• Korean Journal of Microbiology
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• v.51 no.2
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• pp.108-114
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• 2015

We tried to analyze the growth time for secretion of the iron containing superoxide dismutase by comparing the intra-and extracellular enzyme activity from Streptomyces subrutilus P5 and analyze possible genetic information for this enzyme secretion. The mycelial dry weights and glucose concentrations in culture filtrates were determined during growth. Glucose was consumed rapidly during logarithmic growth phase and almost exhausted at 24 h of cultivation. While the intracellular activity of iron containing superoxide dismutase was first appeared at three hours, the extracellular activity of this enzyme appeared from 7.5 h of cultivation, early logarithmic growth phase. This early presence of the superoxide dismutase might not be the result of cell lysis but active secretion pathway. There was no information for signal peptide responsible for the enzyme secretion in sodF. However, we found a type three secretion box in the promoter region of sodF that has been known for the genes of type III secreted proteins in other bacteria. This is the first report on the possible existence of type III secretion in Streptomyces.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.221-226
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• 2008

To produce human ferritins in yeast, human H-chain and L-chain ferritins were amplified from previously cloned vectors. Each amplified ferritin gene was inserted into the pYES2.1/V5-His-TOPO yeast expression vector under the control of the GAL1promoter. Western blot analysis of the recombinant yeast cells revealed that H-and L-chain subunits of human ferritin were expressed in Saccharomyces cerevisiae. Atomic absorption spectrometry (AAS) analysis demonstrated that the intracellular content of iron in the ferritin transformant was 1.6 to 1.8-fold higher than that of the control strain. Ferritin transformants could potentially supply iron-fortified nutrients for food and feed.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1368-1376
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• 2008

In our previous study, the expression of active H-ferritins in Saccharomyces cerevisiae was found to reduce cell growth and reactive oxygen species (ROS) generation upon exposure to oxidative stress; such expression enhanced that of high-affinity iron transport genes (FET3 and FTR1). The results suggested that the recombinant cells expressing H-ferritins induced cytosolic iron depletion. The present study analyzes metabolic changes under these circumstances via proteomic methods. The YGH2 yeast strain expressing A-ferritin, the YGH2-KG (E62K and H65G) mutant strain, and the YGT control strain were used. Comparative proteomic analysis showed that the synthesis of 34 proteins was at least stimulated in YGH2, whereas the other 37 proteins were repressed. Among these, the 31 major protein spots were analyzed via nano-LC/MS/MS. The increased proteins included major heat-shock proteins and proteins related to endoplasmic reticulum-associated degradation (ERAD). On the other hand, the proteins involved with folate metabolism, purine and methionine biosynthesis, and translation were reduced. In addition, we analyzed the insoluble protein fractions and identified the fragments of Idh1p and Pgk1p, as well as several ribosomal assembly-related proteins. This suggests that intracellular iron depletion induces imperfect translation of proteins. Although the proteins identified above result from changes in iron metabolism (i.e., iron deficiency), definitive evidence for iron-related proteins remains insufficient. Nevertheless, this study is the first to present a molecular model for iron deficiency, and the results may provide valuable information on the regulatory network of iron metabolism.

• Journal of Applied Biological Chemistry
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• v.52 no.4
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• pp.157-162
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• 2009

An agar-liquefying bacteria (SC-22), which produces a diffusible agarase that caused agar softening around the colony was isolated from Daecheong lake in Korea. Chemotaxanomic and phylogenetic analyses based on 16S rRNA gene sequences revealed the strain was classified as Cellvibrio mixtus SC-22. The isolate SC-22 showed maximal extracellular agarase activity with 58.5 U/mL after 48 h cultivation in the presence of 0.2% agar. It was observed that the isolate produced two kinds of extracellular and three kinds of intracellular isoenzymes. The major agarase was purified from the culture filtrate of agarolytic bacteria by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The molecular mass of the purified enzyme was estimated to be 25 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 7.0 and $50^{\circ}C$, respectively. The agarase activity was activated by $Fe^{2+}$, $Na^+$ and $Ca^{2+}$ ions while it was inhibited by $Hg^{2+}$, $Mn^{2+}$ and $Cu^{2+}$ at 1 mM concentration. The predominant hydrolysis product of agarose by the enzyme was galactose and disaccharide on TLC, indicating the cleavage of $\beta$-1,4 linkage in a random manner. The enzyme showed high substrate specificity for only agar and agarose among various polysaccharides.

