• The Journal of Korean Medicine
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• v.23 no.1
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• pp.24-36
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• 2002

Objectives: The chemotherapeutic potential of Gagamgilgyung-tang for the treatment of human lung cancer, the antitumorigenic effects of Gagamgilgyung-tang on the proliferation and apoptosis of human lung cancer cell line A427 were investigated using molecular biological approaches, Methods: To determine Gagamgilgyung-tang concentrations which do not evoke cytotoxic damage to the cell line, cell viability was examined by MTT assay. To prove Gagamgilgyung-tang's antitumorigenic potential to human lung cancer, [3H]thymidine incorporation assay, trypan blue exclusion and Cpp32 protease activity assays and quantitative RT-PCR analysis were examined. Results: While A427 cells treated with $0.1-2.0{\mu\textrm{g}}/ml$ of Gagamgilgyung-tang showed no recognizable effect, marked reductions of cell viability were detected at concentrations over $5.0{\;}\mu\textrm{g}/ml$. DNA replication of A427 cells was inhibited by Gagamgilgyung-tang in a dose-dependent manner and Gagamgilgyung-tang induced the G1 cell cycle arrest through inhibition of DNA replication. Gagamgilgyung-tang triggered apoptotic cell death of A427 and enhanced the apoptotic sensitivity of the cells that were injured by a DNA damage-inducing chemotherapeutic drug etoposide. Gagamgilgyung-tang induces expression of growth-inhibiting genes such as p53 and p21/Wafl whereas it inhibited expression of growth-promoting genes such as c-Myc and Cyclin D1. Expression of a representative apoptosis-inducing gene Bax was also found to be induced by Gagamgilgyung-tang while apoptosis-suppressing Bcl-2 expression was not changed. Conclusions: Gagamgilgyung-tang could suppress the abnormal growth of tumor cells by suppressing the survival of genetically altered cells via induction of apoptosis. This study suggests that Gagamgilgyung-tang might have an antitumorigenic potential to human lung cancer cells, which might be associated with its growth-inhibiting and apoptosis-inducing properties.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1614-1623
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• 2010

Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to $41^{\circ}C$ and at pH 11.

• Molecules and Cells
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• v.28 no.5
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• pp.463-472
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• 2009

Although the possible cellular roles of several ubiquitin-specific proteases (UBPs) were identified in Arabidopsis, almost nothing is known about UBP homologs in rice, a monocot model plant. In this report, we searched the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE) and identified 21 putative UBP family members (OsUBPs) in the rice genome. These OsUBP genes each contain a ubiquitin carboxyl-terminal hydrolase (UCH) domain with highly conserved Cys and His boxes and were subdivided into 9 groups based on their sequence identities and domain structures. RT-PCR analysis indicated that rice OsUBP genes are expressed at varying degrees in different rice tissues. We isolated a full-length cDNA clone for OsUBP6, which possesses not only a UCH domain, but also an N-terminal ubiquitin motif. Bacterially expressed OsUBP6 was capable of dismantling K48-linked tetra-ubiquitin chains in vitro. Quantitative real-time RT-PCR indicated that OsUBP6 is constitutively expressed in different tissues of rice plants. An in vivo targeting experiment showed that OsUBP6 is predominantly localized to the nucleus in onion epidermal cells. We also examined how knock-out of OsUBP6 affects developmental growth of rice plants. Although homozygous T3 osubp6 T-DNA insertion mutant seedlings displayed slower growth relative to wild type seedlings, mature mutant plants appeared to be normal. These results raise the possibility that loss of OsUBP6 is functionally compensated for by an as-yet unknown OsUBP homolog during later stages of development in rice plants.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• Microbiology and Biotechnology Letters
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• v.46 no.4
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• pp.313-319
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• 2018

