• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.44-54
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• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• Biomolecules & Therapeutics
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• 제22권3호
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• pp.193-199
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• 2014

The aim of this study was to determine whether britanin, isolated from the flowers of Inula japonica (Inulae Flos), modulates the generation of allergic inflammatory mediators in activated mast cells. To understand the biological activity of britanin, the authors investigated its effects on the generation of prostaglandin $D_2$ ($PGD_2$), leukotriene $C_4$ ($LTC_4$), and degranulation in IgE/Ag-induced bone marrow-derived mast cells (BMMCs). Britanin dose dependently inhibited degranulation and the generations of $PGD_2$ and $LTC_4$ in BMMCs. Biochemical analyses of IgE/Ag-mediated signaling pathways demonstrated that britanin suppressed the phosphorylation of Syk kinase and multiple downstream signaling processes, including phospholipase $C{\gamma}1$ ($PLC{\gamma}1$)-mediated calcium influx, the activation of mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2, c-Jun $NH_2$-terminal kinase and p38), and the nuclear factor-${\kappa}B$ ($NF-{\kappa}B$) pathway. Taken together, the findings of this study suggest britanin suppresses degranulation and eicosanoid generation by inhibiting the Syk-dependent pathway and britanin might be useful for the treatment of allergic inflammatory diseases.

• 미생물학회지
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• 제31권4호
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• pp.261-266
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• 1993

유일한 탄소 및 에어지원으로 메탄올만을 이용하여 성장하는 새로운 절대 메탄올 자화세균을 토양으로 부터 분리하였다. 분리균주는 그람음성의 운동성이 없고 포자를 형성하지 않는 간균으로 정대호기성 세균이었다. 이 균주는 catalase 활성과 oxidase 활성을 가지고 있으며, nitrate를 nitrite로 환원시킬 수 있고 성장시 vitamin이나 특이한 생육인자를 요구하지 않았다. 세대시간은 1.6시간으로 메탄올 동화경로로는 ribulose monophosphate pathway(Entner-Doudoroff 변형경로)를 이용하나 α-ketoglutarate dehydrogenase 활성은 없었다. Ammonium ion은 glutamate dehydrogenase를 이용하여 동화하였다. DNA의 guanine plus cytosine 함량은 61.0 mol%이고 세포내 지방산으로는 누고 straight-chain saturated C$^{16:0}$ acids(palmitie acids)와 unsaturated C$^{16:1}$ acids(palmitoleic acids)를 가지고 있으나 이외에도 두 종류의 unidentified C$_{17}$ branched fatty acid도 포함하고 있었다. Major ubiquinone은 Q-8이나 Q-6와 Q-7을 특이하게 소량 가지고 있었다. Phospatidylethanolamine과 Phosphatidylglycerol이 주요한 phospholip의 구성물질이나 diphosphatidylgycerol도 소량 포함하고 있었다. 위와같은 생리적, 생화학적 자료로 부터 분리균주를 새로은 종 즉, Methylohacillus methanolovorus sp. nov.로 명명하였다.

• Molecules and Cells
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• 제47권1호
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• pp.100001.1-100001.8
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• 2024

In eukaryotes, a primary protein quality control (PQC) process involves the destruction of conformationally misfolded proteins through the ubiquitin-proteasome system. Because approximately one-third of eukaryotic proteomes fold and assemble within the endoplasmic reticulum (ER) before being sent to their destinations, the ER plays a crucial role in PQC. The specific functions and biochemical roles of several E3 ubiquitin ligases involved in ER-associated degradation in mammals, on the other hand, are mainly unknown. We identified 2 E3 ligases, ubiquitin protein ligase E3 component N-recognin 1 (UBR1) and ubiquitin protein ligase E3 component N-recognin 2 (UBR2), which are the key N-recognins in the N-degron pathway and participate in the ER stress response in mammalian cells by modulating their stability. Cells lacking UBR1 and UBR2 are hypersensitive to ER stress-induced apoptosis. Under normal circumstances, these proteins are polyubiquitinated through Lys48-specific linkages and are then degraded by the 26S proteasome. In contrast, when cells are subjected to ER stress, UBR1 and UBR2 exhibit greater stability, potentially as a cellular adaptive response to stressful conditions. Although the precise mechanisms underlying these findings require further investigation, our findings show that cytoplasmic UBR1 and UBR2 have anti-ER stress activities and contribute to global PQC in mammals. These data also reveal an additional level of complexity within the mammalian ER-associated degradation system, implicating potential involvement of the N-degron pathway.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.540-549
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• 2021

