• Molecular & Cellular Toxicology
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• v.1 no.3
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• pp.172-178
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• 2005

[ $21{\alpha}$ ]- and ${\beta}$-Methylmelianodiol were isolated as the inhibitor of IL-5 bioactivity from Poncirus tripoliata. To develope as an anti-septic drug, the genotoxicity of $21{\alpha}\;-and\;{\beta}-methylmelianodiol$ was subjected to high throughput toxicity screening (HTTS) because they revealed strong IL-5 inhibitory activity and limitation of quantity. Mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), single cell gel electrophoresis (Comet) assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. These compounds are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. Before performing the comet assay, $IC_{20}$ of $21{\alpha}-methylmelianodiol$ was determined the concentration of $25.51\;{\mu}g/mL\;and\;21.99\;{\mu}g/mL$ with and without S-9, respectively. Also $21{\beta}-methylmelianodiol$ was determined the concentration of $24.15\;{\mu}g/mL\;and\;\;22.46\;{\mu}g/mL$ with and without S-9, respectively. In the comet assay, DNA damage was not observed both $21{\alpha}-methylmelianodiol\;and\;21{\beta}-methylmelianodiol$ in mouse lymphoma cell line. Also, the mutant frequencies in the treated cultures were similar to the vehicle controls, and none of $21{\alpha}\;-and\;{\beta}-methylmelianodiol$ with and without S-9 doses induced a mutant frequency over. twice the background. It is suggests that $21{\alpha}\;-and\;{\beta}-methylmelianodiol$ are non-mutagenic in MOLY assay. The results of this battery of assays indicate that $21{\alpha}\;-and\;{\beta}-methylmelianodiol$ have no genotoxic potential in bacterial or mammalian cell systems. Therefore, we suggest that $21{\alpha}\;-and\;{\beta}-methylmelianodiol$, as the optimal candidates with both no genotoxic potential and IL-5 inhibitory effects must be chosen.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.14-21
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• 2011

Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.259-268
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• 2000

Senescence is a sequence of biochemical and physiological events that lead to death of a cell, organ, or whole organism. Senescence is now clearly regarded as a genetically determined and evolutionarilly acquired developmental process comprising the final stage of development. However, in spite of the biological and practical importance, genetic mechanism of senescence has been very limited. Through forward and reverse genetic approaches, we are trying to reveal the molecular and genetic mechanism of senescence in plants, employing leaf organs of Arabidopsis as a model system. Using forward genetic approach, we have initially isolated several delayed senescence mutants either from T-DNA insertional lines or chemical-mutagenized lines. In the case of ore 4 and ore 9 mutants, the mutated genes were identified. The recent progress on characterization of mutants and identification of the mutated genes will be reported. We are also screening mutations from other various sources of mutant pools, such as activation tagging lines and promoter trap lines. Two dominant senescence-delayed mutants were isolated from the activation tagging pool. Cloning of the genes responsible for this phenotype is in progress. For reverse genetic approach, the genes that induced during leaf senescence were first isolated by differential screening method. We are currently using PCR-based suppression subtractive hybridization, designed to enrich a cDNA library for rare differentially expressed transcripts. Using this method, we have identified over 35 new sequences that are upregulated at leaf senescence stage. We are investigating the function of these novel genes by systemically generating antisense lines.

• Journal of Technologic Dentistry
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• v.22 no.1
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• pp.145-152
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• 2000

In order to select an effective antibiotic substance against oral resident bacteria, we were isolated from soil and texonomically analyzed. Seven hundred and eighteen strains were isolated on humic acid- vitamin agar(HV agar) and 220 strains were on methanol medium from three each paddy forest, field and riverside soil samples. So, during the screening of antibiotics from soil, we isolated microorganisms showing powerful antagonistic activity against oral resident bacteria. Microorganism was tested against 25 strains of bacteria, yeast and fungi. Among them, No. 248 strain exhibited the most strongly growth inhibition. So, the taxonomical analysis the isolated strain was found to be unknown Actinomyces sp. and was named No 248. A production of the antibiotics from No. 248 begins at the early exponential phase developed at the 72th hour under the optinum conditions. The property of No. 248 antimicrobial compound was very stable under acid(pH 3.0) and alkali(pH 10.0) treatment, but it was instable in heat treatment at $120^{\circ}C$. For the improvement of antibiotic activity, two mutants were isolated from strain No. 248 by the treatment of mutagenic agents, NTG and hydroxylamine. As a result, the mutant strains excreted the potent antibiotics to inhibit the growth of Candida albicans.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.6 no.1
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• pp.96-107
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• 2006

