• Journal of the Korean Society of Radiology
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• v.13 no.1
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• pp.125-132
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• 2019

Potassium cyanate (KCN) is an inorganic reagent and can induce the post-translation carbamylation of proteins. The carbamylated reaction in the body is involved in cell death in various diseases. According the results in our previous study, KCN enhances the radiosensitivity of human colorectal cancer cell line, HCT 116 cells. However, it was not enough to confirm the mechanism that KCN works in these cells. To determinated the mechanisms of KCN in the cells with increased radiosensitivity, HCT 116 cells were treated KCN with low-dose gamma-radiation. And then, we examined alteration of the cell cycle, cell proliferation, cytokine level and the activation of cell signaling protein. As a result, cell cycle arrest and cell death were induced by the activation of caspase-3 and PARP in the irradiated cells with KCN treatment. These changes of the irradiated cell with KCN treatment were induced by the release of $TNF-{\alpha}$ via $NF-{\kappa}B$ activation. In conclusions, enhanced radio-sensitivity mediated by KCN induced cell death and it occurs by $NF-{\kappa}B$-dependent $TNF-{\alpha}$ production.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.52-52
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• 2001

Many nucleoside diphosphate (NDP) kinases are ubiquitous enzymes responsible for the exchange of ${\gamma}$-phosphates between tri- and diphosphonucleosides. The catalytic Many nucleoside diphosphate (NDP) kinases are ubiquitous enzymes responsible for the exchange of ${\gamma}$-phosphates between tri- and diphosphonucleosides. The catalytic reaction follows a ping-pong mechanism in which the enzyme is transiently phosphorylated on a histidine residue conserved in all nucleoside diphosphate kinases. Beside their role in nucleotide synthesis, these enzymes present additional functions, possibly independent of catalysis, in processes such as differentiation, cell growth, tumor progression, metastasis and development. To clone murine nm23-M5, several expressed sequence tags (ESTs) of the GenBank data base, selected according to their homology to nm23-H5 cDNA, reconstituted a complete open reading frame (GenBank AF222750). To test whether murine NDPKs (1, 2, 3, 4, 5, and 6) can inhibit Bax-mediated toxicity in yeast, co-transformation was performed respectively. The yeast S.cerevisiae was transformed with a copy expression plasmid containing the histidine selection marker and expressing murine Bax under the control of a galactose-inducible promoter. Several clones were selected and found to be growth inhibited when Bax expression was induced with galactose. A representative clone was transformed again with a copy expression plasmid containing the tryptophane selection marker and expressing either murine Bcl-xL or NDPK under the control of a galactose-inducible promoter. Several subclones of the double-transformants were selected and characterized. The ability of Bcl-xL and NDPKs to suppress Bax-mediated toxicity was determined by growing yeast cells overnight in galactose media and spot-testing on galactose plates starting with an equal number of yeast cells as determined by taking the OD$_{600}$. Ten-fold serial dilutions were used in the spot-test. Plates were grown at 3$0^{\circ}C$ for 2-3 days. All murine NDPKs suppressed Bax dependent apoptosis. Futher study will be peformed whether Bax-toxicity inhibition was caused by NDP kinase activity or additional function.n.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.337-346
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• 2023

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-I to LC3-II and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.46-46
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• 2003

Interleukin-10 (IL-10) is a homodimeric protein with a wide spectrum of anti-inflammatory and immune activities. It inhibits cytokine production and expression of immune surface molecules in various cell types. The transgenic mice carrying the human IL-10 gene in conjunction with the bovine $\beta$-casein promoter produced the human IL-10 in milk during lactation. Transgenic mice were generated using a standard method as described previously. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues with a primer set. In this study, stability of germ line transmission and expression of IL-10 gene integrated into host chromosome were monitored up to generation F15 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to F15 mice showed similar transmission rates (66.0$\pm$20.13%, 61.5$\pm$16.66%, 41.1$\pm$8.40%, 40.7$\pm$20.34%, 61.3$\pm$10.75%, 49.2$\pm$18.82%, and 43.8$\pm$25.91%, respectively), implying that the IL-10 gene can be transmitted stably up to long term generation in the transgenic mice. For ELISA analysis, IL-10 expression levels were determined with an hIL-10 ELISA and a mIL-10 ELISA kit in accordance with the supplier's protocol. Expression levels of human IL-10 from milk of generation F9 to F13 mice were 3.6$\pm$1.20 mg/ml, 4.2$\pm$0.93 mg/ml, 5.7$\pm$1.46 mg/ml, 6.3$\pm$3.46 mg/ml, and 6.8$\pm$4.52 mg/ml, respectively. These expression levels are higher than in generation F1 (1.6 mg/ml) mice. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations, although there was a little fluctuation in the transmission frequency and expression level between the generations.