Halophilic lactic acid bacteria (LAB) were isolated from myeolchi-jeotgal (23% NaCl, w/v) fermented in jangdok (Korean earthenware) located outside a house. They were identified as Tetragenococcus halophilus by 16S rRNA and recA gene sequencing. Four T. halophilus isolates showing high protease activities were selected for further studies. Four strains grew well, reaching $OD_{600}$ values of 0.75-0.92 at 18% NaCl content (w/v) and 0.28-0.44 at 23% salt. They showed rapid growth, attaining $OD_{600}$ values of 1.1-1.2 at $20-30^{\circ}C$, but did not grow at $4^{\circ}C$. At $15^{\circ}C$, the highest $OD_{600}$ values, which exceeded 0.6, were observed at 20 days, and were higher than those of cultures at $37^{\circ}C$ and $42^{\circ}C$ (approximately 0.5). Four isolates grew best in broth where the initial pH was adjusted to 8 and did not grow at $pH{\leq}4$. T. halophilus BS2-36 showed the highest survival ratio of 18.7% after 2 hours of exposure at pH 3. BS2-36 showed the highest survival ratio (1.29%) in presence of 0.3% bile salts. T. halophilus BS2-36 seems a promising candidate as a starter for jeotgal and other fermented foods with high salinities.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.813-819
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• 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe respiratory impairment with a reported mortality rate of ~36% in humans. The absence of clinically available MERS-CoV vaccines and treatments to date has resulted in uncontrolled incidence and propagation of the virus. In vaccine design, fusion with the IgG Fc domain is reported to increase the immunogenicity of various vaccine antigens. However, limited reports have documented the potential negative effects of Fc fusion on vaccine antigens. To determine whether Fc fusion affects the immunogenicity of MERS-CoV antigen, we constructed a Fcassociated MERS-CoV spike protein (eS770-Fc, 110 kDa), whereby human IgG4 Fc domain was fused to MERS-CoV spike protein (eS770) via a Gly/Pro linker using baculovirus as the expression system. For comparative analyses, two eS770 proteins lacking the IgG4 Fc domain were generated using the IdeS protease ($eS770-{\Delta}Fc$) or His tag attachment (eS770-His) and the immunogenicity of the above constructs were examined following intramuscular immunization in mice. Contrary to expectations, non-Fc spike proteins ($eS770-{\Delta}Fc$, eS770-His; 90 kDa) showed higher immunogenicity than the Fc fusion protein (eS770-Fc). Moreover, unlike non-Fc spike proteins, eS770-Fc immunization did not elicit neutralizing antibodies against MERS-CoV. The lower immunogenicity of Fc-fused eS770 was related to alterations in the structural conformation of the spike protein. Taken together, our results indicate that IgG Fc fusion reduces the immunogenicity of eS770 by interfering with the proper folding structure.

• The Plant Pathology Journal
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• v.37 no.6
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• pp.632-640
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• 2021

Cucumber mosaic virus (CMV) and Pepper mild mottle virus (PMMoV) causes severe economic loss in crop productivity of both agriculture and horticulture crops in Korea. The previous surveys showed that naturally available biopolymer material - chitosan (CS), which is from shrimp cells, reduced CMV accumulation on pepper. To improve the antiviral activity of CS, it was synthesized to form phosphate cross-linked chitosan (PCS) and compared with the original CS. Initially, the activity of CS and PCS (0.01%, 0.05%, and 0.1% concentration) compound against PMMoV infection and replication was tested using a half-leaf assay on Nicotiana glutinosa leaves. The total number of local lesions represented on a leaf of N. glutinosa were counted and analyzed with phosphate buffer treated leaves as a negative control. The leaves treated with a 0.1% concentration of CS or PCS compounds exhibited an inhibition effect by 40-75% compared with the control leaves. The same treatment significantly reduced about 40% CMV accumulation measured by double antibody sandwich enzyme-linked immunosorbent assay and increased the relative expression levels of the NPR1, PR-1, cysteine protease inhibitor gene, LOX, PAL, SRC2, CRF3 and ERF4 genes analyzed by quantitative reverse transcriptase-polymerase chain reaction, in chili pepper plants.

• BMB Reports
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• v.54 no.12
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• pp.592-600
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• 2021

Parkinson's disease (PD) is one of the most common neurodegenerative diseases in the elderly population and is caused by the loss of dopaminergic neurons. PD has been predominantly attributed to mitochondrial dysfunction. The structural alteration of α-synuclein triggers toxic oligomer formation in the neurons, which greatly contributes to PD. In this article, we discuss the role of several familial PD-related proteins, such as α-synuclein, DJ-1, LRRK2, PINK1, and parkin in mitophagy, which entails a selective degradation of mitochondria via autophagy. Defective changes in mitochondrial dynamics and their biochemical and functional interaction induce the formation of toxic α-synuclein-containing protein aggregates in PD. In addition, these gene products play an essential role in ubiquitin proteasome system (UPS)-mediated proteolysis as well as mitophagy. Interestingly, a few deubiquitinating enzymes (DUBs) additionally modulate these two pathways negatively or positively. Based on these findings, we summarize the close relationship between several DUBs and the precise modulation of mitophagy. For example, the USP8, USP10, and USP15, among many DUBs are reported to specifically regulate the K48- or K63-linked de-ubiquitination reactions of several target proteins associated with the mitophagic process, in turn upregulating the mitophagy and protecting neuronal cells from α-synuclein-derived toxicity. In contrast, USP30 inhibits mitophagy by opposing parkin-mediated ubiquitination of target proteins. Furthermore, the association between these changes and PD pathogenesis will be discussed. Taken together, although the functional roles of several PD-related genes have yet to be fully understood, they are substantially associated with mitochondrial quality control as well as UPS. Therefore, a better understanding of their relationship provides valuable therapeutic clues for appropriate management strategies.

• Parasites, Hosts and Diseases
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• v.60 no.2
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• pp.117-126
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• 2022

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.

• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1462-1470
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• 2022

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70℃ and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.