• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.24
• /
• pp.10647-10652
• /
• 2015

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

• Microbiology and Biotechnology Letters
• /
• v.43 no.1
• /
• pp.22-30
• /
• 2015

CSF is a major concern for the swine industry, representing currently the most epizootically dangerous disease to the species. Numerous CSFV isolates with various degrees of virulence have already been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe per acute hemorrhagic fever with very high mortality. The molecular epidemiology of CSFVs has proven to be an essential tool for effective disease control and the development of safe and effective vaccines. Therefore, this study cloned and sequenced local CSFV isolates, and conducted a phylogenetic analysis based on the E2 glycoprotein encoding sequences.The RNA was extracted from PK15 cell culture passaged CSFV isolates, the cDNA prepared, and the complete E2 gene amplified with a product size of 1186 bp. The gelpurified PCR product was cloned into a pGEMT easy vector and the positive clone commercially sequenced. Aligning the nucleotide (1119 bp) and amino acid (373) sequences with 29 reference strains revealed nucleotide and amino acid sequence identities of 82.60-97.80% and 88.70-98.70%, respectively, indicating a higher mutation rate of the field CSFV strains. The phylogenetic analysis based on the complete E2 amino acid sequences also revealed a reliable differentiation of all the analyzed strains into specific genetic groups and subgroups, plus the local isolate (CSFV-E2) was found to cluster with the CSFV subgroup 2.2. Thus, the full-length E2 cds proved to be most suitable for a reliable and statistically significant phylogenetic analysis of CSFV isolates.

• Microbiology and Biotechnology Letters
• /
• v.14 no.3
• /
• pp.239-244
• /
• 1986

To develop the potential use as new host strain for gene cloning, alkaline-tolerant isolates from soil were examined for amylase activity, protease activity, antimicrobial activity and transformability by using plasmid pUB 110. Of these strains, one was selected and identified as Bacillus sp. YA-14. in the enzymatic properties of Bacillus sp. YA-14 the optimal conditions for the reaction of amylase and protease were at pH 0.8 and pH 7.5 respectively. The antimicrobial activity of Bacillus sp. YA-14 was also found. For the transformation, Bacillus sp. YA-14 was cultured to late logarithmic growth phase ai 37$^{\circ}C$ in modified SPI medium (pH 8.0) containing 0.4% MgSO$_4$. The presence of pUB 110 plasmid DNA in transformants was confirmed by electrophoresis and stably maintained in the new host.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1996.11a
• /
• pp.153-159
• /
• 1996

${\alpha}$-Melanotropin (Ac-Ser-Tyr- Ser-Met-Glu$\^$5/-His-Phe-Arg-Trp-Gly$\^$10/-Lys-Pro-Val-NH$_2$) is one of the first peptide hormones to be isolated and have its structure determined. It was early recognized to have essentially the same N-terminal tridecapeptide sequence as adrenocorticotropic hormone (ACTH) except that the N-terminal was acetylated in the case of ${\alpha}$-MSH but not in the case of ACTH, indicating that their biosyntheses were different (Figure 1). Subsequently it was discovered that ${\alpha}$-MSH and ACTH were derived from the same gene, currently referred to as proopiomelanocortin (POMC). Its original bioactivity was pigmentation, but it also was recognized that it may have activity in the central nervous system, though the precise nature of these central activities have been controversial. The recent cloning and expression of five melanocortin receptors, with the MC3 and MC4 receptors found primarily in the brain and the MC5 receptor (MC5-R) found throughout the body, has provided new impetus to understand the structure-activity relationships of ${\alpha}$-MSH at these receptors. The effects of ${\alpha}$-MSH on pigmentation are mediated by the MC1-R expressed specifically on the surface of melanocytes. Similarly the MC2-R is involved in the regulation of adrenal steroidogenesis by ACTH. However, given the complexity of expression of the MC3, MC4, and MC5 receptors, it has not been possible to identify any simple correlations between these receptors and the reported biological activities of the melanocortin peptides. Consequently, potent and receptor specific agonists and especially antagonists would be extremely valuable tools for the determination of the physiological roles of the MC3, MC4, and MC5 receptors. Though the extensive structure-activity relationships have provided much information on agonist activity related to pigmentary effects, only recently has it been possible to begin to systematically develop potent and selective antagonists.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1996.11a
• /
• pp.65-73
• /
• 1996

LPS/LOS, the compound found only in gram-negative bacterial outer membrane, plays important roles in bacterial maintenance as well as its pathogenesis. We isolated and characterized several genes required for NTHi 2019 LOS biosynthesis, which encode enzymes required for sugar substrate synthesis or the transfer of substrates to receptor molecules. The htrB gene, however, appears to have more complex role. It has acryltransferase activity as well as various other activity, which may control regulation of LOS biosynthesis as well as its pathogenicity. Evidences supporting the latter come from the observations that the lipid A of the B29 induced significantly less TNF ${\alpha}$ from macrophages than that of the wild type LOS (unpublished data). H. influenzae A2-htrB mutant strain was also significantly less invasive than the wild type strain. The structural similarities of the enterobacterial LPS and the Haemophilus LOS enabled us to isolate the NTHi 2019 genes involved in LOS biosynthesis genes by using the S. typhimurium LPS deep core mutants. While a similar approach has been used for E. coli, this technique for selection of an LPS phenotype has not been applied to nonenterobacterial species. The difficulties inherent in the molecular manipulation of organism such as Neisseria and Haemophilus species make this approach particularly attractive in the identification and cloning LOS genes. Studies on genetic features of LPS/LOS biosynthesis would be useful for understanding bacterial pathogenesis as well as for developing vaccines for these gram-negative pathogenic bacteria.