Citrin deficiency resulting from mutations of SLC25A13is associated with two major clinical phenotypes; neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and adult-onset type 2 citrullinemia (CTLN2). In Korea, 7 cases of citrin deficiency have been diagnosed based on biochemical and molecular findings. Four NICCD cases were identified by newborn screening using MS/MS or presenting symptoms like cholestatic jaundice. They are all males, presenting with conjugated hyperbilirubinemia, elevated liver enzymes, hypoalbuminemia, mild hyperammonemia, elevated citrullin, methionine and threonine. All of them have been spontaneously recovered from hepatic manifestation by the age of 6-8 months. Mutation analysis has been performed using their genomic & cDNAs obtained from skin fibroblasts. They turned out to be compound heterozygotes carrying each of 851del4, IVS11+1G>A, and IVS13+1G>A. Three CTLN2 patients were identified. Two adult male patients presented with a sudden loss of consciousness, seizure, vomiting, hyperammonemia and citrullinemia in their twenties. They carried an IVS13+1G>A, 851del4, and IVS11+1G>A mutant alleles. The other CTLN2 patient was 52 year old female patient, manifesting lethargy, altered consciousness, irritability and hyperammonemia. Similar clinical symptoms had recurred at the delivery of first and second babies in her past medical history. She was managed by hemodialysis and survived with neurological sequellae. Also, we screened the presence of 9 common mutations in 500 Korean newborns using dried blood spot of filter papers. Only a allele carried 854del4 mutation. In conclusion, the entire picture of citrin deficiency in Korea including incidence, genotype, clinical features and natural courses, is still vague at the present time.

• Korean Journal of Environmental Agriculture
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• v.22 no.3
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• pp.203-209
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• 2003

In the course of screening for antifungal agents, a bacterial strain, DM3 was isolated from a mud sample collected at Daechon in Chungnam province and identified as Bacillus licheniformis based on API 50CHB test. It has antifungal activity against 12 plant pathogenic fungi in paper disc assay. At the 95% lethal dose of gamma radiation ($^{60}Co$, 10 kGy, $D_{10}=2.32\;kGy$), the antifungal activity deficient mutant (mDM3) against Colletotrichum gloeosporioides was induced From 2-D electrophoresis analysis, serine hydroxymethyltransferase (45.0 kDa), hypothetical protein(40.7 kDa), NifU protein homolog(15.4 kDa), and resolvase(12.5 kDa) homologous proteins were detected only in B. licheniformis DM3. Lysozyme(18.1 kDa) and alkyl hydroperoxide reductase(15.6 kDa) homologous proteins were expressed uniquely in B. licheniformis mDM3. Further studies are needed to reveal that these proteins from B. licheniformis DM3 could be closely related to the antifungal activity against plant pathogenic fungi.

• Journal of Environmental Health Sciences
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• v.31 no.5 s.86
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• pp.360-364
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• 2005

When rapeseed oil as the carbon source was used for tylosin production from Streptomyces fradiae TP-1239 was very sensitive to oleic acid. Cell growth was restrained by adding 0.8 g/l of oleic acid to the culture broth. Mutant strain TM-224-1 resistant to 1.2 g/l of oleic acid was obtained by screening in solid and liquid media containing oleic acid. The uptake rate of oleic acid by TM-224-1 was approximately 3.8 fold higher than the parent strain. For comparing the TM-224-1 and the parent strain, batch cultures were carried out in a jar fermentor. Cell growth of TM-224-1 strain was higher than the parent strain after two days of culturing. However, after four days of culturing, it was similar to that of the parent strain. The amount of rapeseed oil consumed by TM-224-1 and the parent strain were 60.5 and 78.2 g/l, respectively. The production and yield of tylosin was aproximately 2.0 and 3.2 fold higher than the parent strain, respectively. From these results, it was concluded that this mutant, which was resistant to oleic acid, has improved tylosin production.

