• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.260-267
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• 2006

The Liquid Culture Assay using a recombinant Agrobacterium tumefaciens strain has been developed as a means for quorum sensing autoinducer screening. However, the low aqueous solubility of marine natural product extracts used as potential autoinducers has been a hindrance in the screening process. Although the addition of organic solvents and/or surfactants could increase aqueous solubility, errors in data interpretation including false positive results could be a serious problem. Therefore, determining the best possible solvent and surfactant at the optimum concentration is crucial. Evaluating methanol, ethanol, 1-propanol, DMSO and DMF at concentration ranges of 0~10% revealed < 2% methanol to be most favorable when tested for ${\beta}$-gal activity and growth inhibition of the recombinant A. tumefaciens strain. On the other hand, among surfactants tested, Triton X-100 was similarly effective in increasing the delivery of autoinducers for activity at less than 0.05% concentration.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.363-368
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• 1988

Molecular cloning of gene for $\alpha$-acylamino-$\beta$-lactam acylhydrolase (ALAH) III from Acetobacter turbidans has been attempted by immunochemical detection method, in which polyclonal antibody from mouse Balb/c against this enzyme was employed as a probe. As a cloning vector, λ gtll was chosen for this purpose. Two positive clones has been selected from genomic libraries of A. turbidans, which had somewhat different binding affinities on anti-ALAH III umm and anti-$\beta$-galactosidase. By restriction analysis, both clones has been turned out to lose one of EeoRI sites. From these results, it concluded that deletion of DNA between lacZ gene and inserted DNA has occurred during replication of these clones in host cells.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.98-103
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• 2007

To isolate probiotic lactic acid bacteria for animal, we have screened the microorganisms from chicken feces, by random selection and agar well diffusion assay. Among them, CPM-7 strain showing superior inhibitory activity against Escherichia coli was selected. By examining carbohydrates utilization, morphologic property and 16S rRNA gene sequence, CPM-7 strain was identified as Lactobacillus salivarius, then named L. salivarius CPM-7. L. salivarius CPM-7 produced thirteen enzymes in the test using API ZYM kit, and showed resistance to low pH and bile salts. It survived at pH 2 for 30 min. and pH 3 for 6 hr. And, it was able to grow in MRS medium containing 0.2% (w/v) bile salts. L. salivarius CPM-7 adhered to the jejunal epithelium cells of pig. Both the supernatant of L. salivarius CPM-7 and the its neutralized one showed high inhibitory activity against E. coli K88.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.102-110
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• 2006

Background: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific $CD8^+$ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of ${\beta}$-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using ${\beta}$-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

• International Journal of Oral Biology
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• v.36 no.3
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• pp.135-141
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• 2011

Hertwig's epithelial root sheath (HERS) consists of bi-layered cells derived from the inner and outer dental epithelia and plays important roles in tooth root formation as well as in the maintenance and regeneration of periodontal tissues. With regards to the fate of HERS, and although previous reports have suggested that this entails the formation of epithelial rests of Malassez, apoptosis or an epithelial-mesenchymal transformation (EMT), it is unclear what changes occur in the epithelial cells in this structure. This study examined whether HERS cells undergo EMT using a keratin-14 (K14) cre:ROSA 26 transgenic reporter mouse. The K14 transgene is expressed by many epithelial tissues, including the oral epithelium and the enamel organ. A distinct K14 expression pattern was found in the continuous HERS bi-layer and the epithelial diaphragm were visualized by detecting the ${\beta}$-galactosidase (lacZ) activity in 1 week postnatal mice. The 2 and 4 week old mice showed a fragmented HERS with cell aggregation along the root surface. However, some of the lacZ-positive dissociated cells along the root surface were not positive for pan-cytokeratin. These results suggest that the K14 transgene is a valuable marker of HERS. In addition, the current data suggest that some of the HERS cells may lose their epithelial properties after fragmentation and subsequently undergo EMT.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• KSBB Journal
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• v.28 no.1
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• pp.18-23
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• 2013