• Korean Journal of Acupuncture
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• v.26 no.3
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• pp.55-68
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• 2009

Objectives : To investigate the anti-inflammatory effects of Scutellaria baicalensis Georgi pharmacopuncture in lipopolysaccharide (LPS)-induced inflammatory rat model. Methods : Sprague-Dawley rats were divided into 4 groups; LPS control (n=6), LPS+Scutellaria baicalensis Georgi pharmacopuncture at BL23 (n=6, BL23), LPS+Scutellaria baicalensis Georgi pharmacopuncture at CV12 (n=6, CV12), and LPS+Scutellaria baicalensis Georgi pharmacopuncture at GV4 (n=6, GV4). Pharmacopuncture was given every two days for 4 weeks followed by inflammation induction by intraperitoneal LPS injection (5mg/kg). Blood, liver tissue, and peritoneal lavage fluid were taken and proinflammatory cytokines and other related factors were analysed. Results : For proinflammatory cytokines, CV12 pharmacopuncture group was significantly different compared with the control group in plasma IL-$1{\beta}$, IL-6, TNF-$\alpha$, and IL-10 5 h after LPS injection (P<0.05). For plasma IL-$1{\beta}$ and IL-6, CV12 pharmacopuncture group also showed significant difference at 2 h compared with the control (P<0.05). GV4 pharmacopuncture group was significantly different compared with the control at 5 h in plasma IL-$1{\beta}$, IL-6, and TNF-$\alpha$ and at 2 h in IL-10 (P<0.05). Liver cytokines were analyzed at 5 h after LPS injection; only CV12 pharmacopuncture group showed significant difference in IL-$1{\beta}$ (P<0.05) and others including IL-6, TNF-$\alpha$, and IL-10 had no difference compared with the control group. CD4/CD8 ratio and the phagocytic activities of polymorphonuclear neutrophils were not different from those of control group in all pharmacopuncture groups (P>0.05). Plasma NO3-/NO2- and intracellular adhesion molecule-1 of CV12 pharmacopuncture group were significantly lower than that of the control group (P<0.05). In the plasma concentration of prostaglandin E2, all 3 pharmacopuncture groups had significantly lower values than that of the control group (P<0.05), but there was no difference among pharmacopucnture groups. Monocyte chemoattractant protein-1 and cytokine-induced neutorphil chemoattractant-1 in peritoneal lavage fluid was significantly decreased in CV12 pharmacopuncture group compared with the control group (P<0.05). Conclusions : These results indicate that Scutellaria baicalensis Georgi pharmacopuncture at CV12 may have a potent anti-inflammatory effect in an LPS-induced inflammatory rat model.

• Journal of Life Science
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• v.14 no.3
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• pp.501-508
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• 2004

A bacterial strain CJ-3 which produced chitinase was isolated from Korean traditional soy sauce. Using 16S rDNA analysis, the strain CJ-3 was identified as Bacillus atrophaeus. The approximate molecular weight of the putative chitinase enzyme was 31.0 kDa and the enzyme activity was remarkably induced by addition of colloidal chitin (0.5, 1.0, 2.0%). The antioxidant activity was increased 53% by the browning reaction products of B. atrophneus CJ-3. Escherichia. coli lipopolysaccharides (LPS)-induced production of nitric oxide(NO) was reduced up to 45% by the browning reaction product in RAW264.7 macrophage. Inhibition of cell viability in the presence of LPS was recovered to normal level by the browning reaction product. These results suggest that browning reaction of B. atrophaeus CJ-3 plays an important role for activation of immune system. B. atrophaeus CJ-3 exhibited optimum temperature and pH of 37$^{\circ}C$ and pH 7.0∼8.0, respectively. The major intracelluar free amino acid was determined to be glutamate.