• Korean Journal of Microbiology
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• v.17 no.3
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• pp.97-130
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• 1979

Many industrial processes those employ bacteria are subjected to phage infestations. In L-glutamic acid fermentions using acetic acid, the phage infestations of the organisms have been recently recognized. In efforts to elucidate the sources of phage contamination involved in the abnormal fermentation, a series of study was conducted to isolate the phages both from the contents of abnormally fermented tanks and the soil or sewage samples from the surroundings of a fermentation factory, to define major charateristics of the phage isolates, and finally to determine the correlation between the phage isolates and temperate phages originating from the miscellaneous bacterial species isolated from the soil or sewage samples. The results are summarized as follows; 1) All phages were isolated from the irregular fermentation tanks and soil or sewage samples, and they were designated as phage PR-1, PR-2, PR-3, PR-4, PR-5, PR-6, and PR-7, in the order of isolation. These PR-series phages were proved to be highly specific for the variant strains of Br. lactofermentum only, namely, phage PR-1 and PR-2 for Br. lactofermentum No. 468-5 and phage PR-3~PR-7 for Br. lactofemrentum No. 2256. By cross-neutralization test, the 7 phagescould be subdivided into 3 groups, i. e., phage PR-I and PR-2 the first, phage PR-3, PR-4, PR-5, PR-6 the second, and the phage PR-7 the third. 2) The 7 phages were virulent under the experimental conditions. They produced plaques with clear and relatively sharp margins without distinct halo. The mean sizes of plaques were 1.5mm in diameter for phage PR-1 and PR-2, and 1. Omm for phages PR-3~PR-7. Double layer technique modified by Hongo and described by Adams, was applied to assay of the PR-series phages. The factors influencing the plaques were as follows;young age cells of host bacteria cultured for 3-6 hours represented the largest number and size, optimum was pH 7.0, incubation temperature was $30^{\circ}C$, and agar concentration and amount of overlayer medium were 0.6% and 0.2ml, respectively. 3) PR-series phages were stable in 0.05M tris buffer and 0.1M ammonium acetate buffer solution. The addition of $5{\times}10^{-3}M$ magnesium ion effectively increased the stability. Thermostability experiments indicated that PR-series phages were stable at the teinperture between $50^{\circ}{\sim}55^{\circ}C$ in nutrient medium, $45^{\circ}{\sim}50^{\circ}C$ in buffer solution. However, the phages mere completely inactivated at 603C and 65$^{\circ}$C within 10 minutes. The phages were stable at the range of pH6~9 in nutrient medium and of pH 8-9 in buffer solution, respectively. Exposure of the phages to UV for 25, 60 and 100 seconds resulted in the complete loss of infectivily, respectively. 4) Electron microscopy showed that PR-series phage particles exhibited rather similar morphology, differing in the size All of PR-series phages had a multilateral head and had a simple long tiil about three to five times long as compared with head. By the size, phage PR-1 and PR-2, PR-3, PR-4, PR-5, and PR-6 and PR-7 were classified into same groups, respectively. The head and tail size of phage PR-1, PR-5, PR-5(T) and PR-7 were 85nm, 74nm and 235nm and 350mm, and 72nm and 210nm, respectively. 5) Nucleic acids of PR-series phages were double stranded DNA. The G+C contents of phage PR-1, PR-5 and PR-7 were 56.1, 52.9 and 53.7, respectively. The values of G+C contents derived from the $T_m$ were in agreement with the chemically determined values. 6) PR-series phages effectively adsorbed on their host bacteria at the rate of more than 90% during 5 min. K value for phage PR-1, PR-5 and PR-7 were calculated to be $6{\times}10^9 ml$ per minute, respectiveky. The pH of the medium did effect adsorption rate, but both temperature and age of host cells did not. Generally, optimum adsorption condition of phages seemed to be almost same as optimum growth conditions of host bacteria. 7) In one-step growth experiments, the latent periods at $30^{\circ}C$ for PR-1, and PR-7 were about 70, 50 and 55 min, respectively. The corresponding average burst size was 200, 70 and 90, respectively. Lpsis period according to the multiplicity of infection and a phage series. In case of m. o. i. 100, strain No. 2256 (PR-5) and No. 468-5(PR-1) failed to grow and turbidity decreased after 50 and 70min, respectively. 8) In the lysate of a plaque purified phage PR-5 infected bacteria, there observed 2 types ofphage particles, i. e., phage PR-5 and PR-5 (T) of similar morphology but differing at the length of phage tail, and phage tail like particles. The phage taillike particles could be divided into 4 types by the length. Induction experiments of Br. lactofermentum with UV irradiation, mitomycin C or bacitracin treatment produced neither phage PR-5 (T) or phage tail-like particles. 9) No lysis occured when the growth of 7 strains of miscellaneous bacteria, isolated from soil and sewage samples, were inoculated with either phage PR-5 (T) or phage tail-like particles the inoculation of phage PR-5 pellet resulted in the growth inhibition of the orgainsms in the spot test. The lysates obtained from 3 miscellaneous soil derived bacteria following mitomycin C treatment the growth of Br. lactofermentum, but did not lyze the bacterium.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.12 no.2
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• pp.140-149
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• 2009