The Wnt/β-catenin signaling pathway is involved in breast cancer and Myxococcus fulvus KYC4048 is a myxobacterial strain that can produce a variety of bioactive secondary metabolites. Although a previous study revealed that KYC4048 metabolites exhibit anti-proliferative effects on breast cancer, the biochemical mechanism involved in their effects remains unclear. In the present study, KYC4048 metabolites were separated into polar and non-polar (ethyl acetate and n-hexane) fractions via liquid-liquid extraction. The effects of these polar and non-polar KYC4048 metabolites on the viability of breast cancer cells were then determined by MTT assay. Expression levels of Wnt/β-catenin pathway proteins were determined by Western blot analysis. Cell cycle and apoptosis were measured via fluorescence-activated cell sorting (FACS). The results revealed that non-polar KYC4048 metabolites induced cell death of breast cancer cells and decreased expression levels of WNT2B, β-catenin, and Wnt target genes (c-Myc and cyclin D1). Moreover, the n-hexane fraction of non-polar KYC4048 metabolites was found most effective in inducing apoptosis, necrosis, and cell cycle arrest, leading us to conclude that it can induce apoptosis of breast cancer cells through the Wnt/β-catenin pathway. These findings provide evidence that the n-hexane fraction of non-polar KYC4048 metabolites can be developed as a potential therapeutic agent for breast cancer via inhibition of the Wnt/β-catenin pathway.

• IMMUNE NETWORK
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• 제1권2호
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• pp.95-103
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• 2001

T cells play a central role in the initiation and regulation of the immune response to foreign antigens. Full activation of T cells requires the engagement of T cell receptor complex (TCR) and the binding of a second costimulatory receptor to its ligand expressed on antigen presenting cells (APC). Among the molecules known to provide costimulatory function, CD28 has been the most dominant and potent costimulatory molecule. However, the function of CD28 is becoming more complex due to the recent discovery of its structural homologue, CTLA-4 and ICOS. This review summarizes the biology and physiologic function of each of these receptors, and further focuses on the biochemical mechanism underlying the function of these receptors. Complete understanding of the CD28/CTLA-4/ICOS costimulatory pathway will provide the basis for developing new therapeutic approaches for immunological dieseases.

• 미생물학회지
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• 제23권3호
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• pp.230-234
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• 1985

Malonate를 유일한 탄소원으로 활용할 수 있는 세균을 토양으로부터 분리하였다. 이 세균은 행태, 배양, 생리 그리고 생화학적 연구를 통하여 Acinetobacter calcoaceticus임이 확인되었다. 이 미생물을 malonate를 유일한 탄소원으로 하는 배지에서 배양하였을 갱우, malonyl CoA synthetase, isocitrate lyase 및 malate, synthase가 유도 되었다. 따라서 이 미생물에서도 Pseudomonas fluorescens에서 제안되었던 대사경로 즉 $malonate{\rightarrow}malonyl-CoA{\rightarrow}acetyl-CoA{\rightarrow}glyoxylate\;cycle$을 통하여 malonate를 이용하는 것으로 판단된다.

• 생명과학회지
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• 제9권4호
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• pp.408-413
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• 1999

Extracellular capsidiol, sesquiterpenoid phytoalexin, in the medium of pepper (Capsicum annuum L.) suspension cells was not identified from control cells, but highly accumulated in the elicitor-induced cells within 6 hours after the addition of 0.05$\mu\textrm{g}$/$m\ell$ cellulase. Capsidiol production in elicitor-induced cells was markedly suppressed by cytochrome P450 inhibitors, such as ancymidol and ketoconazole demonstrating that biosynthesis of capsidiol is catalyzed by at least on hydroxylation enzyme in the biochemical pathway. Based on protein electrophoresis, two bands, 23.0kDa and 27.5kDa, were identified as newly synthesized polypeptides in the elicitor-induced suspension cells, suggesting that pepper cells which were subjected to elicitor treatment activate specific gene(s) for capsidiol biosynthesis in cultured cells.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.334-337
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• 2008

A tightly regulated gene expression system composed of a single-copy target gene under the control of a lac promoter derivative and lacI gene in a multicopy plasmid is proposed, and its ability to control the flux of a metabolic pathway is demonstrated. A model system to control the flux of acetyl-CoA to acetyl phosphate was constructed by integrating pta, a gene encoding phosphotransacetylase, under a tac promoter into the chromosome of E. coli with a pta-negative background and transforming a multicopy plasmid containing the $lacI^Q$ gene into the strain. The production rate of acetate was shown to be tightly controlled when varying the concentration of the inducer (IPTG) in he model system.