• Parasites, Hosts and Diseases
• /
• v.47 no.4
• /
• pp.359-367
• /
• 2009

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.

• Journal of Applied Biological Chemistry
• /
• v.44 no.2
• /
• pp.59-64
• /
• 2001

Pepper plants (Nogkwang, 60-day old) were inoculated with Phytophthora capsici to induce sesquiterpene cyclase associated with the biosynthesis of phytoalexin (capsidiol), a substance related to the defense against pathogens in plants. One day after inoculation, mRNA was isolated from the root, cDNA synthesized, and a library constructed in a ZAP express XR vector. The efficiency was $2{\times}10^6pfu/{\mu}g$. Sesquiterpene cyclase cDNA from Hyoscyamus muticus was labeled with $^{32}P$ and used as a probe for screening the cDNA library. After the third screening, 25 positive clones were selected. Through restrictive digestion and DNA gel-blot analysis, six different cyclase gene expressions were identified. PSC1B sequences of the six clones were determined, which were 1966 base pairs encoded 556 amino acids with an expected molecular weight of 63.8 kDa. Response against the pathogen was different between the resistant and susceptible peppers. After the infection of the pathogen, the expression of PSC genes continued in the resistant peppers while the plants were alive. The expression in the susceptible peppers lasted for only 4 days.

• The Plant Pathology Journal
• /
• v.16 no.6
• /
• pp.297-305
• /
• 2000

Weeds are widely grown in the field and are infected by many viruses. A survey was conducted to identify viruses infecting weeds in Korea. Virus-infected weed samples including Rorippa indica (L.) Hiern, R. islandica (Oed.) Bord, Crepidiastrum denticulatum (Houtt.) Pak & Kawanno, Achyranthes japonica (Miq.) Nakai, and Chrysanthemum boreale (Makino) Makino were collected in Kyonggi Province. These weeds were grown in the greenhouse and were isolated on 10 test plants. Several virus isolates were isolated fron infected tissues and were further studied by host range assay, serological test, electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Each isolated virus strain was mechanically transmitted to weeds and various hosts including Nicotiana spp., Brassica spp., Vigna unguiculata, Capsicum annuum, and Cucumis sativus and showed systemic mosaic, vein clearing, necrosis, mottle, malformation, chlorosis, and/or death of host plants in some cases. Each virus was then purified using infected leaves and observed by EM. From these results three viruses were isolated and identified as Turnip mosaic virus (TuMV), Broad bean wilt virus (BBWV), and Cucumber mosaic virus (CMV). RT-PCR using virus-specific oligonucleotide primers and the cloning were conducted to determine the nucleotide sequences of coat proteins of the three viruses their amino acid sequence were deduced. The amino acid sequence homologies were about 92.7 to 99.7%, 96.2 to 97.7%, and 93.9 to 98.6% to other reported TuMV, BBWV, and CMV strains, respectively. These results suggest that many weeds may serve as primary inoculum source of diseases caused by TuMV, BBWV, CMV and that the management of these viral diseases can be achieved through weed control.

• Journal of the Korean Society of Tobacco Science
• /
• v.23 no.2
• /
• pp.123-132
• /
• 2001

In order to transform pokeweed antiviral protein cDNA to tobacco plant, total RNA was extracted from Phytolacca americana. PAP-II cDNA was synthesized from purified total RNA via RT-PCR and subcloned to recombinant vector pBluescript II SK-. 10 deletion mutant PAP-II cDNA fragments which were sequentially deleted from N-terminal by 90bp were synthesized from PAP-II cDNA except leading frame by PCR with primers designed in our laboratory. To select non-cytotoxic clone, pAc55M was constructed with yeast expression vector pAc55 and multicloning site(MCS). Sequentially deleted mutant PAP-II cDNAs were cloned on downstream of gall promoter of pAc55M. 6 non-cytotoxic deletion mutant PAP-II cDNA were selected. Selected cDNAs were cloned into plant expression vector pKGT101BH for transformation of these clones to plant through Agrobacterium tumefacience. After cloning, recombinant pKGT101BH carrying deleted mutant PAP-IIcDNA were transformed to Nicotiana tabacum cv. NC567. Transformed tobacco plants cultured on shooting and rooting media were transfered to green-house. About four weeks later, these plants were infected with physically infection using carborandum with PVY-VN strain. After 4 weeks, plants resistant to virus were selected , and seeds of these plants were gathered. Southern blot hybridization showed deleted fragments by 220bp and 420bp, so resistant ability of these plants is due to mutant PAP-II cDNA.

• Journal of Plant Biotechnology
• /
• v.30 no.4
• /
• pp.307-313
• /
• 2003

Fluorescent differential display (FDD) is a method for identifying differentially expressed genes in eukaryotic cells. The mRNA FDD technology works by systematic amplification of the 3' terminal regions of mRNAs. This method involve the reverse transcription using anchored primers designed to bind 5'boundary of the poly A tails, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences. The amplified cDNA subpopulations are separated by denaturing polyacrylamide electrophoresis. To identify the genes involved in the development of first trap leaf, we applied a FDD method using mRNAs from leaf base, first trap leaf and flower tissue, respectively. We screened several genes that expressed specifically in first trap leaf. Nucleotide sequence analysis of these genes revealed that these were protease inhibitor (PI), myo-inositol-1-phosphate synthase and lipocalin-type prostaglandin D synthase. Northern blot analysis showed that these genes were expressed specifically in first trap leaf (in vivo and in vitro). FDD could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.