Purpose: Helicobacter pylori infection is probably acquired in childhood and persists as an asymptomatic infection for decades in most individuals. It is unclear why only a minority of those infected develop a clinical manifestation, even in childhood, such as peptic ulcer disease. H. pylori infection activates local immune responses and causes lymphocyte infiltration in the gastric mucosa. We have previously reported that both T and B cells in the lamina propria play important roles in the local immune response of H. pylori-infected children. The aim of this study was to investigate the association between H. pylori genotypes and gastric mucosal lymphocytes. Methods: Twenty-five H. pylori-infected children (10 with peptic ulcer disease and 15 with gastritis) were enrolled in this study. We investigated the genotypes (cagA, cagE, vacA, and babA2) and evaluated the association with clinical manifestations, histopathology, and gastric mucosal lymphocytes. Results: The prevalence of cagA, cagE, vacA s1m1, and babA2 was 80%, 60%, 84%, and 88%, respectively. The most prevalent (68%) combination of cagA, vacA, and babA2 genotypes was cagA+/vacA s1m1+/babA2+. H. pylori genotypes were not associated with clinical manifestations, histopathology, or gastric mucosal lymphocytes. Conclusion: There was no association between the cagA, cagE, vacA, or babA2 status and gastric mucosal lymphocytes. The role of the host immune response in relation to H. pylori genotypes and disease potential in children needs further studies.

• Childhood Kidney Diseases
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• v.7 no.1
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• pp.52-59
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• 2003

Purpose : The renin-angiotensin system(RAS) plays an important role in renal growth and development. We have studied the prevalence of renal anomalies and documented the association between karyotype and renal anomalies using IVP and ultrasonography. Furthermore, to investigate the impact of RAS gene polymorphism on renal anomaly in Turner syndrome, we examined the ACE I/D genotype, angiotensinogen(AGT) gene M235T, angiotensin receptor type 1(ATR) gene A1166C. Methods : Cytogenetic analysis was performed in 33 Turner syndrome patients on peripheral blood lymphocytes. Ultrasonography(US) of the kidneys and collecting system and intravenous pyelography(IVP) were perfomed in all patients. Nuclear scintigraphy{Tc 99m dimercaptosuccinic acid(DMSA) scan} was also performed for the definite renal diagnosis if indicated. And, ACE I/D genotype, angiotensinogen(AGT) gene M235T, angiotensin receptor type 1(ATR) gene A1166C were examined by PCR amplification of genomic DNA samples. Results : The prevalence of renal anolmalies in Turner syndrome was 36.4%(12/33). The Karyotype 45, X was observed in 18 of the 33 girls(54.5%), of whom 8(44.4%) had renal anomalies. Mosaic karyotypes were observed in 11(33.3%) and four(12.2%) had a non-mosaic structural aberration of the X chromosome. In this group 4(25.7%) had renal anomalies. More renal anomalies were associated with the 45, X karyotype than those with mosaic/structural abnormalities of X chromosome, but the difference was not statistically significant(P>0.05). And, there was no significant differences in the RAS gene polymorphism and allele frequencies between renal anomaly group and normal group in Turner syndrome. Conclusion : The prevalence of renal anolmalies in Turner syndrome was 36.4%. There is no significant differences in the RAS gene polymorphism and allele frequencies between the renal anomaly group and the normal group in Turner syndrome.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2004.05a
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• pp.89-119
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• 2004

This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.

• Tuberculosis and Respiratory Diseases
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• v.41 no.3
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• pp.215-221
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• 1994

Background: It is well known that oxygen free radicals(OFR) play a vital role in the various type of acute lung injury. Among various antioxidant defense mechanisms, the superoxide dismutases(SOD) are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen. Eukaryotic cells contain two types of intracellular SOD : cytosolic, dimeric copper/zinc- containing enzyme(CuZnSOD) and mitochondrial, tetrameric manganese-containing enzyme(MnSOD). The purpose of this study is to evaluate the time-dependent gene expression of MnSOD and CuZnSOD in the endotoxin-treated rats, and to compare with the manifestations of LPS-induced acute lung injury in rats. Methods: Total RNA from rat lung was isolated using single step phenol extraction 0, 1, 2, 4, 6, 12, 18, 24 hours after E. coli endotoxin injection(n=3, respectively). RNA was separated by formaldehyde-containing 1.2% agarose gels elctrophoresis, transblotted, baked, prehybridized, and hybridized with $^{32}P$-labeled cDNA probes for rat MnSOD and CuZnSOD, which were kindly donated by Dr. Ho(Duke University, Durham, NC, USA). The probes were labeled by nick translation. Blots were washed and autoradiography were quantitated using laser densitometry. Equivalent amounts of total RNA/gel were assessed by monitoring 28S and 18S rRNA. Results: Endotoxin caused a rise in steady-state MnSOD mRNA levels by 4h with peak mRNA accumulation by 6h. Continued MnSOD mRNA expression was observed at 12h. CuZnSOD mRNA expression was observed from 1h to 24h with peak levels by 18h. Conclusion: These results suggest that SOD palys an important defensive role in the endotoxin-induced acute lung injury in rats.