• Journal of Life Science
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• v.12 no.6
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• pp.812-820
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• 2002

In order to understand stress response of Leuconostoc mesenteroides against lactic acid, a new Leuconostoc sp. which has acid tolerance was isolated from various Kimchi samples. And it identified as Leuconostoc mesenteroides LM3 by comparing its fatty acid composition with reference strain. Its growth pattern was investigated by adding a given concentration of lactic acid at the lag phase to the stationary phase. In the DeMan, Rogosa, Sharpe (MRS) media containing over 0.4% (final v/v) lactic acid, this strain slowed slowly After exposure of the stationary phase cells to 4% of lactic acid for 60 min, this strain could survive, whereas a reference strain, Leuconostoc mesenteroides KCTC3505, showed no survival. And changes of trehalose concentration, the activity of trehalase and ATPase in the growth of Leuconostoc mesenteroides LM3 after addition of 0.6% (final v/v) lactic acid were investigated : After exposure to lartic acid, trehalose concentration in this strain was increased in comparison with no treatment, but its trehalase activity was not changed. And its ATPase activity was constant, and intracellular pH was almost constant. This result meant Leuconostoc mesenteroides LM3 should have a tolerance against lactic acid. It remains to further study the mechanism of this acid tolerance.

• Journal of Veterinary Clinics
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• v.35 no.2
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• pp.39-45
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• 2018

We evaluated the effects of green tea extract (GTE) supplementation at different dilution steps on boar sperm freezing and in vitro fertilization. Sperm intracellular hydrogen peroxide ($H_2O_2$), motility, viability, acrosome integrity and morphology were determined. In addition, sperm IVF parameters (penetration and monospermy) and glutathione (GSH) levels of presumptive zygotes (PZs) were evaluated. Semen was diluted in lactose egg yolk (LEY) and cooled at $5^{\circ}C$ for 3 h (first dilution step) and then diluted in LEY with 9% glycerol and maintained at $5^{\circ}C$ for 30 min (second dilution step). Four experimental groups were compared: first and second dilution steps without GTE (control), first dilution step with GTE (Step 1), second dilution step with GTE (Step 2) and first and second dilution step with GTE (Step 1+2). The spermatozoa were frozen in nitrogen vapor. Higher sperm motility, viability and acrosome integrity after thawing were observed in Step 1, Step 2 and Step 1+2 groups compared with the control (P < 0.05). Lower $H_2O_2$ level was observed in Step 1+2 compared with control and Step 1 (P < 0.05). For IVF, matured oocytes were co-cultured with spermatozoa frozen according to the experimental groups. GSH levels of PZs were significantly higher in Step 2 and Step 1+2 than in control and Step 1 (P < 0.05) without a significant difference in IVF parameters. In conclusion, supplementation with GTE in both first and second dilution steps during the freezing process resulted in better boar sperm cryopreservation and might be beneficial for further embryo development.

• Nutrition Research and Practice
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• v.14 no.1
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• pp.3-11
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• 2020

BACKGROUND/OBJECTIVES: Oxidative stress causes cell damage and death, which contribute to the pathogenesis of neurodegenerative diseases. Urolithin A (UA), a gut microbial-derived metabolite of ellagitannins and ellagic acid, has high bioavailability and various health benefits such as antioxidant and anti-inflammatory effects. However, it is unknown whether it has protective effects against oxidative stress-induced cell death. We investigated whether UA ameliorates H₂O₂-induced neuronal cell death. MATERIALS/METHODS: We induced oxidative damage with 300 μM H₂O₂ after UA pretreatment at concentrations of 1.25, 2.5, and 5 μM in SK-N-MC cells. Cytotoxicity and cell viability were determined using the CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using a 2,7-dichlorofluorescein diacetate assay. Hoechst 33342 staining was used to characterize morphological changes in apoptotic cells. The expressions of apoptosis proteins were measured using Western blotting. RESULTS: UA significantly increased cell viability and decreased intracellular ROS production in a dose-dependent manner in SK-N-MC cells. It also decreased the Bax/Bcl-2 ratio and the expressions of cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved PARP. In addition, it suppressed the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: UA attenuates oxidative stress-induced apoptosis via inhibiting the mitochondrial-related apoptosis pathway and modulating the p38 MAPK pathway, suggesting that it may be an effective neuroprotective agent.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.493-502
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• 2020

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (ΔΨm), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)-histone H2A.X. and caused a transition delay at the G₂/M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosisand necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved ΔΨm loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.