• Journal of the Korean Applied Science and Technology
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• v.30 no.1
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• pp.173-182
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• 2013

Atopic dermatitis is a chronic, relapsing inflammatory skin disease associated with dysfunction of skin barrier and cutaneous hyper-reactivity to environmental triggers. In the previous study, cytotoxicity, antioxidant, anti-inflammatory and anti-allergic activities were investigated for various herbal extracts such as Aloe vera L. (AV), Viola mandshurica W. Becker (VM), Punica granatum L. (PG), and Dendrobium nobile L. (DN) in order to develop effective therapeutic herbal extracts for atopic dermatitis, In this study, anti-inflammatory activities of these herb extracts in lipopolysaccharide (LPS)-induced macrophage RAW264.7 cells were further examined to find the underlying molecular mechanisms. The RT-PCR (reverse transcription polymerase chain reaction) analysis showed that PG, DN and AV inhibited effectively the gene expression of pro-inflammatory cytokines IL-6 and IL-$1{\beta}$ in LPS-stimulated macrophages, while VM did not. The transfection and luciferase analysis exhibited that all herbal extracts hindered the activation of transcription nuclear factor kappa B (NF-${\kappa}B$). The western blot analysis indicated that AV blocked the activation of only JNK MAP (c-Jun N-terminal kinase mitogen-activated protein) kinase not p38 MAP kinase, while VM, PG and DN did not show the activation of both JNK and p38 MAP kinases. These results suggest that AV, VM, PG, and DN have anti-inflammatory activities and thus have the potential to reduce and alleviate the symptoms of atopic dermatitis.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1291-1299
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• 2007

Two ${\beta}$-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS 1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were $^1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr^{15}$-for DI and $^1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys^{15}$-for DIII. The apparent sequences exhibited high sequence similarities with other bacterial ${\beta}$-1,4-glucanases as well as ${\beta}$-1,4-xylanases.

• Journal of Microbiology
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• v.42 no.4
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• Journal of Microbiology
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• v.35 no.4
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• pp.347-353
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• 1997

We have characterized a phage library displaying random 22mer peptides which were produced as N-terminal fusions to the pIII coat protein of M13 filamentous phages. Among the sixty phages randomly picked from the library, 25 phages had the 22mer peptide inserts. The DNA sequence analysis of the 25 inserts showed the following results: first, each nucleotide was represented almost equally at each codon position except that there were some biases toward G bases at the first position of the codons. Secondly, the expected 47 sense codons were represented. The deduced amino acid sequences of the 25 inserts were analyzed to examine its diversity. Glycine and glutamate were the two most overrepresented residues above the expected value, whereas cysteine and threonine residues were underrepresented. The range of dicersity in dipeptide sequences showed that the amino acid residues were randomly distributed along the peptide insert. Acidic, basic, polar, and nonpolar amino acid residues were represented to the extent expected at most positions of the peptide inserts. The predicted isoelectric points and hydropathy indices of the 25 peptides showed that a variety of the peptide were represented in the library. These results indicate that this phage display library could be useful in fiuding ligands for a broad spectrum of receptors by affinity screening.

• Animal cells and systems
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• v.6 no.3
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• pp.271-275
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• 2002

Rph1 and Gisl are damage-responsive repressors involved in PHR1 expression. They have two $C_{2}$H/ sub 2/ zinc finger motifs as putative DNA binding domains and N-terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji- encoded protein. The repressors are able to bind to A $G_{4}$ sequence within a 39-bp sequence called upstream repressing sequence of PHR1 promoter (UR $S_{PHR1}$) responsible for the damage-response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP-Rph1 fusion protein. On the basis of the fact that the A $G_{4}$ sequence that is recognized by Rph1 and Gisl is also recognized by Msn2 and Msn4 in a process of stress response, we a1so tried to examine the in vivo function of A $G_{4}$ and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When A $G_{4}$ sequence was inserted into the minimal promoter of the cyc1-LacZ reporter, the increased LacZ expression was observed indicating its involvement in transcriptional activation. The data suggest that the A $G_{4}$ is primarily required for basal transcriptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the A $G_{4}$ is also involved in the repression of PHR1 via Rphl and Gisl, it is proposed that A $G_{4}$ functions as either URS or upstream activating sequence (UAS) depending on the promoter context.t.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.501-508
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• 1998

