• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.389-398
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• 2009

Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

• Tuberculosis and Respiratory Diseases
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• v.42 no.4
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• pp.492-501
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• 1995

Background: Transforming growth factor- alpha(TGF-$\alpha$) may play important roles in carcinogenesis, tumor growth, and angiogenesis. Transforming growth factor-beta(TGF-$\beta$) are known to be involved in cell-cycle control and regeneration. TGF-$\alpha$ positively acts on growth control of many epithelial cells in contrast to the negative role of TGF-$\beta$. Method: To evaluate the possible role of TGF-$\alpha$ and TGF-$\beta$ in human primary lung cancers, the expression of TGF-$\alpha$ and TGF-$\beta$ were immmunohistochemically investigated in tissue sections from forty seven cases with lung cancers and ten cases with non-cancerous lung tissues. Recombinant cloned monoclonal antibody of TGF-$\alpha$ and neutralizing antibody of TGF-$\beta$ were employed as primary antibodies after dewaxing the formalin-fixed, paraffinized tissue sections. Results: TGF-$\alpha$ was expressed in the cytoplasms of tumor cells in thirty five cases of forty seven(74.5%) primary lung cancers, whereas the control expressed in two of ten brochial epithelial cells. The expression of TGF-$\alpha$ was disclosed in four cases of eleven(36.4 %) small cell carcinomas and thirty one cases of thirty six(86.1%) non-small cell carcinomas of the lung. Expressions of TGF-$\beta$ was discernible in bronchial epithelium in eight of ten non-cancerous lung tissues. The expression of TGF-$\beta$ was noted in the cytoplasms of tumor cells in eight cases of forty seven(17.0%) primary lung cancers. The expression of TGF-$\beta$ disclosed in two cases of eleven(18.2%) small cell carcinomas and six cases of thirty six(16.7%) non- small cell carcinomas of the lung. Conclusion: These findings suggest that up-regulation of TGF-$\alpha$ and down-regulation of TGF-$\beta$ are involved during development and growth of primary lung cancers.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.87-95
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• 2009

The recombinant DNAs, pGBF, pGTF, and pZ4F, using soybean ferritin gene have constructed with the promoters derived from seed proteins, glutelin, globulin, and zein. The recombinant ferritin genes were transformed into rice plant by Agrobacterium-mediated transformation. Iron contents and agronomic traits have been evaluated in the transgenic progenies. The embryogenic calli survived from second selection medium were regenerated at the rates of 19.2% with pGBF, 15.0% with pGTF, and 18.4% with pZ4F in Donganbyeo and 6.7% with pGBF, 11.7% with pGTF, and 3.4% with pZ4F in Hwashinbyeo. The introduction of ferritin gene in putative transgenic rice plants was confirmed by PCR and Southern blot analysis and also the expression of ferritin gene was identified by Northern blot and Western blot analysis. The iron accumulation in transgenic rice grains of the transgenic rice plant, T1-2, with zein promoter and ferritin gene contained 171.4 ppm showing 6.4 times higher than 26.7 ppm of Hwashinbyeo seed as wild type rice, but the transgenic plants with globulin and glutelin showed a bit higher iron contents with a range from 2.1 to 3.0 times compare to wild type grain. The growth responses of transgenic plants showed the large variances in plant height and number of tillers. However, there were some transgenic plants having similar phenotype to wild type plants. In the T1 generation of transgenic plants, plant height, culm length, panicle length, and number of tillers were similar to those of wild type plants, but ripened grain ratio ranged from 53.3% to 82.2% with relatively high variation. The transgenic rice plants would be useful for developing rice varieties with high iron content in rice grains.

• Neonatal Medicine
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• v.16 no.2
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• pp.221-233
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• 2009

Purpose: Erythropoietin (EPO) has neuroprotective effects in many animal models of brain injury, including hypoxic-ischemic (HI) encephalopathy, trauma, and excitotoxicity. Current studies have demonstrated the neuroprotective effects of EPO, but limited data are available for the neonatal periods. Here in we investigated whether recombinant human EPO (rHuEPO) can protect the developing rat brain from HI injury via modulation of NMDA receptors. Methods: In an in vitro model, embryonic cortical neuronal cell cultures from Sprague-Dawley (SD) rats at 19-days gestation were established. The cultured cells were divided into five groups: normoxia (N), hypoxia (H), and 1, 10, and 100 IU/mL rHuEPO-treated (H+E1, H+ E10, and H+E100) groups. To estimate cell viability and growth, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. In an in vivo model, left carotid artery ligation was performed on 7-day-old SD rat pups. The animals were divided into six groups; normoxia control (NC), normoxia Sham-operated (NS), hypoxia-ischemia only (H), hypoxia-ischemia+vehicle (HV), hypoxia-ischemia+rHuEPO before a HI injury (HE-B), and hypoxia-ischemia+rHuEPO after a HI injury (HE-A). The morphologic changes following brain injuries were noted using hematoxylin and eosin (H/E) staining. Real-time PCR using primers of subunits of NMDA receptors (NR1, NR2A, NR2B, NR2C and NR2D) mRNA were performed. Results: Cell viability in the H group was decreased to less than 60% of that in the N group. In the H+E1 and H+E10 groups, cell viability was increased to >80% of the N group, but cell viability in the H+E100 group did not recover. The percentage of the left hemisphere area compared the to the right hemisphere area were 98.9% in the NC group, 99.1% in the NS group, 57.1% in the H group, 57.0% in the HV group, 87.6% in the HE-B group, and 91.6% in the HE-A group. Real-time PCR analysis of the expressions of subunits of NMDA receptors mRNAs in the in vitro and in vivo neonatal HI brain injuries generally revealed that the expression in the H group was decreased compared to the N group and the expressions in the rHuEPO-treated groups was increased compared to the H group. Conclusion: rHuEPO has neuroprotective property in perinatal HI brain injury via modulation of N-methyl-D-aspartate receptors.

