• Bulletin of the Korean Chemical Society
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• v.26 no.6
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• pp.916-920
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• 2005

Acetohydroxy acid synthase catalyzes the first common step in the biosynthesis of branched chain amino acids. AHAS plays two distinct metabolic roles, and is designated as anabolic AHAS and catabolic AHAS, depending on its function. Anabolic AHAS is FAD-dependent, while its catabolic counterpart is not. In this work, a conserved motif was identified in the $\beta$-domain of anabolic AHASs, but not in catabolic AHAS ($_{372}RFDDR_{376}$). In order to determine the functions of this motif, we replaced the motif with the corresponding sequence in FAD-independent AHAS, SPVEY. None of these three mutants (SPV, SPVE, and SPVEY) was detected with bound FAD. However, two of these mutants (SPVE and SPVEY) were active at a low level of specific activity. Although they exhibited pyruvate- and ThDP- dependent characteristics, the activity of the two active mutants appears to be FAD-independent. The SPVEY mutant was completely insensitive to the three tested herbicides, even at extremely high concentrations and is also somewhat more thermolabile than the wild type enzyme. The data provided in this work suggest that the RFDDR motif is a possible determinant of the FAD-dependent and herbicide-resistant properties of tobacco AHAS. The SPVEY mutant appears to exhibit catabolic AHAS-like activity.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.659-659
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• 2003

In this study, we have investigated the potent anti-aging effect of DA-3711, a cosmetic ingredient derived from artificial skin culture. The artificial skin was originally developed as a skin replacement for the treatment of chronic skin wounds. To produce DA-3711, neonatal human fibroblasts were seeded into biocompatible collagen/chitosan/glycosaminoglycan (GAG) scaffolds and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum and nonessential amino acids. Analysis of the culture broth (DA-3711) showed that growth factors such as VEGF, TGF-$\beta$, KGF were present at significantly higher levels that in the culture broth of fibroblasts cultured in monolayer. The biological activity of DA-3711 was assessed by measuring in vitro cell proliferation and collagen synthesis of normal human fibroblasts. Fibroblasts treated with 10％ DA-3711 showed a 2-fold higher proliferation and 2 to 4-fold higher collagen synthesis than untreated cells. DA-3711 also exhibited anti-oxidative effects, since cells under peroxide-induced oxidative stress showed a 30％ higher viability in DA-3711-containing medium than in medium without DA-3711 addition. The results suggest that DA-3711 may have anti-aging effects by stimulating skin regeneration and protecting against oxidative stress.

• BMB Reports
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• v.43 no.2
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• pp.97-102
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• 2010

Clenbuterol, a $\beta_2$-adrenoceptor agonist, has been proven to be a powerful repartition agent that can decrease fat deposition. Based on results from our previous cDNA microarray experiment of pig clenbuterol administration, a novel up-regulated EST was full-length cloned (4859 bp encoding 1041 amino acids) and found to be the pig homolog of large tumor suppressor 2 (Lats2). We mapped pig Lats2 to chromosome 11p13-14 by using FISH, and western blotting demonstrated that pig Lats2 protein was most abundant in adipose. In Drosophila, Lats2 ortholog was reported as a key component of the Hippo pathway which regulates cell differentiation and growth. Here, we show that pig Lats2 exhibit inverted expression to YAP1, another member of the Hippo pathway which positively regulates cell growth and proliferation, during the differentiation of 3T3-L1 preadipocytes. Our results suggested that Lats2 may involve in Hippo pathway regulating the fat reduction by inhibiting adipocyte differentiation and growth.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.1-6
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• 2011

Inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches have been described to improve gene transfer efficiency but suffer from a number of limitations. Here we tested an adenovirus carrying the small peptide ligand derived from heregulin${\beta}$ EGF-like domain onto fiber, the adenoviral capsid protein, to deliver transgene to ovarian cancer cells which overexpress ErbB, the cognate receptors for heregulin. The attachement of 53 amino acids to fiber didn't affect on the fiber's trimer structure that is critical for the viral entry to cells. The fiber-modified adenovirus can mediate entry and expression of a ${\beta}$-galactosidase into cancer cells in an increased efficiency compared the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to ErbB receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.102-108
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• 2006