An alkalophilic mi.croorganism (strain YB380), which produces yeast cell wall hydrolase extracellulary, was isolated from Korean soil. The rod-shaped cells were 0.3~0.4 by 2~4${\mu}{\textrm}{m}$ long, motile, aerobic, gram-positive, and spore-forming. The color of the colony was light yellow. The temperature range for growth at pH 9.0 was 25 to $45{\circ}C, with optimum growth at $35{\circ}C. The pH range for growth at $35{\circ}C was 8 to 11 with an optimum pH of 9.0. Therefore, the strain YB380 is an obligate alkalophile. The 16S rRNA of strain YB380 has a 99% sequence similarity with that of Bacillus alcalophilus. On the basis of physiological properties, cell wall fatty acid composition, and phylogenetic analysis, we propose that the isolated strain is Bacillus alcalophilus. The yeast cell wall hydrolase from Bacillus alcalophilus subsp. YB380 has been purified and partially characterized. The molecular weight was estimated to be 27,000 daltons with an optimum temperature and pH of $60{\circ}C and 9.0, respectively. The N-terminal amino acid sequence of the enzyme was analyzed as Gln- Thr- Val- Pro- Trp- Gly- Ile- Asn- Arg- Val.

• Journal of Acupuncture Research
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• v.31 no.2
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• pp.87-98
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• 2014

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in $HT_{29}$ human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.

• Korean Journal of Medicinal Crop Science
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• v.26 no.2
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• pp.113-118
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• 2018

Background: Gastrodia elata Blume is a saprophytic perennial plant in the Orchidaceae family, because of its agricultural and medicinal effectiveness, researchers focus on its genome and chemical components. However, cytogenetic information based on the chromosome structure and composition to construct chromosomal backbone for genome sequencing research and for the development and breeding of plants is very limited. Methods and Results: We determined the metaphase chromosome composition of the G. elata genome by fluorescence in situ hybridization (FISH) using 5S and 45S rDNAs and telomeric repeat probes. The nuclear genome of G. elata was organized into 2 n = 36, with relatively small ($2.71-5.50{\mu}m$)chromosomes that showed gradual decrease in size. Conglutination phenomenon was observed among the metaphase chromosomes, and it was distinguished from that in other plant metaphase chromosome spreads. One pair of signal was detected for each 5S and 45S rDNA in the pericentromeric region and interstitial region on the short arm of chromosomes 10 and 4, respectively, and telomeric DNA signals were detected in the terminal region of most chromosomes. Conclusions: To our knowledge, this is the first FISH chromosome composition result in G. elata and could be useful in more comprehensive molecular cytogenetic and genomic analyses as well as breeding programs of the medicinal plant G. elata.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.5
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• pp.2637-2641
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• 2016

Tumor necrosis factor ($TNF-{\alpha}$), an inflammatory cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis, has previously been used in anti-cancer therapy. However, the therapeutic applications of $TNF-{\alpha}$ are largely limited due to its general toxicity and anti-apoptotic influence. To overcome this problem, the present study focused on the effect of active constituents isolated from a medicinal plant on $TNF-{\alpha}$-induced apoptosis in human colon adenocarcinoma (HT-29) cells. Nimbolide from Azadirachta indica was evaluated for cytotoxicity by methyl tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and phase contrast microscopy. Effects on apoptotic signaling proteins were investigated using Western blot analysis. Nimbolide showed cytotoxicity against HT-29 cells that was significantly different from the control group (p<0.01), a concentration of $10{\mu}M$ significantly inducing cell death (p<0.01). In combination with $TNF-{\alpha}$, nimbolide significantly enhanced-induced cell death. In apoptotic pathway, nimbolide activated c-Jun N-terminal kinase (JNK) phosphorylation, BH3 interacting-domain death agonist (Bid) and up-regulated the death receptor 5 (DR5) level. In the combination group, nimbolide markedly sensitized $TNF-{\alpha}$-induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance $TNF-{\alpha}$-mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway.

• BMB Reports
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• v.43 no.8
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• pp.567-572
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• 2010

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCC-binding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3-hy15.