• Journal of Plant Biotechnology
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• v.3 no.1
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• pp.7-12
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• 2001

An antiserum against the carrot HSP17 (17 KDa heat shock protein) was raised using the HSP17 purified after being expressed in a recombinant E.coli in order to develop an assay system for thermotolerance in crops. The DCHsp17.7 including the coding sequence corresponding to a carrot HSP17 protein was recombined within pET-32(b) vector and achieved a maximum expression in 4 hours after an induction in E.coli. The purified DCHsp17.7 was used as an antigen to generate the corresponding antibody. The polyclonal antiserum was confirmed for it's specificity only to the low molecular weight (1mw) HSP. Besides, the possibilities to use the antiserum to interact with 1mwHSPs from other plants such as rice, cucumber, tomato, and hot pepper were examined to be plausible. To reveal any specific correlation between the amounts of 1mwHSP expressed upon HS conditions and an acquisition of thermotolerance two different approaches have been applied. first, it has been shown that only the pre-HS conditions inducing the synthesis of HSP17 allowed for the seedlings to achieve an thermotolerance and to survive the following lethal condition. Second, a western analysis using 15 different collected lines of hot peppers was performed to distinguish each other in terms of the amount of 1mwHSP. The results indicated that all 14 hot pepper lines were able to synthesize HSPs in response to an exposure to HS conditions and the amounts of the proteins synthesized at different HS temperatures were variable among the lines. There are several different patterns of 1mwHSP synthesized as a function of temperature increase observed and their correlation to physiological aspects of thermotolerance remains to be analyzed.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1242-1248
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• 2007

Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to ${\alpha}$-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the ${\alpha}$-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at $80^{\circ}C$ and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life ($t_{1/2}$) values of 10 min at $90^{\circ}C$, despite the high similarity to ${\alpha}$-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermo stability. The presence of $Ca^{2+}$ seemed to be critical, significantly changing $t_{1/2}$ at $90^{\circ}C$ to 153 min by the addition of 0.5 mM $Ca^{2+}$. On the other hand, the thermostability was not enhanced by the addition of $Zn^{2+}$ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermo stability, indicating that the residues involved in metal binding is very critical for the thermostability.

• Journal of Microbiology
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• v.41 no.3
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• pp.212-218
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• 2003

Citrus phytoene synthase (CitPsy) and ${\beta}$-carotene hydroxylase (CitChx), which are involved in caroteinoid biosynthesis, are distantly related to the corresponding bacterial enzymes from the point of view of amino acid sequence similarity. We investigated these enzyme activities using Pantoea ananatis carotenoid biosynthetic genes and Escherichia coli as a host cell. The genes were cloned into two vector systems controlled by the T7 promoter. SDS-polyacrylamide gel electrophoresis showed that CitPsy and CitChx proteins are normally expressed in E. coli in both soluble and insoluble forms. In vivo complementation using the Pantoea ananatis enzymes and HPLC analysis showed that ${\beta}$-carotene and zeaxanthin were produced in recombinant E. coli, which indicated that the citrus enzymes were functionally expressed in E. coli and assembled into a functional multi-enzyme complex with Pantoea ananatis enzymes. These observed activities well matched the results of other researchers on tomato phytoene synthase and Arabidopsis and pepper ${\beta}$-carotene hydroxylases. Thus, our results suggest that plant carotenoid biosynthetic enzymes can generally complement the bacterial enzymes and could be a means of carotenoid production by molecular breeding and fermentation in bacterial and plant systems.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.449-456
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• 2008

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a $Ni^{2+}$-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately $40^{\circ}C$. The enzyme maintained approximately 70% of its maximum activity even at $60^{\circ}C$, whereas its activity rapidly decreased at below $37^{\circ}C$. The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.

• The korean journal of orthodontics
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• v.43 no.6
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• pp.294-301
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• 2013

Objective: To determine the interleukin (IL)-6 levels in gingival crevicular fluid (GCF) of patients with severe root resorption after orthodontic treatment and investigate the effects of different static compressive forces (CFs) on IL-6 production by human periodontal ligament (hPDL) cells and the influence of IL-6 on osteoclastic activation from human osteoclastic precursor (hOCP) cells in vitro. Methods: IL-6 levels in GCF samples collected from 20 patients (15 and 5 subjects without and with radiographic evidence of severe root resorption, respectively) who had undergone orthodontic treatment were measured by ELISA. The levels of IL-6 mRNA in hPDL cells and IL-6 protein in conditioned medium after the application of different uniform CFs (0, 1.0, 2.0, or 4.0 $g/cm^2$ for up to 72 h) were measured by real-time PCR and ELISA, respectively. Finally, the influence of IL-6 on mature osteoclasts was investigated by using hOCP cells on dentin slices in a pit-formation assay. Results: Clinically, the IL-6 levels were significantly higher in the resorption group than in the control group. In vitro, IL-6 mRNA expression significantly increased with increasing CF. IL-6 protein secretion also increased in a time- and magnitude-dependent manner. Resorbed areas on dentin slices were significantly greater in the recombinant human IL-6-treated group and group cultured in hPDL cell-conditioned medium with CF application (4.0 $g/cm^2$) than in the group cultured in hPDL cell-conditioned medium without CF application. Conclusions: IL-6 may play an important role in inducing or facilitating orthodontically induced inflammatory root resorption.