The IscA gene, encoding a levansucrase of 424 amino acids (aa) residues, was cloned from the genomic DNA of Pseudomonas aurantiaca S-4380, and overexpressed in Escherichia coli. The recombinant levansucrase overexpressed in E. coli was then used to produce levan from sucrose. Levan crystals with 98% purity could be obtained from the reaction mixture with $62\%$ yield using an alcohol precipitation method. The molecular weight of the levan was $7\times10^5$ daltons. Methylation studies showed that the levan was branched: main linkage C-2,6; branched linkage C-2,1; and degree of branching $6\%$. Three bacterial levans from different strains were incubated with levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032, which produced $di-\beta-D-fructofuranose$ dianhydride IV (DFA IV); final conversion yields from the levans to DFA IV were $39\%$ in Zymomonas mobilis, $53\%$ in Serratia levanicum, and $59\%$ in P. aurantiaca S-4380 levansucrase. The levan from P. aurantiaca S-4380 levansucrase gave the highest conversion yield of levan to DFAIV so far reported.

• Journal of Microbiology and Biotechnology
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• v.8 no.2
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• pp.152-157
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• 1998

To analyze the role of the C and D domains in the cyclization activity of cyclodextrin glucanotransferase (CGTase), two plasmids, pKB1ΔC300 and pKB1ΔD96, were constructed in which DNA regions encoding 100 and 32 amino acids, respectively, from the C and D domains of B. stearothermophilus NO2 CGTase were deleted. The mutated CGTase from the pKBlΔC300 produced much lower amounts of ${\alpha}$-, ${\beta}$-, and $\gamma$-cyclodextrin (CD) than the parental CGTase. However, the mutated CGTase from the pKBlΔD96 showed a similar production pattern of CDs to wild-type CGTase. The production ratios of the ${\alpha}$-, ${\beta}$- and $\gamma$-CDs were not affected by the deletions, when compared to those of parental CGTase. The optimum temperature of the mutated CGTase from the pKBlΔC300 was decreased from $60^{\circ}C$ to $55^{\circ}C$. The optimum pH of the mutated CGTase from the pKB1D96 was shifted from 6.0 to 7.0. The thermostability of the two mutant CGTases were not changed. From these results, it is suggested that the C and D domains are not related to cyclization activity directly because mutant-enzymes deleted C or D domains still possessed their activity. However, they are important for other enzymatic properties such as productivity and pH optimum as a partition of CGTase tertiary structure.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.

• BMB Reports
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• v.36 no.4
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• pp.409-416
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• 2003

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-$\beta$-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants ($k_{inactivation}$) of $29.5\;M^{-1}s^{-1}$. The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of $3,200\;M^{-1}cm^{-1}$. It was discovered that methyl-$\beta$-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with $R_t$ 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.

• BMB Reports
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• v.50 no.6
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• pp.308-317
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• 2017

Bone morphogenetic protein type 2 receptor (BMPR2) is one of the transforming growth $factor-{\beta}$ ($TGF-{\beta}$) superfamily receptors, performing diverse roles during embryonic development, vasculogenesis, and osteogenesis. Human BMPR2 consists of 1,038 amino acids, and contains functionally conserved extracellular, transmembrane, kinase, and C-terminal cytoplasmic domains. Bone morphogenetic proteins (BMPs) engage the tetrameric complex, composed of BMPR2 and its corresponding type 1 receptors, which initiates SMAD proteins-mediated signal transduction leading to the expression of target genes implicated in the development or differentiation of the embryo, organs and bones. In particular, genetic alterations of BMPR2 gene are associated with several clinical disorders, including representative pulmonary arterial hypertension, cancers, and metabolic diseases, thus demonstrating the physiological importance of BMPR2. In this mini review, we summarize recent findings regarding the molecular basis of BMPR2 functions in BMP signaling, and the versatile roles of BMPR2. In addition, various aspects of experimentally validated pathogenic mutations of BMPR2 and the linked human diseases will also be discussed, which are important in clinical settings for diagnostics and treatment.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.6
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• pp.734-737
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• 2006

This study was carried out to establish the manufacturing process of barley leaf powder tea. The optimal manufacturing process among many trials was determined with sensory evaluation. Finally established process and operation conditions were as follows: pretreatment (cutting and washing), steaming ($100^{\circ}C$, 30 sec), primary drying and roasting ($130^{\circ}C$, 40 min), rolling (RT, 25 min), middle drying and roasting ($60^{\circ}C$, 30 min), final drying and roasting ($55^{\circ}C$, 25 min), drying ($60^{\circ}C$, 20 min), roasting ($85^{\circ}C$, 20 min), and powdering (120 mesh). The barley leaf powder tea produced by this process mainly consisted of dietary fiber (33.8%), amino acids (12.9%), minerals (4.7%) and ${\beta}-carotene$ (6.9 mg%).