• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2305-2309
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• 2012

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin ($IC_{50}$ 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 ${\mu}g$ protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the $T_m$ telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.

• Biomedical Science Letters
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• v.8 no.1
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• pp.29-37
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• 2002

Listeria monocytogenes poses an increasing health risk, which in part is due to increasing health risk, consumption of ready-to-eat food products and the introduction of increasing numbers of food products from regions with different dietary habits. L. monocytogenes can be present in meat, shellfish, vegetables, unpasteurised milk and soft cheese and poses a risk if food containing these products is stored at refrigeration temperature and is not properly heated before consumption, as L. monocytogenes is psychrophilic. Amplified-fragment length polymorphism (AFLP) analysis is the method of genotypic techinique in which adaptor oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification. The amplified fragments are electrophoretically separated to give strain-specific band profiles. Single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains of Listeria monocytogenes was developed. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of Salmonella, Shigella, Yersinia and E. coli. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level. The AFLP patterns of L. monocytogenes are divided by the kinds of specimens in which were isolated. SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method. These features make SE-AFLP suitable for use in either field or laboratory applications.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.168-172
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• 2006

In this study. the transcriptional features of a 2.8 kb region spanning the sigH and rplA genes of Bacillus subtilis were clarified using synthetic oligonucleotides complementary to the transcripts of the rpmG, secE, nusG, and rplK genes. The 5' ends of three transcripts corresponding to this region were located and mapped on the chromosome via primer extension analysis. Three regions, designated Prg, Pn, and Prk, which partially share the consensus sequence recognized by ${\sigma}^A$ RNA polymerase, were theorized to function as promoter elements. The rpmG and secE genes of B. subtilis were cotranscribed from the designated prg promoter, whereas the nusG and rplK genes were transcribed separately from the Pn and Prk promoters, respectively. Accordingly, the transcriptional features, as well as the gene organization, of the region encompassing the sigH and rplA genes of B. subtilis, including the rpmG-secE-nusG-rplK genes, were determined to be distinct from those of Escherichia coli. Divergences in terms of gene organization and transcriptional features within the relevant region would serve as excellent criteria for the delineation of phylogenetic relationships among bacteria.

• Development and Reproduction
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• v.23 no.4
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• pp.377-384
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• 2019

Genomic DNA extracted from representatives of two populations, Gunsan and Chinese, of Urechis spp. was amplified using PCR with several primers. The band-sharing (BS) value between individuals no. 05 from the Gunsan population and no. 22 from the Chinese population was 0.206, which was the lowest recognized value. Oligonucleotides primer OPC-04 revealed 44 unique loci, which distinguished the Chinese population. Primer OPB-17 allowed the discovery of 22 loci shared by the two populations, which were present in all samples. Based on the average BS results, individuals from the Gunsan population demonstrated lower BS values (0.661±0.012) than did those from the Chinese population (0.788±0.014; p<0.05). The shortest genetic distance (GD) displaying a noteworthy molecular difference was between individuals CHINESE no. 12 and no. 13 (GD=0.027). Individual no. 06 from the Gunsan population was most distantly related to CHINESE no. 22 (GD=0.703). A group tree of the two populations was constructed by UPGMA Euclidean GD analysis based on a total of 543 fragments generated using six primers. The explicit markers recognized in this study will be used for genetic analysis, as well as to evaluate the species security and proliferation of echiuran individuals in intertidal regions of the Korean Peninsula.

• Development and Reproduction
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• v.22 no.2
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• pp.193-198
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• 2018

The author undertook PCR-founded genetic platform to investigate the hierarchical dendrogram of Euclidean genetic distances of one razor clam population, particularly for Solen corneus, which was further associated with those of the other clam population, by engaging with the precisely designed oligonucleotide primer sets. Seven oligonucleotides primers were used producing a total of 639 counted bands in population A and 595 in population B, respectively, ranging in size of DNA fragments from larger than approximately 50 bp to less than 1,100 bp. Their primers generated 39 specific fragments (6.10%) in population A and 47 (7.90%) in population B, respectively Comparatively, individuals of one razor clam population were fairly related to that of the other clam population, as shown in the hierarchical dendrogram of Euclidean genetic distances. The analysis of genetic variation between razor clam populations could offer important statistics for fisheries and mariculture. Generally the results showed specific and/or conserved genetic loci between razor clam populations. Specific markers established by the author will be valuable for the genetic analysis, species protection and increase of razor clam individuals in coastal region of the Korean Peninsula.