The purpose of this work was to investigate the several functional characteristics of Lactobacillus sakei JK-17 isolated from long-term fermented kimchi, Muk Eun Ji. Initially, phylogenetic analysis using 16S rRNA sequencing was performed to identify the isolate JK-17, and the strain could be assigned to Lactobacillus sakei and designated as L. sakei JK-17. The strain was registered in GenBank as [JX841311]. The changes of bacterial growth and residual organic acids were monitored and HPLC was used to measure quantitatively two organic acids, lactic acid and acetic acid, produced in the culture during 84 hours of incubation. During the incubation period, several functional characteristics of L. sakei JK-17 were examined. L. sakei JK-17 culture depleted nitrite concentration 94.75%. Antioxidant activity of cultural supernatants of L. sakei JK-17 was approx. 53.8%, and ${\beta}$-galactosidase activities were 0.243 units/mL at pH 7.0 and 0.387 units/mL at pH 4.1, respectively. The antibacterial activities against food-poisoning causing bacteria were examined with 20-fold concentrated culture supernatants from L. sakei JK-17 and the antibacterial effects were clearly observed against all bacteria tested in this work.

• Journal of the East Asian Society of Dietary Life
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• v.14 no.4
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• pp.363-369
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• 2004

Three different doengjang(fermented soybean paste) were prepared by using B. subtilis SS103 with high angiotensin converting enzyme(ACE) inhibitory activity, Asp. oryzae and mixed culture of two organisms, respectively. Quality characteristics and the inhibitory activity were compared. $\beta$-Amylase activity rapidly increased during 30 days of fermentation, but lower activity was found in doenjang prepared with B. subtilis. However, $\alpha$-Galactosidase activity decreased for 15 days of fermentation and little activity was observed after that fermentation period. Protease production tended to increase during the fermentation. High protease activity was found only in doengjang prepared with Asp. oryzae, but the doengjang prepared with B. subtilis SS103 showed very low activity. Total isoflavone content at 90 day fermentation was the highest in doengjang prepared with mixture of Asp. oryzae and B. subtilis. ACE inhibitory activity increased during the fermentation period. The highest activity was found in doengjang made with mixture of Asp. oryzae and B. subtilis. Sensory evaluation on doengjang itself and its soup indicated that doengjang made with mixture of Asp. oryzae and B. subtilis showed higher acceptability on taste, flavor and color than those prepared with only Asp. oryzae or B. subtilis, respectively.

• BMB Reports
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• v.40 no.1
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• pp.119-124
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• 2007

Adenoviral vectors are among the most promising vectors available for human gene therapy. However, the use of recombinant adenoviral vectors, including replicationcompetent adenovirus (RCA), raises a variety of safety concerns in relation to the development of new therapies based on gene therapy. To examine how organic compounds change in rat plasma following the injection of adenovirus, $\beta$-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA, we investigated the content of fatty acids (FAs), which are important biochemical indicators in pathological conditions. Pattern recognition analysis on the level of FAs in rat plasma is described for the visual discrimination of adenovirus infection groups from normal controls. Plasma FAs from four control rats (normal group), and from four rats with rAdLacZ infection and six rats with RCA infection (the two abnormal groups), were examined by gas chromatography-mass spectrometry in selected ion monitoring modes as their tert-butyldimethylsilyl derivatives. In total, 20 FAs were positively detected and quantified. The results of the Student's t-test on the normal mean of two abnormal groups, the levels of three FAs (p<0.05) from rAdLacZ group and eleven FAs (p<0.05) from RCA group were significantly different. When star symbol plotting was applied to the group mean values of 20 FAs after normalization to the corresponding normal mean values, the resulting eicosagonal star patterns of the two infected groups were distorted into similar shapes, but were distinguishable from each other. Thus, these approaches will be useful for screening and monitoring of diagnostic markers for the effects of infection following the use of adenoviral vectors in gene therapy.

• Journal of Ginseng Research
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• v.36 no.1
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• pp.78-85
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• 2012

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside $Rg_3$ prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by ${\beta}$-galactosidase (${\beta}$-gal) staining. Staining with 4'-6-Diamidino-2-phenylindole verified that most adherent cells (93${\pm}$2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of ${\beta}$-gal-positive EPCs was decreased from 93.8${\pm}$2.0% to 62.5${\pm}$3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms.