• Korean Journal of Microbiology
• /
• v.52 no.3
• /
• pp.278-285
• /
• 2016

The bifunctional PheA protein, having chorismate mutase and prephenate dehydratase (CMPD) activities, is one of the key regulatory enzymes in the aromatic amino acid biosynthesis in Escherichia coli, and is negatively regulated by an end-product, phenyalanine. Therefore, PheA protein has been thought as useful for protein engineering to utilize mass production of essential amino acid phenylalanine. To obtain feedback resistant PheA protein against phenylalanine, we mutated by using random mutagenesis, extensively screened, and obtained $pheA^{FBR}$ gene encoding a feedback resistant PheA protein. The mutant PheA protein contains substitution of Leu to Phe at the position of 118, displaying that higher affinity (about $290{\mu}M$) for prephenate in comparison with that (about $850{\mu}M$) of wild type PheA protein. Kinetic analysis showed that the saturation curve of $PheA^{FBR}$ against phenyalanine is hyperbolic rather than that of $PheA^{WT}$, which is sigmoidal, indicating that the L118F mutant enzyme has no cooperative effects in prephenate binding in the presence of phenylalanine. In vitro enzymatic assay showed that the mutant protein exhibited increased activity by above 3.5 folds compared to the wild type enzyme. Moreover, L118F mutant protein appeared insensitive to feedback inhibition with keeping 40% of enzymatic activity even in the presence of 10 mM phenylalanine at which the activity of wild type $PheA^{WT}$ was not observed. The substitution of Leu to Phe in CMPD may induce significant conformational change for this enzyme to acquire feedback resistance to end-product of the pathway by modulating kinetic properties.

• Journal of Oral Medicine and Pain
• /
• v.34 no.3
• /
• pp.261-276
• /
• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

• Tuberculosis and Respiratory Diseases
• /
• v.53 no.2
• /
• pp.113-126
• /
• 2002

Background : DNA repair plays a crucial role in protection from cancer-causing agents. Therefore, a reduced DNA repair capacity can increase the susceptibility to lung cancer. The XPC gene contains 15 exons and encodes a 940 amino acid protein that plays a central role in DNA damage recognition of the nucleotide excision repair pathway, which is a major DNA repair mechanisim removing the bulky-helix distorting DNA lesions caused by smoking. Recently several polymorphisms in the XPC gene were identified. In addition, it is possible that these polymorphisms may affect the DNA repair capacity, which modulate cancer susceptibility. The relationship between codon 499 and 939 polymorphisms, and a poly(AT) insertion/deletion polymorphism in the XPC gene, and the lung cancer risk were investigated. Materials and Methods : The genotypes were determined using either PCR or PCR-RFLP analysis in 219 male lung cancer patients and 150 healthy males controls. Results : The frequencies of the genotypes (Val499Ala, PAT and Lys939Gln) among the cases were not significantly different from those of the controls. There was no significant associantion between these polymorphi는 and the lung cancer risk when the analyses were stratified according to age, smoking status and the pack-years of smoking. Moreover, the genotypes had no apparent relationship with any of the histological types of lung cancer. There was a linkage disequilibrium among the Val499Ala, PAT and Lys939Gln polymorphisms. The PAT polymorphism had a strong linkage disequilibrium with the Lys939Gln polymorphism (kappa value=0.87). The XPC haplotypes showed no significant association with the lung cancer risk. Conclusion : These results suggest that XPC Val499Ala, PAT and Lys939Gln polymorphisms are not major contributors to the individual lung cancer susceptibility in Koreans.

• Journal of Life Science
• /
• v.25 no.6
• /
• pp.693-702
• /
• 2015

Blocking new blood-vessel formation (angiogenesis) is now recognized as a useful approach to the therapeutic treatment of many solid tumors. The best validated approach to date is to target the vascular endothelial growth-factor (VEGF) pathway, a key regulator of angiogenesis. Many natural products and extracts that contain a variety of chemopreventive compounds have been shown to suppress the development of malignancies through their anti-angiogenic properties. Phellodendron amurense, which is widely used in Korean traditional medicine, has been shown to possess antitumor, antimicrobial, and anti-inflammatory properties, among others. The present study investigated the effects of P. amurense hot-water extract (PAHWE) on angiogenesis, a key process in tumor growth, invasion, and metastasis. To investigate PAHWE’s anti-angiogenic properties, this study’s authors performed an analysis of angiogenesis and endothelial-cell proliferation, migration, invasion, and tube formation, as well as zymogram assays and the rat aortic ring-sprouting assay. PAHWE inhibited cell growth, mobility, and vessel formation in response to VEGF in vitro and ex vivo. Furthermore, it reduced VEGF-induced intracellular signaling events, such as the activation of matrix metalloproteinases (MMPs) -2 and -9. These results indicate that PAHWE’s anti-angiogenic properties might lead to the development of potential drugs for treating angiogenesis-associated diseases such as cancer.