• Journal of Biosystems Engineering
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• v.39 no.2
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• pp.111-114
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• 2014

Purpose: This review aims to introduce aptamers and the methods of its development to improve the sensitivity and selectivity to target bacteria. In this review, we have highlighted current developments and directions in the pathogen detection based on aptamers. Background: Aptamers, the specific nucleic acid sequences, can bind to targets with high affinity and specificity. Some of researches on the use of aptamers for the detection of pathogen have been reported in recent years. Aptamers have more applicability than antibodies for the development of pathogen detection using biosensor; such as easy to synthesis and labeling, lack of immunogenicity, and a low cost of production. However, only few reports on the development and use of aptamers for the detection of pathogen have been published. Review: Aptamers specific to pathogen are obtained by whole-cell systematic evolution of ligands by exponential enrichment (SELEX) process. SELEX process is composed of screening random oligonucleotide bound with target cells, multiple separation and amplification of nucleic acids, final identification of the best sequences. For improving those affinity and selectivity to target bacteria, optimization of multiple separating process to remove unbounded oligonucleotides from aptamer candidates and sorting process by flow cytometry are required.

• Journal of Life Science
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• v.10 no.4
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1998.11a
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• pp.121-121
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• 1998

Artemisia annua, an indigenous plant in Korea, contains a clinically important potent antimalarial principle, artemisinin. Artemisinin is a cadinane-type sesquiterpene endoperoxide. Cadinane synthase catalyzes the first committed step in artemisinin biosynthesis by cyclizing farnesyldiphosphate. In hopes of finding a cadinane synthase gene involved in artemisinin biosynthesis, oligonucleotides were synthesized on. the basis of the consensus nucleotide sequences and Nco I restriction sites for convenience in cloning. Specifically, nucleotide sequences of two highly conserved regions were deduced from the genes of similar function of Hyoscyamus muticus, Nicotiana tabacum, Abies grandis, Lycopersicon esculentum, and Gossypium hirsutum to construct a set of primers for polymerase chain reation (PCR). A 184 bp fragment was found to be amplified by PCR, and subsequently cloned. The gene revealed 62.8% identity in nucleotide and 55.6% in amino acid sequence to correspondent gene of N. tabacum. The gene was different from another sesquiterpene cyclase gene of A. annua, germacranadiene synthase gene, recently reported by Mercke and Bordelius (1998).

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.310-320
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• 2005

A membrane-based oligonucleotide array was used to detect predominant bacterial species in human fecal samples. Digoxygenin-labeled 16S rDNA probes were generated by PCR from DNA that had been extracted from fecal samples or slurries. These probes were hybridized to an array of 120 oligonucleotides with sequences specific for 40 different bacterial species commonly found in human feces, followed by color development using an alkaline phosphatase-conjugated antibody and NBT /BCIP. Twenty of the species were detected by this method, but E. coli, which was present at $\~$1 $\times 10$^5$ CFU per gram feces, was not detected. To improve the sensitivity of this assay, reverse transcriptase-PCR was used to generate probes from RNA extracted from fecal cultures. Coupled with a chemiluminescence detection method, this approach lowered the detection limit for E. coli from $\~1$ $\times 10$^6$ to ${\leq}$ 1 $\times 10$^5$ These results indicate that the membrane-array method with reverse transcriptase-PCR and chemiluminescence detection can simultaneously identify bacterial species present in fecal samples at cell concentrations as low as${\leq}$ 1 $\times 10$^5$ CFU per gram.

• Development and Reproduction
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• v.16 no.1
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• pp.47-51
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• 2012

Genomic DNAs (gDNAs) were isolated from the hard clam ($Meretrix$ $lusoria$, Roding, 1798) populations of Gunsan located in the Yellow Sea of the Korean peninsula. Genetic distances among different individuals of the LSCP (light shell color population) population of the hard clam (lane 1-11), GSCP (grey shell color population) population of the hard clam (lane 12-22) and DSCP (dark shell color population) population of the hard clam (lane 23-33), respectively, were generated using Systat version 10 according to the bandsharing values and similarity matrix. The dendrogram, generated by seven reliable oligonucleotides primers, indicates 3 genetic clusters. LSCP population could be evidently discriminated with the other two populations among three populations. The longest genetic distance (0.801) was found to exist between individuals in the two populations, between individuals' no. 33 of the DSCP population and no. 06 of the LSCP population. The higher fragment sizes (>2,000 bp) are much more observed in the GSCP population. Three hard clam populations can be clearly distinguished, especially, by their morphological characters and PCR-based approach.