• Journal of Physiology & Pathology in Korean Medicine
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• v.35 no.4
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• pp.117-123
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• 2021

Gefitinib, a first generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), provides obvious clinical benefit in patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, patients ultimately develop gefitinib resistance which mainly caused by EGFR T790M secondary mutation. In the current study, we investigated whether the root extract of Scutellaria baicalensis (SB) overcomes gefitinib resistance. Gefitinib-resistant H1975 human NSCLC cells (EGFR L858R/T790M double mutant) were treated with gefitinib and/or ethanol extract of SB (ESB) to evaluate the effect of ESB on the gefitinib sensitivity. The cell viability was measured by MTT assay and trypan blue exclusion assay. The colony-forming ability was evaluated by anchorage-dependent colony formation assay. Combined treatment with gefitinib and ESB markedly decreased the cell viability and colony formation than single treatment with gefitinib or ESB in H1975 cells. In addition, cells treated with both gefitinib and ESB exhibited a significant increase of sub-G1 DNA content which indicates apoptotic cells compared with those treated with gefitinib or ESB alone. As a molecular mechanism, combined treatment with gefitinib and ESB strongly downregulated the phosphorylation of ERK and JNK than single treatment with gefitinib or ESB. Taken together, our results demonstrate that ESB sensitizes H1975 cells to gefitinib treatment. We cautiously propose that ESB can be used in combination with gefitinib for the advanced NSCLC patients with acquired resistance to EGFR TKIs.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.453-459
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• 2000

Three rice (Oryza sativa L. var Dong-Jin) mutants (DTR1, DTR2, DTR3) resistant to S-methyltryptophan (5MT) were selected by mutagenized M3 seeds. The frequency of chlorophyll mutations induced by the EMS (0.2%) treatment performed 2 hours after flowering is clearly higher than that induced by other treatments in M1 generation. Progeny obtained from the self-pollinating of 5MT-resistant lines segregated with 3 : 1 of resistant to sensitive ratio. Furthermore, the ratio of homozygote to heterozygote in 5MT-resistant plants of the M4 generation was 1：2. These results show that 5MT resistance was inherited as a single dominant nuclear gene. The resistance was also expressed in callus derived from seeds. Total free amino acid content in homozygous seeds of DTR1 and DTR2 showed about 1.7 fold-increased compared to the wild-type seeds. In particular, the levels of phenylalanine and Iysine were, respectively, 6.2 and 3.2 times higher than those in the wild-type seeds. However, seeds of DTR3 had lower levels of free amino acid than the wild-type seeds. This result indicate that these mutants as a significant step towards the production of new rice with balanced amino acid content.

• Korean Journal of Breeding Science
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• v.42 no.4
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• pp.413-418
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• 2010

A total of 424 plants was regenerated from the seed-culture of a rice cultivar, 'Ilpum'. The regenerated plants were grown in a greenhouse. The 297 plants with high fertility were selected among 424 plants. The harvested seeds from each plant were planted to each line at experiment field in 2008 and 2009. The each line was evaluated for the agronomic and morphological traits, also. The 64 lines (21.5%) showed significant differences in agronomic and morphological traits from donor cultivar 'Ilpum' among 297 lines. The heading date different from donor cultivar 'Ilpum' showed highest frequency in 297 lines, and accounts for 9.1% (29 lines). The phenotype of opaque endosperm and rolling leaf account for 1.7% and 1.3% in 297 lines, respectively. The genetic segregation was observed in dwarf/semi-dwarf, rolling leaf and opaque endosperm at $S_1$ generation, but not in $S_2$ generation. These results suggest that the mutant derived from a tissue-culture will be one of the promising genetic resources, due to its wide variation and high frequency of mutation, comparatively.

• Journal of Radiation Industry
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• v.5 no.3
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• pp.211-220
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• 2011

This study was carried out to develop salt tolerant varities of rapeseed (Brassica napus L.) which can be grown in the high salty reclaimed land. The seeds of three varieties 'Naehan', 'Tammi', and 'Halla' were treated by proton ion beams and gamma rays with 0 to 2,000 Gy. For the selection of salt tolerant lines, emergence and survival rate, and growth characteristics of $M_2$ to $M_4$ generations were investigated in the Saemangeum reclaimed fields with the different salt concentrations. The lines with potential salt tolerance were selected in the $M_4$ generation and tested indoor for their growth characteristics. There was no significant changes in the soil pH for $M_4$ generation during growth period. However, soil EC was higher in early spring than sowing period (mid October). In $M_4$ generation test, the seeds of original and selected line showed high rates of emergence and survival, as determined one month after sowing. After wintering, however, the original varieties showed the significant reduction in the survival rate, while the selected lines showed a higher survival rate and good growth, leading to the completion of their life cycle. Consequently we selected 9 lines from $M_4$ generation with better performance in growth and yield. Soil EC was $2.8{\sim}4.3dS{\cdot}m^{-1}$ during $M_4$ generation growth period. The laboratory test of the lines selected from $M_4$ generation was made for their salt tolerance potential. The selected lines showed higher chlorophyll and proline contents than the original varieties. There was also no significant difference in the emergence rate of seed between the original and selected varieties. In 200 mM natural sea salt, the $N{\gamma}600-21-1-641$ line derived from 'Naehan' was the highest in growth rate, leaf chlorophyll and proline contents. $T{\gamma}800-20-2-461$ line derived from 'Tammi' didn't show significant difference in growth rate compared to original variety in 200 mM and withered in 250 mM like other lines as time passed. $H{\gamma}200-7-1-740$ line showed similar growth and chlorophyll content compared to its original variety.