• Journal of Life Science
• /
• v.30 no.11
• /
• pp.999-1006
• /
• 2020

The suppression of neuroinflammatory responses in microglial cells, well known as the main immune cells in the central nervous system (CNS), are considered a key target for improving the progression of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Teleogryllus emma is widely consumed around the world for its broad-spectrum therapeutic effect. In a previous work, we performed transcriptome analysis on T. emma in order to obtain the diversity and activity of its antimicrobial peptides (AMPs). AMPs are found in a variety of species, from microorganisms to mammals. They have received much attention as candidates oftherapeutic drugs for the treatment of inflammation-associated diseases. In this study, we investigated the anti-neuroinflammatory effect of Teleogryllusine (VKWKRLNNNKVLQKIYFVKI-NH₂) derived from T. emma on lipopolysaccharide (LPS) induced BV-2 microglia cells. Teleogryllusine significantly inhibited nitric oxide (NO) production without cytotoxicity, and reducing pro-inflammatory enzymes expression such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, Telegryllusine also inhibited the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) through down-regulation of the mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathway. These results suggest that T. emma-derived Teleogryllusine could be a good source of functional substances that prevent neuroinflammation and neurodegenerative diseases.

• Journal of the Korean Applied Science and Technology
• /
• v.37 no.3
• /
• pp.448-462
• /
• 2020

Dietary nutrition is a critical lifestyle factor that can reduce the risk of future cognitive impairments caused by dementia. Accumulating evidence suggests that dietary supplementation with Sulforaphane may help the prevention of cognitive impairments and dementia. Thus, Sulforaphane-enriched broccoli extract would hold promise to improve cognitive impairments of dementia patients. Here, we have used broccoli extracts, prepared from broccoli cultivated in Magma Seawater, to test if the broccoli extracts can be dietary supplement to improve cognitive impairments. Magma Seawater originated from Jeju Island, Korea is unique in terms of containing high concentrations of usable minerals (Zinc, Vanadium and Germanium etc.). Broccoli, grown in Magma Seawater, would contain Sulforaphane and the extra amount of usable minerals. The chemical compositions of the broccoli extracts were analyzed using LC-Q-orbitrap to detect Sulforaphane and Glucoraphanin. Analysis method based on HPLC was developed for measurement of sulforaphane levels in the broccoli extracts. We have tested if the broccoli extracts have anti-apoptotic and anti-inflammatory effects on neuron-like SH-SY5Y cells. In addition, we examined if the broccoli extracts are able to upregulate expression of synaptic plasticity-associated proteins (BDNF and phospho-CREB) and to inhibit acetylcholine esterase (AchE) activity. We have shown that the broccoli extracts inhibited the apoptotic pathway and inflammatory responses. Finally, we present evidence showing that AchE activity was inhibited by the broccoli extracts, but expression of BDNF and phospho-CREB was upregulated. Taken together, these findings suggest that the broccoli extracts from Magma Seawater-grown broccoli would be a good source of dietary nutrition to improve cognitive impairments in the future.

• Tuberculosis and Respiratory Diseases
• /
• v.44 no.3
• /
• pp.601-610
• /
• 1997

Background : Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis, cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-negative sepsis. In animals exposed to nonlethal doses of endotoxin, a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was known as 'LPS tolerance'. However, little information is available regarding the underlying mechanism of LPS tolerance. Method : Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate the conditions to obtain LPS tolerance, preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation, culture plates were washed two times and were stimulated with LPS at $1{\mu}g/ml$ for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or $PGF_2$ were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF-$\alpha$ and IL-8 and mRNA of TNF-$\alpha$ and IL-8 were determined by Northern blot analysis. Results : The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-$\alpha$, but not IL-8. Anti-CD14 Ab partially recovered production of TNF-$\alpha$ which was suppressed by preculture with low dose LPS. The preculture with PMA induces LPS tolerance, as preculture with low dose LPS. Conclusion : LPS tolerance to TNF-$\alpha$ is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.

