In these results, we recognized that is important to provide the suitable surface environment according to cell types and culture condition for improvement of cell adhesion and proliferation.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.2
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• pp.241-246
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• 2004

To develop novel anti-aging peptides from the germinated black rice, we treated with bromelain, papain and Pronase E. And we investigated the effects of the germinated black rice peptide (GBRP) as anti-aging cosmetic ingredients, and compared with the non-germinated black rice protein (NBRP). We investigated the effects on in vitro inhibition of matrix-metalloprotease (MMP), proliferation of human skin fibroblasts, stimulation of collagen synthesis and expression of UVA-induced MMPs in human skin fibroblasts, UVA induced MMP-1 expression and collagen contents in human skin fibroblasts were analyzed by enzyme-linked immunosorbent assay (ELISA). As a result, the molecular weight distributions of GBRP and NBRP were determined by gel permeation chromatography to be approximately 900 and 10,000 daltons. GBRP increased skin cell proliferation about 40% and reduced UVA-induced MMP-1 expression about 50%. Also the collagen protein level of cells, which were cultured with GBRP, was increased about 25%. These results suggest that the geminated plant seed peptides can be novel anti-aging ingredients for cosmetics.

• Food Science and Preservation
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• v.24 no.1
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• pp.145-152
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• 2017

In this study, near infrared (NIR) spectroscopy known as a non-destructive analysis technique was applied to investigate peptide cleavage and consequent release of amino acids in soybean lumps as affected by its moisture content and incubation time during fermentation at 25 for 3 weeks. The NIR spectra of the soybean lump semi-dried and soaked in saline water showed that absorption intensity around 1,400 nm originating from hydrogen bonds of water decreased and absorption band shifted to 1,430 nm as moisture content decreased during incubation at 25 for 3 weeks. In addition, absorption around 2,050 nm which was assigned to amino groups increased as incubation time increased. NIR spectra data from 1,000 to 2,250 nm showed higher accuracy in the discriminant analysis between outside and inside parts of fermented soybean lumps than visible spectra result. NIR spectroscopy for the amino acid and moisture contents in traditional fermented soybean lumps showed relatively good accuracy with the multiple correlation coefficient ($R^2$) of 0.91 and 0.81, respectively, and root mean square error of cross validation (RMSECv) of 0.23 and 0.83%, respectively, in partial least square regression (PLSR). These results indicate that NIR spectral observations could be applicable to control the fermentation process for preparation of soybean products.

• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

• KSBB Journal
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• v.23 no.3
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• pp.205-212
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• 2008

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.