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.123-132
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• 2005

Melittin,cationic 26-amino acid, is the principal component of the bee venom (BV) which has been used for treatment of inflammatory disease such as arthritis rheumatism NF-kB is activated by subsequent release of inhibitory IkB via activation of a multisubunit IkB kinase (IKK). We previously found that melittin bind to the sulfhydryl group of p50, a subunit of NF-kB. Since sulfhydryl group is present in kinase domain of IKKa and IKKb, melittin could modify IKK activity by protein-protein interaction. We therefore examined effect of melittin on IKK activities in sodium nitroprusside (SNP)-stimulated synoviocyte obtained from RA patients. Melittin suppressed the SNP-induced release of IkB resulted in inhibition of DNA binding activity of NF-kB and NF-kB-dependent luciferase activity. Consistent with the inhibitory effect on NF-kB activation, IKKa and IKKb activities were also suppressed by melittin. Surface plasmon resonance analysis realized that melitin binds to IKKa $(Kd\;=\;1.34{\times}10-9M)$ and IKKb$(Kd\;=\;1.0{\times}10-9M)$. Inhibition of IKKa and IKKb resulted in reduction of the SNP-induced production of inflammatory mediators NO and PGE2 generation. The inhibitory effect of melittin on the IKKs activities, binding affinity of melittin to IKKs, and NO and PGE2 generation were blocked by addition of reducing agents dithiothreitol and glutathione. In addition, melittin did not show inhibitory effect in the transfected Synoviocytes with plasmid carrying dominant negative mutant IKKa (C178A) and IKKb (C179A). These results demonstrate that melittin directly binds to sulfhydryl group of IKKs resulting in IkBrelease, thereby inhibits activation of NF-kB and expression of genes involving in the inflammatory responses.

• Plant Resources
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• v.8 no.1
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• pp.60-66
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• 2005

Lysine is the first essential amino acid for optimal nutrient quality in rice grain. For the narrow genetic diversities of lysine contents in rice, somaclonal variation was the source of mutation in our breeding program. Biochemical selection was conducted using 1 mM S-(2-aminoethyl) cysteine followed by two passages of 5 mM lysine plus threonine in the callus subculture medium. The lysine contents in endosperm of all progenies recovered from the biochemical selection were higher than those of their donor cultivar 'Hwayeongbyeo'. These elevated lysine levels of mutants were successfully transmitted to $M_4$ generation. The lysine contents in endosperm varied 3.85 to $4.80\%$ compare to their donor cultivar 'Hwayeongbyeo' was $3.85\%$. Three of high-lysine germplasms, Lys-l, Lys-2 and Lys-7 were selected by biochemical selection and rapid screening methods. DNA analysis showed that a new insertion of Tos 17 which mapped to rice chromosome 11 on the high-lysine mutant, Lys-2.

• Environmental Mutagens and Carcinogens
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• v.25 no.1
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• pp.19-24
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• 2005

The Swedish double mutation (KM670/671NL) of amyloid precursor protein (Swe-APP) is associated with early-onset familial Alzheimer's disease (FAD) and increases amyloid beta peptide production. Although APP/A/3 mediated neurotoxicity is observed both in vitro and in vivo, the relationship between mutant APP expression, A/3 production, and neuronal death observed in the brains of FAD patients remains to be elucidated. In this study, we investigated the mechanisms of Swe-APP-induced cell death in HEK293 and NGF-differentiated PC 12 cells. We found that the expression of Swe-APP induced cytochrome C relase, activation of caspase 3 in HEK 293 and NGF-differentiated PC 12 cells. We also show that the reactive oxygen species (ROS) was detected in Swe-APP expressing HEK 293 cells and NGF-differentiated PC 12 cells and that pretreatment with vitamine E attenuated the cellular death, cytochrome C release induced by Swe-APP expression, indicating the involvement of free radical in these processes. These results suggest one of possible apoptotic mechanisms of Swe-APP which could occur through cytochrome C release from mitochondria and this apoptosis inducing effects could be at least in part, due to ROS generation by Swe-APP expression.

• International Journal of Industrial Entomology and Biomaterials
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• v.39 no.1
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• pp.14-21
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• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• Genomics & Informatics
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• v.15 no.4
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• pp.136-141
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• 2017

Accurate detection of genomic alterations, especially druggable hotspot mutations in tumors, has become an essential part of precision medicine. With targeted sequencing, we can obtain deeper coverage of reads and handle data more easily with a relatively lower cost and less time than whole-exome or whole-genome sequencing. Recently, we designed a customized gene panel for targeted sequencing of major solid cancers. In this study, we aimed to validate its performance. The cancer panel targets 95 cancer-related genes. In terms of the limit of detection, more than 86% of target mutations with a mutant allele frequency (MAF) <1% can be identified, and any mutation with >3% MAF can be detected. When we applied this system for the analysis of Acrometrix Oncology Hotspot Control DNA, which contains more than 500 COSMIC mutations across 53 genes, 99% of the expected mutations were robustly detected. We also confirmed the high reproducibility of the detection of mutations in multiple independent analyses. When we explored copy number alterations (CNAs), the expected CNAs were successfully detected, and this result was confirmed by target-specific genomic quantitative polymerase chain reaction. Taken together, these results support the reliability and accuracy of our cancer panel in detecting mutations. This panel could be useful for key mutation profiling research in solid tumors and clinical translation.