• Korean Journal of Weed Science
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• v.13 no.2
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• pp.89-95
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• 1993

The Inhibiting activity of newly synthesized phenol (E-series) and triazine (T-series) derivatives was evaluated by using thylakoid membranes extracted from cyanobacteria (Anacystis nidulans $R_2$). There were no significant differences between phenol derivatives and dinoseb to the thylakoid membrane extracted from wild type in the Hill reaction. However, a phenol derivative, E-24 which has no -Cl at phenyl ring, did not show any activity. The longer the length of R substituents was in phenol derivatives, the lower inhibiting activity was in the Hill reaction. Triazine derivatives, T-27, T-28, T-40, T-41, T-47 and T-48 were also compared with diuron and atrazine. Among triazine compounds, T-27 and T-28 showed 10 and 30 times activity as high as atrazine to wild type, respectively. Other triazine derivatives, T-40, T-41, T-47 and T-48 showed low inhibiting activity to wild and mutant type. A structural difference of T-27 and T-28 from T-40, T-41, T-47 and T-48 was the presented of -C-NH-. Both T-27 and T-28 were very closely associated with serine, an amino acid located at the 264th position of D1 protein because of the resistant ratio(R/S) to mutant G-264 were higher than that of atrazine.

• Journal of Life Science
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• v.28 no.10
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• pp.1140-1146
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• 2018

Dual-specificity phosphatases (DUSPs) play a role in cell growth and differentiation by modulating mitogen-activated protein kinases. DUSPs are considered targets for drugs against cancers, diabetes, immune diseases, and neuronal diseases. Part of the DUSP family, DUSP19 modulates c-Jun N-terminal kinase activity and is involved in osteoarthritis pathogenesis. Here, we report screening of cavity-creating mutants and the crystal structure of a cavity-creating L75A mutant of DUSP19 which has significantly enhanced enzyme activity in comparison to the wild-type protein. The crystal structure reveals a well-formed cavity due to the absent Leu75 side chain and a rotation of the active site-bound sulfate ion. Despite the cavity creation, residues surrounding the cavity did not rearrange significantly. Instead, a tightened hydrophobic interaction by a remote tryptophan residue was observed, indicating that the protein folding of the L75A mutant is stabilized by global folding energy minimization, not by local rearrangements in the cavity region. Conformation of the rotated active site sulfate ion resembles that of the phosphor-tyrosine substrate, indicating that cavity creation induces an optimal active site conformation. The activity enhancement by an internal cavity and its structural information provide insight on allosteric modulation of DUSP19 activity and development of therapeutics.

• Experimental and Molecular Medicine
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• v.50 no.11
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• pp.8.1-8.14
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• 2018

We previously demonstrated that the direct transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) into the dentate gyrus ameliorated the neurological symptoms of Niemann-Pick type C1 (NPC1)-mutant mice. However, the clinical presentation of NPC1-mutant mice was not fully understood with a molecular mechanism. Here, we found 14,15-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P450 (CYP) metabolite, from hUCB-MSCs and the cerebella of NPC1-mutant mice and investigated the functional consequence of this metabolite. Our screening of the CYP2J family indicated a dysregulation in the CYP system in a cerebellar-specific manner. Moreover, in Purkinje cells, CYP2J6 showed an elevated expression level compared to that of astrocytes, granule cells, and microglia. In this regard, we found that one CYP metabolite, 14,15-EET, acts as a key mediator in ameliorating cholesterol accumulation. In confirming this hypothesis, 14,15-EET treatment reduced the accumulation of cholesterol in human NPC1 patient-derived fibroblasts in vitro by suppressing cholesterol synthesis and ameliorating the impaired autophagic flux. We show that the reduced activity within the CYP system in the cerebellum could cause the neurological symptoms of NPC1 patients, as 14,15-EET treatment significantly rescued cholesterol accumulation and impaired autophagy. We also provide evidence that the intranasal administration of hUCB-MSCs is a highly promising alternative to traumatic surgical transplantation for NPC1 patients.