• Tuberculosis and Respiratory Diseases
• /
• v.44 no.6
• /
• pp.1285-1295
• /
• 1997

Background : EGFR is one of the initial step in signal transduction pathway about multistep carcinogenesis. It is homologous to oncogene erbB-2 and is the receptor for EGF and TGF alpha. EGFR has important role in the growth and differentiation of tumor cells. So, EGFR in non-small cell lung cancer was examined to search for possible evidence as clinical prognostic factor. Methods : To investigate the role of EGFR in lung cancer, the author performed immunohistochemical stain of EGFR on 57 resected primary non-small cell lung cancer specimens. And the author analyzed the correlation between EGFR expression, clinical parameters, Sand $G_1$ phase fraction and survival. Results : 1) EGFR were detected in 56% of total 57 patients (according to histologic type, squamous cancer 50%, adenocarcinoma 63%, large cell cancer 75%) (according to TNM stage, stage I 64%, stage II 38%, stage III 55%) (according to cellular differentiation, well 50%, moderately 52%, poorly 65%). All differences were insignificant 2) Using the flow cytometric analysis, mean S-phase fraction of EGFR (+) and (-) group were 22.3(${\pm}10.5$)%. 18.0(${\pm}10.9$)% (p>0.05), mean $G_1$-phase fraction of EGFR (+) and (-) group were 68.4(${\pm}11.6$)%, 71.1(${\pm}12.8$)%, (p>0.05) 3) Two-year survival rate of EGFR (+) and (-) group were 53%, 84%, median survival time of EGFR (+) and (-) group were 26, 53 months. (p<0.05, Kaplan-Meier, generalized Wilcox) Conclusion : EGFR immunostaining may be a simple and useful method for survival prediction in non-small cell lung cancer.

• Tuberculosis and Respiratory Diseases
• /
• v.54 no.5
• /
• pp.542-550
• /
• 2003

Background : Synthetic glucocorticoids are widely used in many chronic inflammatory diseases because of their excellent anti-inflammatory activity. Enhancing the transcription of $I{\kappa}B$ and preventing activated NF-${\kappa}B$ from binding to ${\kappa}B$ sites are thought to be the underlying mechanisms. But these data are largely derived from in vitro studies using cell lines. In this study, after administrating a steroid to volunteers, we evaluated the effect on the NF-${\kappa}B$ system. Methods : Prednisolone(0.5mg/kg/d) was orally administered to 5 healthy volunteers for 7 days. Before and after the administration, we sampled their peripheral blood monocytes, and performed western blot analysis both with stimulation, using IL-$1{\beta}$, LPS, TNF, and without stimulation(baseline). We also performed EMSA after stimulation with LPS. Results : After ingestion of the steroid, baseline expressions of $I{\kappa}B{\alpha}$ were increased in two of the subjects, while suppressed degradations of $I{\kappa}B{\alpha}$ to stimulations were observed in all five. In addition, the binding capacity of NF-${\kappa}B$ after the administration was decreased. Conclusion : Steroid plays such roles as enhancing the transcription of $I{\kappa}B{\alpha}$, suppressing the DNA binding capacity of NF-${\kappa}B$, and suppressing the degradation of $I{\kappa}B{\alpha}$.

• Korean Journal of Environmental Biology
• /
• v.27 no.1
• /
• pp.66-72
• /
• 2009

Diabetic Retinopathy (DR) is a leading cause of blindness among adults in the western countries. Hyperglycemia is a condition, that induces apoptotic cell death in a variety of cell types in diabetes, but the mechanism remains unclear. The aim of the study is to understand the effects of high Glucose on Human Retinal Endothelial Cells. Retinal endothelial cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 5, 25 and 50 mM Glucose, incubated for 24, 36 and 48 hours in humidified 5 % CO$_2$ incubator at 37$^{\circ}C$. Human Retinal Endothelial Cell Line (HREC) were characterized for morphology with different treatment by phase contrast microscopic analysis. Number of dead and viable cells was counted by trypan blue exclusion and supported by MTT assay. The intracellular Hydrogen peroxide (H$_2$O$_2$), a Reactive Oxygen Species (ROS) generation in high glucose conditions was assessed by FOX II assay and apoptosis by caspase-3 assay. The high glucose treated cells undergoing DNA fragmentation was witnessed by Agarose gel electrophoresis. We found that the cells incubated with 25 and 50 mM glucose containing medium for 48 hours altered the morphology of the cell, induced apoptosis and DNA fragmentation. The dead cell number were high in 25 and 50 mM when compared to the cells incubated with 5 mM glucose for 24, 36, and 48 hours. Also, the H$_2$O$_2$ levels and the activity of caspase-3 were increased in high glucose treated cells. Conclusions/interpretation: Our results demonstrated that elevated glucose induces apoptosis in cultured HREC. The hyperglycemia-induced increase in apoptosis may be dependent on caspase activation. The association between ROS generation and caspase-3 activation on high glucose treated cells is yet to be investigated.