• Journal of Microbiology
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• v.44 no.5
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• pp.548-555
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• 2006

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, $C_{83907}$ (K88ad, $CT^+,\;ST^+$). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

• Molecules and Cells
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• v.26 no.2
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• pp.146-151
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• 2008

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

• Molecules and Cells
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• v.27 no.5
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• pp.563-570
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• 2009

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

• Korean Journal of Veterinary Service
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• v.38 no.3
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• pp.145-153
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• 2015

In this study, we developed a loop-mediated isothermal amplification (LAMP) with hydroxynaphtol blue dye (HNB) for rapid and direct visual detection of porcine circovirus 2 DNA with high sensitivity and specificity. The LAMP was completed in 40 min at $63^{\circ}C$, and the results of the LAMP can be confirmed by naked eye without any detection process. The sensitivity of the LAMP was 10-fold higher than that of the commercial PCR (cPCR) and real time PCR (rPCR) previously reported. In clinical application, the PCV2 detection rate of the LAMP was the same on porcine tissue samples (75.0%, 36/48) between porcine blood samples (75.0%, 39/52). The PCV2 detection rate (75.0%) of LAMP was higher than those of the cPCR and rPCR (67.3%, 35/52) in blood samples. In conclusion, the LAMP developed in the study could be an useful alternative method for the detection of PCV2 in the swine disease diagnostic laboratories.

• Journal of Life Science
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• v.25 no.8
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• pp.925-931
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• 2015

We performed a molecular marker-based analysis of quantitative trait loci (QTLs) for traits that determine the quality of the appearance of grains, using 120 doubled-haploid (DH) lines developed by another culture from the F1 cross between ‘Cheongcheong’ (Oryza sativa L. ssp. Indica) and ‘Nagdong’ (Oryza sativa L. ssp. Japonica). The traits studied included length, width, and thickness of the grains, as well as length-to-width ratio and 1,000-grain weight. The objective of this study was to determine the genetic control of these traits in order to formulate a strategy for improving the appearance of this hybrid. Within the DH population, five traits exhibited wide variation, with mean values occurring within the range of the two parents. Three QTLs were identified for grain length on chromosomes 2, 5, and 7. Three QTLs were mapped for grain width on chromosome 2: qGW2-1, qGW2-2, and qGW2-3. Six chromosomes were identified for the grain length-to-width ratio; four of these were on chromosome 2, whereas the other two were on chromosomes 7 and 12. One QTL influencing 1,000-grain weight was identified and located on chromosome 8. The results presented in the present study should facilitate rice-breeding, especially for improved hybrid-rice quality.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.9-19
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• 2011

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

• The Korean Journal of Mycology
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• v.41 no.4
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• pp.218-224
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• 2013

Of 1,100 yeast strains which were isolated from various Korean fermented foods, screened for phosphate solubilization, five strains showed the ability to solubilize tricalcium phosphate. The 26S rDNA domain D1-D2 sequence analysis revealed the identification of strain Y393 and Y524 as Pichia anomala (99.8 and 100% identity, respectively), Y669 as Pichia farinosa (100% identity), Y901 as Candida versatilis (100% identity), and Y1101 as Pichia subpelliculosa (100% identity). All the phosphate solubilizing strains showed mesophilic characteristics. The temperature range for growth of 4 strains was $20{\sim}35^{\circ}C$ and P. farinosa Y669 was able to grow up to $45^{\circ}C$. The strain C. versatilis Y907 was able to grow at pH range of 5.0~6.0 and showed halophilic characteristics with tolerance to 15% of NaCl concentration. The Phosphate solubilizing yeast strains were survived well in bed soil for 8 weeks which were maintained densities of $10^7{\sim}10^8$ cfu/g. The highest phosphate solubilizing activity was observed in P. subpelliculosa Y1101. It solubilized 697.2 ug/mL of phosphorus from tricalcium phosphate with decrease in pH from 6.8 to 4.37 after 11 days of inoculation.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.292-301
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• 2010

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and betasubunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, $FSH{\beta}$ subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the $FSH{\beta}$ subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal specificity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified $FSH{\beta}$ protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p<0.05). These observations suggested that the expressed $FSH{\beta}$ protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

• IMMUNE NETWORK
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• v.9 no.6
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• pp.236-242
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• 2009

Background: Melanoma is the most fatal form of skin cancer due to its rapid metastasis. Recently, several studies reported that selenium can induce apoptosis in melanoma cells. However, the precise mechanism remains to be elucidated. In this study, we investigated the effect of selenium on cell proliferation in murine melanoma and on tumor growth and metastasis in C57BL/6 mice. Methods: Cell proliferation was measured by MTT assay in selenium-treated melanoma cells. Cell cycle distribution was analysized by staining DNA with propidum iodide (PI). mRNA and protein expression related to cell cycle arrest was measured by reverse transcription PCR and western blot. Tumor growth and metastasis was measured by in vivo model. Results: Selenium was suppressed the proliferation of melanoma cells in a dose dependent manner. The growth inhibition of melanoma by selenium was associated with an arrest of cell cycle distribution at G0/G1 stage. The mRNA and protein level of CDK2/CDK4 was suppressed by treatment with selenium in a time-dependent manner. In vivo, tumor growth was not suppressed by selenium; however tumor metastasis was suppressed by selenium in mouse model. Conclusion: These results suggest that selenium might be a potent agent to inhibit proliferative activity of melanoma cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4555-4561
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• 2014

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and $IC_{50}$ values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down-regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.